Trends Sci. 202 6 ; 23 (2): 11549

Molecular Mechanisms and Experimental Protocols in the Study of Vasorelaxant Activity in Aortic Smooth Muscle Cells

Sirojiddin Omonturdiev ^1,4,5,* , Izzatullo Abdullaev ¹ , Alikhon Khasanov ³ , Azizbek Abdullaev ¹ , Dolimjon Inomjanov ³ , Ulugbek Gayibov ¹ , Sabina Gayibova ¹ , Kuzijon Baratov ¹ ,

Yulduzkhon Mirzaeva ² , Mirtaza Allamuratov ⁴ , Pulat Usmonov ² and Takhir Aripov ¹

¹ A. S. Sadykov Institute of Bioorganic Chemistry, Science Academy of Uzbekistan, Laboratory of Plant Cytoprotectors, Tashkent 100007, Uzbekistan

² Institute of Biophysics and Biochemistry, National University of Uzbekistan, Tashkent 100174 , Uzbekistan

³ Department of Anatomy and Physiology, Namangan State University, Namangan, Uzbeksitan

⁴ Department of Human and Animal Physiology, National University of Uzbekistan, Tashkent 100174, Uzbeksitan

⁵ Oriental University, Tashkent, Uzbekistan

( ^* Corresponding author’s e-mail: [email protected])

Received: 9 August 2025, Revised: 19 September 2025, Accepted: 1 October 2025, Published: 10 November 2025

Abstract

Vasorelaxation is a key process in cardiovascular regulation and a promising target for treating hypertension and vascular dysfunctions. This study outlines a comprehensive experimental framework for evaluating vasorelaxant mechanisms in isolated rat aortic rings using isometric tension recording. A combination of pharmacological tools and ionic modulation was used to explore the roles of voltage-dependent calcium channels, GPCR signaling, intracellular calcium stores (SERCA and ryanodine receptors), ion exchangers (NCX, Na⁺/K⁺-ATPase) and endothelium-derived factors. Computational docking complemented experimental data by identifying ligand-binding sites on key ion channels. Findings revealed that vasorelaxation involves multifaceted modulation of calcium influx, receptor-mediated signaling, and intracellular calcium cycling, with both endothelium-dependent and -independent pathways contributing. This integrative approach provides novel insights into vascular smooth muscle regulation and supports the development of targeted cardiovascular therapies.

Keywords: Vasorelaxation, Calcium channels, SERCA, Ryanodine receptors, Na⁺/Ca²⁺ exchanger

Introduction

Vasorelaxation, the process by which blood vessels decrease their tone and widen, plays a fundamental role in the regulation of blood pressure, tissue perfusion, and vascular homeostasis. It is a tightly controlled physiological response governed by a complex interplay of neural, humoral and local signals that act on the vascular smooth muscle [1]. Therapeutically, enhancing vasorelaxation is central to the management of hypertension, ischemic heart disease and other cardiovascular disorders. Agents that promote smooth muscle relaxation—either by interfering with calcium signaling, modulating ion channels, or activating endothelial-derived pathways—form the pharmacological backbone of antihypertensive and vasoprotective therapies [2,3].

Among the various vascular segments, the aorta stands out as a crucial structure in the maintenance of systemic hemodynamics. As the largest elastic artery, the aorta buffers pulsatile cardiac output and ensures continuous blood flow during diastole [4]. The smooth muscle cells (SMCs) embedded within its medial layer not only provide structural integrity but also respond dynamically to vasoactive stimuli. Aortic SMCs regulate vascular tone via calcium influx, intracellular calcium mobilization and actomyosin cross-bridge cycling—processes that are highly sensitive to pharmacological agents and pathological stimuli. Importantly, alterations in aortic SMC reactivity are strongly associated with the pathogenesis of hypertension, arterial stiffness, and cardiovascular remodeling [5].

Due to their well-characterized contractile behavior and accessibility in ex vivo setups, aortic smooth muscle cells (particularly in isolated aortic rings) have become a widely accepted experimental model for studying vasorelaxation [6]. This model allows for reproducible investigation of vascular responses under controlled physiological conditions, enabling the dissection of both endothelium-dependent and -independent mechanisms [7].

To unravel the intricate molecular mechanisms underlying vasorelaxation, structured experimental protocols are essential. These include protocols designed to isolate specific ion channel activities (e.g., L-type Ca²⁺ channels, store-operated calcium entry, Na⁺/Ca²⁺ exchangers), investigate receptor-mediated signaling pathways and study intracellular calcium handling via SERCA pumps and ryanodine receptors [8,9]. Combining pharmacological tools with functional assays in the aorta not only aids in identifying molecular targets of candidate vasorelaxant compounds but also provides a translational platform for developing next-generation antihypertensive drugs [10].

In this review, we aim to present a detailed overview of the molecular mechanisms that govern aortic smooth muscle tone, alongside the experimental strategies commonly employed to evaluate them. Emphasis is placed on ion transport systems, receptor-mediated signaling, and intracellular calcium dynamics, with a view toward understanding how these pathways can be modulated to promote vascular relaxation under physiological and pathological conditions.

Aortic smooth muscle cell structure and functional mechanisms

Aortic smooth muscle cells (ASMCs) are elongated, spindle-shaped, non-striated cells organized in concentric layers within the tunica media of the aorta. Unlike skeletal and cardiac muscle fibers, these cells do not form sarcomeres and are instead embedded in a complex extracellular matrix composed predominantly of elastin, collagen and glycosaminoglycans [11]. This matrix not only provides mechanical support and elasticity but also plays a crucial role in mechanotransduction and cellular signaling. The cells are interconnected via gap junctions, allowing for coordinated contraction across the vascular wall [12].

Each smooth muscle cell contains a single, centrally located nucleus, surrounded by abundant contractile filaments composed of actin and myosin. These filaments are anchored to dense bodies and dense bands—cytoplasmic and membrane-associated structures, respectively—that serve a function analogous to Z-lines in striated muscle [13]. The spatial arrangement of these filaments allows for a unique contractile behavior characterized by slow, sustained contractions and high energy efficiency, critical for maintaining vascular tone over prolonged periods [14].

Aortic smooth muscle cells also exhibit significant phenotypic plasticity, transitioning between a "contractile" state—marked by high expression of contractile proteins such as α-smooth muscle actin, myosin heavy chain and calponin—and a “synthetic” state, in which the cells show increased proliferative, migratory, and secretory activity [15]. This transition plays a key role in vascular development, remodeling, and disease progression, particularly in conditions such as atherosclerosis and hypertension [16] ( Figure 1 ).

The functional activity of ASMCs is primarily governed by complex ion transport systems and intracellular signaling cascades that control the excitation-contraction coupling process. Intracellular calcium (Ca²⁺) serves as the central second messenger regulating contraction [17]. Under physiological conditions, contraction is initiated when an external stimulus—mechanical stretch, neurotransmitter, or hormone—activates membrane-bound receptors or causes membrane depolarization. This leads to the opening of L-type voltage-dependent calcium channels, allowing extracellular Ca²⁺ influx into the cytoplasm [18].

In addition to voltage-gated channels, receptor-operated calcium channels (ROCCs) are activated by ligands such as norepinephrine or angiotensin II, which bind to G protein-coupled receptors (GPCRs) on the cell membrane [19]. Activation of GPCRs stimulates phospholipase C (PLC), leading to the generation of inositol trisphosphate (IP₃), which diffuses to the sarcoplasmic reticulum (SR) and triggers Ca²⁺ release through IP₃ receptors (IP₃Rs). Parallelly, ryanodine receptors (RyRs) on the SR membrane can also release stored Ca²⁺, particularly through calcium-induced calcium release mechanisms [20].

As intracellular Ca²⁺ levels rise, Ca²⁺ binds to calmodulin, forming a Ca²⁺–calmodulin complex that activates myosin light chain kinase (MLCK). MLCK phosphorylates the regulatory light chains of myosin, enabling cross-bridge formation with actin filaments and initiating contraction. Relaxation occurs when intracellular Ca²⁺ levels fall, either through active reuptake into the SR via SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase) or extrusion across the plasma membrane by plasma membrane Ca²⁺ ATPase (PMCA) and Na⁺/Ca²⁺ exchangers (NCX) [21].

Membrane potential dynamics are also critical in regulating vascular tone. Potassium channels—such as BK_Ca (large-conductance Ca²⁺-activated K⁺ channels), voltage-gated K⁺ (KV) channels and ATP-sensitive K⁺ (K_ATP) channels—mediate membrane hyperpolarization, thereby inhibiting voltage-gated calcium channels and promoting relaxation [22]. Conversely, closure of these K⁺ channels leads to depolarization and contraction. Chloride channels and non-selective cation channels may further modulate membrane excitability and ion fluxes [23].

Figure 1 Mechanisms of intracellular Ca²⁺ regulation and smooth muscle contraction in vascular smooth muscle cells. Activation of G protein-coupled receptors (GPCRs) leading to myosin light chain phosphorylation and smooth muscle contraction.

Importantly, smooth muscle contractility is not solely determined by [Ca²⁺]ᵢ, but also by Ca²⁺ sensitization mechanisms, such as the inhibition of MLCP via RhoA/ROCK and PKC pathways. These pathways enhance contractility without altering intracellular calcium concentration, contributing to sustained vascular tone often seen in pathological states [24].

The coordination between ion channel activity, receptor signaling, calcium handling, and cytoskeletal dynamics defines the contractile behavior of aortic smooth muscle. Dysregulation of any of these components can contribute to vascular dysfunction, increased arterial stiffness, and elevated blood pressure. Thus, understanding the structural and functional basis of ASMC physiology is vital for developing targeted therapeutic interventions in cardiovascular diseases [25].

Experimental setup and required equipment

Tissue preparation and tension recording

Functional studies of vascular smooth muscle, particularly for assessing vasorelaxant or vasoconstrictor responses, are typically conducted using isolated aortic ring preparations mounted in organ bath systems. This classic ex vivo approach allows for precise control of the extracellular environment and facilitates the investigation of direct effects of pharmacological agents on vascular tone under isometric conditions [26].

Freshly excised thoracic aortae from adult rodents (commonly rats) are carefully cleaned of connective tissue and fat under a stereomicroscope in cold, oxygenated physiological saline solution. The aorta is then sectioned into 3 - 5 mm wide ring segments ( Figure 2 ). In some experiments, the endothelium is mechanically removed by gently rubbing the luminal surface with a stainless steel wire, which enables separate analysis of endothelium-dependent and -independent mechanisms [27].

Figure 2 Dissection and isolation of the thoracic aorta and spinal cord in a rodent model (A - D) .

Sequential steps of thoracic aorta isolation: (A) The chest cavity is opened and the heart is exposed; (B - D) The thoracic aorta is carefully separated from surrounding connective tissue and excised using micro scissors and forceps. (E - F) Spinal cord exposure: (E) The vertebral column is exposed by removing overlying muscle tissue (black arrow indicates spinal cord); (F) Laminectomy is performed to isolate the spinal cord. (G) The excised thoracic spinal cord segment placed on a clean surface for further analysis.

The aortic rings are suspended between two stainless steel hooks: One fixed to the bottom of the organ chamber and the other connected to a highly sensitive isometric force transducer (e.g., ADInstruments MLT0202 or Grass FT03), allowing continuous recording of changes in vascular tone ( Figure 3 ). Each organ bath contains Krebs-Henseleit buffer solution (composed of NaCl 118.0 mM, KCl 4.7 mM, CaCl₂ 2.5 mM, KH₂PO₄ 1.2 mM, MgSO₄ 1.2 mM, NaHCO₃ 25.0 mM and glucose 11.0 mM) maintained at 37 ± 0.5 °C and continuously bubbled with a gas mixture of 95% O₂ and 5% CO₂ to maintain physiological pH (7.4). Before initiating experiments, aortic rings are allowed to equilibrate under an optimal resting tension of 1 - 2 g for at least 45 - 60 min, during which the buffer is replaced every 15 min [28].

Standardized protocols include precontraction of the tissue using high-potassium depolarizing solution (e.g., 60 mM KCl) or agonists such as phenylephrine (PE, 1 μM), followed by cumulative or single-dose administration of the test compound. Isometric tension changes are continuously recorded using data acquisition software (e.g., LabChart, PowerLab) for analysis of relaxation responses expressed as percentage of initial contraction [29].

This model system remains a gold standard in vascular pharmacology, enabling mechanistic dissection of ion channel modulation, second messenger involvement and endothelial contribution in the vasorelaxant or vasoconstrictor activity of bioactive compounds [30].

Figure 3 Schematic representation of the organ bath system used for isometric tension recording of isolated rat aortic rings. The setup includes: (1) organ bath chamber maintained at 37°C by (3) a circulating water bath (DAIHAN), (2) filled with Krebs-Henseleit solution, (4) continuously aerated with a 95% O₂ and 5% CO₂ gas mixture, and (5) connected to an isometric force transducer to detect contractile responses. The data is acquired through (6) a GoLink interface. The aortic ring (~1 g tension ≈ 10 mN) is suspended between two hooks within the chamber (right), allowing for precise drug addition and real-time measurement of smooth muscle responses under controlled physiological conditions.

Reagents and solutions

Krebs–Henseleit (sources?) buffer (KHB) was used as the physiological solution to maintain the viability and contractile responsiveness of isolated aortic rings during ex vivo studies. The standard composition of KHB (in mM) was as follows: NaCl 118.0, KCl 4.7, CaCl₂·2H₂O 2.5, MgSO₄·7H₂O 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0 and glucose 11.0. The buffer was freshly prepared each day using analytical-grade reagents dissolved in double-distilled water. The solution was continuously gassed with a carbogen mixture (95% O₂ and 5% CO₂) to maintain a stable physiological pH of 7.4 and kept at 37 ± 0.5 °C throughout the experiments [31,32].

Solvent systems for dissolving test compounds included dimethyl sulfoxide (DMSO) and methanol (MeOH), depending on the compound’s solubility profile. To avoid solvent-induced artifacts or tissue toxicity, the final concentration of organic solvents in the organ bath was strictly limited to ≤ 0.1% (v/v). All stock solutions were prepared at high concentrations (e.g., 10 - 100 mM) and stored at −20 °C in airtight amber vials to minimize degradation and photo-oxidation. Immediately prior to application, stock solutions were diluted into KHB to achieve working concentrations [33].

During pre-incubation , isolated aortic rings were allowed to equilibrate in KHB for at least 45 - 60 min under a resting passive tension of 1.0 - 2.0 g. This period ensures stabilization of basal tone and allows recovery from mechanical stress induced during dissection and mounting. During equilibration, the buffer was replaced every 15 - 20 min to maintain optimal ionic composition and oxygenation. In some protocols, a pre-treatment with inhibitors or vehicle control was performed during the last 15 - 30 min of the equilibration phase to assess modulatory effects on contractility or relaxation responses [34].

This rigor in buffer composition, solvent handling, and pre-experimental conditioning is essential to maintain tissue viability and ensure reproducibility of pharmacological responses in vascular reactivity studies.

Voltage-dependent ca²⁺ channels: Membrane influx mechanism

Depolarization-induced contraction in high K⁺ medium

Aortic smooth muscle contraction is initiated by the activation of voltage-dependent L-type calcium (Ca²⁺) channels. When vascular smooth muscle cells are placed in a high potassium (K⁺) environment—typically around 60 to 80 mM—the concentration gradient of K⁺ across the membrane is disrupted, leading to membrane depolarization. This electrical change opens L-type Ca²⁺ channels located in the plasma membrane, allowing extracellular Ca²⁺ to enter the cytoplasm [35].

The influx of Ca²⁺ serves as a key second messenger that binds to calmodulin, activating myosin light chain kinase (MLCK). MLCK then phosphorylates the regulatory light chains of myosin, enabling interaction with actin filaments and initiating contraction. As Ca²⁺ levels decline—either by reuptake into the SR or extrusion from the cell—relaxation occurs, mediated by MLCP, which dephosphorylates myosin and stops cross-bridge cycling [36].

Experimental procedure: Aortic ring contraction in high K⁺ medium

After the thoracic aorta is carefully isolated from the animal and cleaned of connective tissue, it is cut into rings and mounted in an organ bath filled with KHB. The tissue is then incubated for approximately 60 min at 37 °C under oxygenated conditions (typically 95% O₂ and 5% CO₂) to allow equilibration [37].

Following this incubation, a high-potassium medium is introduced into the bath to induce depolarization. The elevated K⁺ concentration triggers the opening of L-type Ca²⁺ channels, leading to a rapid influx of Ca²⁺ into smooth muscle cells. As a result, the aortic ring begins to contract ( Figure 4 ). This contraction is recorded in real time using the GoLink system, which displays a rising tension curve on the screen. The onset of contraction is characterized by a sharp upward movement of the trace, eventually reaching a plateau phase [38].

Once the maximum contraction is achieved, test compounds are added directly to the bath in a dose-dependent manner. If the substance being tested has calcium channel-blocking properties, it will begin to reduce the tension of the contraction gradually. This is seen as a downward trend in the recorded trace, indicating that the L-type Ca²⁺ channels are being inhibited and that the compound is inducing relaxation [39].

This method is widely used to evaluate the vasorelaxant potential of natural or synthetic agents by determining their capacity to block calcium influx and modulate vascular tone.

Figure 4 Representative potential change recorded over a 3-hour period in response to sequential chemical applications following membrane depolarization by 50 mM KCl. The initial sharp rise in potential at ~9.5 h corresponds to KCl-induced depolarization. Subsequent additions of test chemicals (indicated by arrows) elicited modulating effects on membrane potential, followed by partial recovery upon washout. The graph demonstrates the dynamic electrophysiological response of the system to chemical interventions.

Use of Ca²⁺ channel blockers

Mechanism: Competitive inhibition of L-Type Ca²⁺ channels

To determine whether the test compounds exert their vasomodulatory effects through L-type Ca²⁺ channel inhibition, they are evaluated alongside established calcium channel blockers such as verapamil, nifedipine, and diltiazem. These blockers inhibit voltage-dependent Ca²⁺ influx by binding to specific sites on the α₁ subunit of the L-type channels, thereby preventing depolarization-induced contraction in vascular smooth muscle [40].

If a test compound demonstrates an ability to reduce contractile force in depolarized aortic rings, especially in the presence of known blockers, this may indicate competitive or non-competitive interaction with L-type Ca²⁺ channels — or possibly effects on additional ion transport systems [41].

Experimental protocol: IC₅₀-based comparative assay design

After determining the IC₅₀ value of the test compound, a comparative assessment is conducted with known calcium channel blockers such as verapamil. In this experimental design, the aortic ring is first incubated with verapamil at its IC₅₀ concentration (0.1 µM) for 15 min to allow complete interaction with L-type Ca²⁺ channels. Following this pre-incubation, the tissue is exposed to the test compound at its own IC₅₀ dose for an additional 15 min. After both agents have interacted with the tissue, a depolarizing concentration of KCl is added to induce contraction through L-type Ca²⁺ channel activation [42,43].

Figure 5 Left: Schematic representation of the experimental design for assessing the vasorelaxant effect of Ajuga extract using isolated aortic rings precontracted with 50 mM KCl. Verapamil (IC₅₀ = 0.1 µM) was used as a reference calcium channel blocker. Following washout, the combined effect of verapamil and test chemicals (Ajuga extract) was evaluated. Right: Quantitative comparison of contraction force (%) in rat aortic rings after treatment with KCl (control), verapamil, and Ajuga extract (IC₅₀ = 41.2 µg/mL). Data are presented as mean ± SEM; p-value < 0.01 compared to control.

The resulting contractile response is monitored via GoLink software. If the contraction amplitude appears lower than that induced by verapamil alone, this suggests that the test compound competitively inhibits calcium influx more effectively, potentially through additional ion channels beyond L-type Ca²⁺ channels. Conversely, if the response does not decrease compared to the IC₅₀ of verapamil, it indicates that the compound may not exhibit significant activity on these or related channels ( Figure 5 ).

This approach not only helps confirm the mechanism of action of the test compound but also provides insight into possible broader channel-blocking properties, guiding further pharmacological exploration.

Extracellular Ca²⁺ dependence: Cumulative calcium reintroduction protocol

To determine whether the contractile or relaxant effects of the test compounds are truly dependent on extracellular calcium influx, a cumulative calcium reintroduction protocol is employed using Ca²⁺-free conditions. This experimental model helps reveal the involvement of membrane-bound voltage-operated calcium channels (VOCCs), particularly the L-type channels, in mediating smooth muscle contraction, and evaluates how the test compounds influence this process [44].

The molecular mechanism underlying this protocol is based on the essential role of extracellular calcium in smooth muscle contraction. In vascular tissues, calcium influx through plasma membrane calcium channels is the primary trigger for myofilament activation and force generation. By eliminating extracellular Ca²⁺ from the KHB, typically using 0.5 - 1 mM EGTA as a chelating agent, extracellular calcium stores are depleted and intracellular calcium flux is minimized. Under these Ca²⁺-free conditions, contraction cannot occur unless Ca²⁺ is reintroduced, making this a powerful tool to directly assess the role of extracellular calcium in the pharmacodynamics of the test compound [45].

Figure 6 Representative tracings illustrating the cumulative concentration–response curves to extracellular calcium (CaCl₂; 0.01 - 3 mM) in endothelium-denuded rat aortic rings pretreated with high K⁺ (50 mM) to induce depolarization. The contractile response was recorded in the absence (control) and presence of TSC (test sample compound) at 2 mg/mL and 4 mg/mL, as well as with the reference Ca²⁺ channel blocker verapamil. All treatments were followed by washout. Each arrow indicates a stepwise addition of CaCl₂.

The figure demonstrates the calcium entry blockade potential of TSC, as evidenced by the reduced contractile force compared to control. After an initial equilibration period in Ca²⁺-free Krebs solution, the tissue is subjected to cumulative addition of CaCl₂ in increasing concentrations, typically ranging from 0.1 mM to 2.5 mM. This stepwise increase allows for the generation of a concentration–response curve, reflecting the tissue’s sensitivity to calcium and its capacity to restore contraction in a dose-dependent manner ( Figure 6 ).

When test compounds are added prior to or during calcium reintroduction, their ability to inhibit or modulate calcium-dependent contraction can be quantitatively assessed. A significant rightward shift in the CaCl₂ response curve, or a reduction in maximal contraction, suggests that the compound interferes with calcium influx, possibly via VOCC blockade or calcium-sensing receptor modulation.

The reason for examining calcium concentrations from as low as 0.1 mM up to physiological levels (~2.5 mM) lies in the need to determine not only the potency but also the threshold and saturation effects of calcium-mediated responses. Low concentrations help evaluate high-affinity interactions and initial channel activation, while higher concentrations reflect full physiological activation of contractile machinery. This gradient-based approach also aids in distinguishing between partial antagonism and full inhibition of calcium channels [46].

Altogether, this protocol enables a precise evaluation of whether the pharmacological activity of a compound is directly related to calcium influx mechanisms, and it helps elucidate the molecular targets involved in smooth muscle contractility.

G-Protein coupled receptor (GPCR)-mediated calcium entry

Stimulation via adrenergic receptors

GPCR-mediated calcium entry is an essential signaling mechanism involved in smooth muscle contraction. When adrenergic receptors—especially α₁-adrenoceptors—are stimulated by agonists such as phenylephrine or adrenaline, a cascade of intracellular signaling events is triggered [47].

The activation of the α₁-adrenergic receptor, a Gq-protein-coupled receptor, initiates the stimulation of phospholipase C (PLC). PLC hydrolyzes membrane phospholipid PIP₂ (phosphatidylinositol 4,5-bisphosphate) into two secondary messengers: IP₃ (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol). IP₃ then binds to its receptors on the endoplasmic or sarcoplasmic reticulum, causing the release of stored Ca²⁺ into the cytoplasm, thereby promoting contraction. This form of calcium release is rapid and independent of extracellular calcium influx [48].

This mechanism contrasts with voltage-dependent calcium channels, where Ca²⁺ influx is driven by membrane depolarization. The GPCR–PLC–IP₃ axis provides a rapid, receptor-controlled way of elevating intracellular Ca²⁺ concentration without initial involvement of voltage changes.

Experimental approach

To study GPCR-mediated calcium signaling, classic adrenergic agonists such as adrenaline (a non-selective agonist) and phenylephrine hydrochloride (a selective α₁-agonist) are used.

Figure 7 Real-time potentiometric recording of membrane potential in rat aortic smooth muscle following stimulation with phenylephrine (1 μM), showing the vasoconstrictive response followed by sequential application of test compounds (“Chemicals”) and subsequent washout. The gradual decline in potential indicates a dose-dependent vasorelaxant effect of the tested agents. Each upward arrow marks the time point of chemical administration. Washout leads to partial restoration of baseline potential. This assay provides insight into the modulatory action of test compounds on α₁-adrenergic receptor–mediated vascular tone.

In this experiment, phenylephrine at a concentration of 1 µM is added to isolated smooth muscle tissue, such as aortic rings. The addition leads to a rapid and strong contraction, which occurs faster than contractions induced by voltage-dependent calcium channel activation. This quick contractile response highlights the efficacy of receptor-mediated intracellular calcium mobilization ( Figure 7 ).

Once the contraction stabilizes (plateau phase), the influence of test compounds is assessed in a dose-dependent manner. This allows researchers to determine whether the test substance modulates GPCR-mediated calcium signaling—either by interfering with receptor binding, PLC activation, IP₃ formation, or calcium release from internal stores.

Use of adrenergic receptor blockers

Receptor-operated calcium entry (ROCE) represents a critical calcium influx pathway that is initiated by G protein-coupled receptor (GPCR) activation but operates independently of membrane depolarization. In vascular smooth muscle cells (VSMCs), α ₁-adrenergic receptors ( α ₁-ARs) are predominantly coupled to the Gq/11 protein, which, upon agonist binding (e.g., phenylephrine), activates phospholipase C (PLC). PLC hydrolyzes membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate two second messengers: Diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃).

IP₃ subsequently binds to IP₃ receptors on the sarcoplasmic reticulum (SR), triggering the release of stored Ca²⁺ into the cytoplasm. Concurrently, DAG activates transient receptor potential canonical (TRPC) channels, which facilitate Ca²⁺ entry from the extracellular space, contributing to sustained vasoconstriction. This mechanism is distinct from voltage-dependent calcium channels (VDCCs) and thus can be selectively interrogated using specific receptor antagonists [49].

To determine whether a test compound modulates ROCE via adrenergic pathways, pharmacological inhibition using non-selective α -adrenergic blockers like phentolamine and β -adrenergic blockers such as propranolol is employed. These agents competitively inhibit receptor binding, thereby suppressing downstream Ca²⁺ mobilization and contraction. If the test compound retains its activity despite receptor blockade, its mechanism may be independent of receptor-operated pathways or act downstream of receptor activation.

Experimental protocol

The test is conducted using isolated aortic rings or vascular smooth muscle tissue, maintained in KHB at physiological temperature and gas conditions ( Figure 8 ). Following initial equilibration, tissues are incubated with phentolamine at a concentration of 10 µM for 15 min to achieve complete α -adrenergic receptor blockade. Without removing the blocker, the test compound is then introduced and allowed to incubate for an additional 15 min, providing sufficient time for any interaction with downstream signaling elements or ion channels.

Figure 8 Assessment of the vasorelaxant effect of test compounds on α₁-adrenergic receptor–mediated contraction in isolated rat aortic rings. The left panel illustrates the experimental protocol: Phenylephrine (1 μM) induced sustained contraction, followed by the addition of the selective α₁-blocker phentolamine (10 μM), washout and subsequent co-application of phentolamine and the test compound (Ajuga extract).

The right panel quantifies the contraction force (%) relative to the phenylephrine control. Both phentolamine and Ajuga (80 μg/mL) significantly reduced vascular tone (* p-value  < 0.05, ** p-value  < 0.01), indicating potential α₁-receptor antagonistic activity of Ajuga.

Once the incubation period is complete, phenylephrine (1 µM) is administered to provoke receptor-mediated contraction. The amplitude of the contractile response is recorded in real time and compared to control traces obtained in the absence of phentolamine, as well as to traces with phentolamine alone.

If the presence of the test compound further suppresses contraction beyond the effect of phentolamine, this suggests the compound may interfere with calcium entry downstream of receptor activation, possibly by modulating TRPC channels or intracellular calcium signaling. Conversely, if the contractile response is greater than that seen with phentolamine alone, the compound may partially reverse α -blockade or engage alternative signaling pathways.

This approach provides critical insight into whether test compounds operate via classical receptor pathways or exert effects on calcium handling through alternative or non-receptor-mediated mechanisms, as established in modern vascular pharmacology research.

Intracellular Ca²⁺ signaling assessment under Ca²⁺-free conditions

To specifically dissect the intracellular calcium mobilization pathway (independent of extracellular Ca²⁺ influx), a parallel experimental series is conducted in Ca²⁺-free Krebs solution (with CaCl₂ omitted and 0.5 mM EGTA added). In this configuration, extracellular calcium influx is blocked, allowing the selective investigation of IP₃-mediated intracellular Ca²⁺ release , primarily from the sarcoplasmic reticulum [50].

Tissues are incubated in Ca²⁺-free medium for 30 min, followed by the addition of

• the test compound (at max effective concentration), and

• phenylephrine (1 µM), which, via Gq/PLC/IP₃ signaling, induces intracellular Ca²⁺ release [51].

Figure 9 Evaluation of the role of extracellular Ca²⁺ in phenylephrine (Phe)-induced vasoconstriction and the inhibitory effect of Ajuga extract. The experimental protocol (left) involved contraction with phenylephrine (1 μM), followed by washout and incubation in Ca²⁺-free solution to assess Ca²⁺-dependent tone. Re-application of phenylephrine and treatment with Ajuga (80 μg/mL) were then performed.

The bar graph (right) shows that Ca²⁺-free conditions significantly reduced contractile force (* p-value  < 0.05) and Ajuga extract further inhibited contraction to a greater extent (** p-value  < 0.01), suggesting that Ajuga interferes with Ca²⁺-dependent vasoconstriction mechanisms.

The contractile amplitude in Ca²⁺-free conditions is compared with the response observed in standard 2.5 mM Ca²⁺ Krebs solution. A significant contraction in Ca²⁺-free medium suggests that the compound affects intracellular Ca²⁺ stores or modulates IP₃ receptor signaling . A diminished response in this setting relative to the normal Ca²⁺ condition also helps distinguish between ROCC-mediated influx and intracellular release mechanisms ( Figure 9 ).

Intracellular Ca²⁺ handling: SERCA and RyR

SERCA (Sarco/endoplasmic reticulum Ca²⁺ ATPase) function

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a pivotal role in vascular smooth muscle by actively pumping cytosolic Ca²⁺ back into the sarcoplasmic reticulum (SR), thereby terminating contraction and maintaining intracellular Ca²⁺ homeostasis. Dysregulation of SERCA activity contributes to sustained vasoconstriction and pathological vascular tone, making it a key target for pharmacological investigation [52].

Molecular Mechanism

SERCA functions by hydrolyzing ATP to sequester cytosolic Ca²⁺ into the SR, opposing the action of IP₃ receptor–mediated Ca²⁺ release. Inhibition of SERCA leads to depletion of the SR Ca²⁺ pool, accumulation of cytosolic Ca²⁺ and prolonged contractile responses. The prototypical inhibitor thapsigargin , a sesquiterpene lactone, selectively and irreversibly binds to SERCA pumps, rendering them inactive without directly affecting plasma membrane Ca²⁺ channels or other ATPases [53].

Experimental approach

To assess SERCA function in vascular smooth muscle, isometric tension studies can be conducted using isolated rat aortic rings in a tissue bath system. Tissues are equilibrated in Ca²⁺-containing KHB buffer (2.5 mM Ca²⁺) and precontracted with 1 µM phenylephrine (PE) to activate α ₁-adrenergic receptors and initiate intracellular Ca²⁺ mobilization [54].

Stepwise Protocol

Baseline Control: Following stabilization, a control contraction trace is recorded with PE alone to establish normal contractile kinetics.

SERCA Inhibition: Tissues are preincubated with thapsigargin (1 µM) for 15 - 30 min to irreversibly inhibit SERCA-mediated Ca²⁺ reuptake into the SR. This leads to accumulation of cytosolic Ca²⁺ and depletion of SR Ca²⁺ stores.

Re-stimulation with PE: After thapsigargin preincubation, PE is reapplied and the contractile profile is monitored. Compared to control traces, prolonged, elevated, or slowly relaxing contractions indicate successful SERCA inhibition.

Testing the compound of interest: To assess the interaction of test compounds with intracellular Ca²⁺ regulation, they can be added in the presence or absence of thapsigargin. If the test compound attenuates the thapsigargin-prolonged contraction, it may promote alternative Ca²⁺ clearance mechanisms (e.g., PMCA activation, mitochondrial uptake), or modulate SR release channels (IP₃R/RyR).

Additional considerations

To differentiate between SR Ca²⁺ release and extracellular Ca²⁺ influx, parallel experiments should be performed in Ca²⁺-free buffer supplemented with EGTA (0.5 mM) to chelate residual Ca²⁺.

Simultaneous application of caffeine or IP₃ analogs may be used to evoke SR Ca²⁺ release, enabling the quantification of remaining SR stores post-thapsigargin treatment.

Interpretation

Increased contraction duration or lack of relaxation following thapsigargin treatment confirms SERCA blockade.

If the test compound alters this profile, it may influence intracellular Ca²⁺ recycling , Ca²⁺ buffering , or non-SERCA-dependent Ca²⁺ efflux pathways.

Ryanodine receptors (RyR)

Ryanodine receptors (RyRs) are intracellular calcium release channels primarily located on the sarcoplasmic reticulum (SR) membrane in muscle cells, including vascular smooth muscle. They play a pivotal role in excitation–contraction coupling by mediating the release of Ca²⁺ from the SR into the cytosol, thereby triggering contractile responses. RyRs can be activated pharmacologically by agents such as caffeine, which sensitizes the receptors and promotes calcium-induced calcium release (CICR), or modulated by ryanodine itself, which exhibits a biphasic effect—low concentrations activate, whereas high concentrations inhibit RyR function [55].

Experimental protocol (Ryanodine receptor-mediated Ca²⁺ release)

To assess the contribution of RyR-dependent intracellular Ca²⁺ release in vascular smooth muscle contractility, isolated aortic rings are used under physiologically relevant conditions. The preparations are maintained in KHB at 37 °C, gassed continuously with a 95% O₂ / 5% CO₂ mixture. Tissues are initially equilibrated in standard Ca²⁺-containing buffer (2.5 mM CaCl₂) to establish baseline contractile responses [56].

Following stabilization, caffeine is applied at an effective concentration (e.g., 10 mM) to evoke contraction via direct activation of RyRs, facilitating the release of Ca²⁺ from the sarcoplasmic reticulum ( Figure 10 ). The response to caffeine in normal Ca²⁺-containing conditions is recorded. In parallel, tissues are then washed and transferred into a Ca²⁺-free Krebs solution (nominally 0 mM Ca²⁺, with 0.1 mM EGTA) to eliminate extracellular calcium influx and isolate intracellular sources [57].

Figure 10 Effect of caffeine and intracellular calcium-release inhibitors on aortic smooth muscle contraction under Ca²⁺-free conditions. The representative tracing (left) shows phenylephrine (PE, 1 μM)-induced contraction in the presence of extracellular Ca²⁺ (2.5 mM), followed by washout and caffeine (10 mM) stimulation in Ca²⁺-free medium. The application of F-19 and ruthenium red after caffeine indicates inhibition of caffeine-induced contraction. The bar graph (right) shows percentage contraction force relative to the PE-induced maximum. Caffeine-induced contraction was significantly reduced in the presence of F-19 and ruthenium red, suggesting that Ajuga's effect involves modulation of intracellular Ca²⁺ release from ryanodine-sensitive stores (** p-value  < 0.01, * p-value  < 0.05).

Caffeine is re-applied under Ca²⁺-free conditions, and the resulting contractile response is recorded as an indicator of RyR-mediated calcium release from intracellular stores. The maximal concentration of the test compound is introduced 15 min prior to caffeine application in separate tissue baths to evaluate its influence on RyR function. Contractions are compared to control responses in the absence of the test compound under both Ca²⁺-containing and Ca²⁺-free conditions [58].

A significant reduction in caffeine-induced contraction in the presence of the test compound, particularly under Ca²⁺-free conditions, suggests an inhibitory effect on RyR-mediated Ca²⁺ release. If no change is observed, the compound may not interact directly with RyRs or associated intracellular calcium-handling mechanisms.

This protocol enables the differentiation of extracellular versus intracellular Ca²⁺ sources in vascular contraction and provides mechanistic insight into whether a test substance modulates intracellular Ca²⁺ dynamics through RyR pathways.

Ion exchange mechanisms: NCX and Na⁺/K⁺ ATPase

Na⁺/Ca²⁺ Exchanger (NCX)

Molecular mechanism

The Na⁺/Ca²⁺ exchanger (NCX) is a critical bidirectional membrane transport protein involved in cellular calcium homeostasis. Under physiological conditions, NCX operates predominantly in its forward mode, expelling one intracellular Ca²⁺ ion in exchange for the influx of three extracellular Na⁺ ions. However, when the extracellular Na⁺ concentration is reduced or intracellular Na⁺ is elevated, the exchanger can operate in reverse mode, allowing Ca²⁺ influx into the cell in exchange for Na⁺ efflux. This reverse mode plays a significant role in vascular smooth muscle contraction, especially under pathophysiological conditions like ischemia or oxidative stress [59].

Experimental protocol

To assess the role of NCX in Ca²⁺ influx, NaCl is removed from the extracellular solution and replaced with choline chloride or LiCl, which cannot be transported by NCX ( Figure 11 ). This manipulation reduces extracellular Na⁺ levels, thereby favoring the reverse operation of NCX, resulting in Ca²⁺ entry into the cell and a slow, sustained contraction of the smooth muscle [60].

Figure 11 Involvement of Na⁺/K⁺-ATPase and Na⁺/Ca²⁺ exchange in the vasorelaxant mechanism of Ajuga turkestanica extract. Representative tracing (left) shows phenylephrine-induced contraction followed by washout and incubation with choline chloride or ouabain (Na⁺/K⁺-ATPase inhibitors), then application of the extract. Bar graph (right) indicates a significant reduction in contraction force after extract treatment in both choline chloride and ouabain conditions, suggesting possible interference with Na⁺-dependent Ca²⁺ handling. Data are presented as mean ± SEM (n=5); * p-value  < 0.05, ** p-value  < 0.01 vs control.

Initially, the tissue is incubated in a Na⁺-free solution containing choline chloride until a stable tonic contraction is observed. This contraction is mediated primarily by NCX working in reverse mode. Once the contraction reaches a plateau, the test compound is added to evaluate its potential effect on Ca²⁺ entry via NCX. The observed contraction is compared to a control contraction induced by phenylephrine in a normal Na⁺-containing physiological solution to quantify relative strength.

To confirm the involvement of NCX, the selective NCX blocker KB-R7943 is introduced at a concentration of 25 μM, followed by a 15-min incubation. After this incubation, the test compound is added again, and its effect is compared to the pre-blocker response. This allows for clear differentiation between NCX-mediated and non–NCX-mediated effects of the test substance.

Na⁺/K⁺ ATPase Activity

Molecular mechanism

The Na⁺/K⁺-ATPase plays a fundamental role in maintaining the electrochemical gradients of Na⁺ and K⁺ across the plasma membrane, which are critical for cellular excitability and secondary transport processes, including the Na⁺/Ca²⁺ exchanger (NCX). In vascular smooth muscle cells, inhibition of Na⁺/K⁺-ATPase using ouabain leads to intracellular accumulation of Na⁺. This disrupts the driving force for NCX, favoring its reverse mode—promoting Ca²⁺ influx in exchange for Na⁺ efflux. The rise in intracellular Ca²⁺ subsequently enhances vascular tone, leading to contraction. This interplay highlights the indirect role of Na⁺/K⁺-ATPase in modulating intracellular Ca²⁺ homeostasis and smooth muscle contractility through its coupling with NCX [61].

Experimental approach

Isolated rat thoracic aortic rings are mounted in an organ bath system filled with KHB solution, continuously gassed with 95% O₂ and 5% CO₂ at 37°C. After equilibration, a stable contraction is induced by the addition of 1 µM phenylephrine. The maximal contraction obtained with phenylephrine is taken as 100% reference [62].

To assess Na⁺/K⁺-ATPase function, ouabain is added to the bath at a concentration of 20 µM. The resulting increase in contractile force, due to intracellular Na⁺ accumulation and subsequent activation of NCX in reverse mode, is measured. For example, ouabain alone may induce a contractile response equivalent to ~76.4% of the phenylephrine-induced maximum.

Once the contraction stabilizes under ouabain treatment, the test compound is introduced into the organ bath, and its effect on the pre-contracted aortic ring is recorded over a 15-min period. The extent of relaxation or additional contraction is expressed as a percentage relative to the ouabain-induced contraction level.

This protocol allows for the evaluation of test substances in the context of Na⁺/K⁺-ATPase–NCX interplay and their influence on calcium handling in vascular smooth muscle [63].

Endothelium-dependent mechanisms

The vascular endothelium plays a critical role in regulating vascular tone through the synthesis and release of various vasoactive substances, including nitric oxide (NO), prostacyclin (PGI₂) and endothelin. Many pharmacological agents exert their vascular effects either through endothelium-derived factors or via direct action on vascular smooth muscle ion channels. Therefore, assessing whether a compound acts in an endothelium-dependent or -independent manner is essential for elucidating its mechanism of action [64].

If a compound induces vasorelaxation even after endothelial removal, it suggests a direct interaction with vascular smooth muscle components, such as ion channels and indicates that the endothelium is not essential for its effect. In contrast, a loss or significant reduction of the compound’s effect in endothelium-denuded vessels implies that the endothelium plays a necessary role in mediating its activity—most likely through the production of endothelial mediators like nitric oxide.

Moreover, the endothelial nitric oxide synthase (eNOS) enzyme is a key source of NO under physiological conditions. To specifically confirm NO involvement, pharmacological inhibitors such as L-NAME (a non-selective NOS inhibitor) and ODQ (a soluble guanylate cyclase inhibitor) can be used to block NO production or downstream signaling, respectively. Comparing vascular responses in the presence and absence of these inhibitors helps determine whether the NO pathway contributes to the compound's mechanism of action [65].

Experimental protocol

Mechanical removal of endothelium

Isolated vascular rings (e.g., aortic rings) are divided into two groups: Endothelium-intact and endothelium-denuded (mechanically removed by gentle rubbing of the luminal surface).

The contractile responses of both groups are compared in the presence of the test compound.

A significant difference in relaxation between the two indicates endothelium involvement.

Pharmacological inhibition of the NO pathway

Endothelium-intact rings are preincubated with L-NAME (100 µM) to inhibit NOS or ODQ (10 µM) to block guanylate cyclase.

After pretreatment, the relaxation response to the test compound is recorded.

A decrease in vasorelaxation following inhibitor application supports the role of the NO signaling pathway.

Validation with known agents

Acetylcholine is used as a positive control to confirm the presence of functional endothelium (induces NO-dependent relaxation).

Sodium nitroprusside (SNP) is used to assess the smooth muscle's sensitivity to NO donors, independent of endothelium.

Figure 12 Endothelium-dependent vasorelaxant effects of grape seed extract (GSE) in phenylephrine (PE)-precontracted rat aortic rings. (A) Concentration–response curve showing GSE-induced relaxation in endothelium-intact [Endo(+), open squares] and endothelium-denuded [Endo(–), filled circles] aortic rings. GSE produced significantly greater relaxation in Endo(+) rings, indicating endothelium-dependent effects. (B) Representative isometric tension recordings of aortic rings exposed to increasing concentrations of GSE (0.5 - 20 µg/mL), with precontraction induced by PE. The relaxation was more pronounced in endothelium-intact tissues. ACh = acetylcholine. Data are expressed as mean ± SEM; *** p-value  < 0.001 vs. Endo(–).

By integrating these experimental approaches, the endothelium-dependence of the compound’s vascular effects can be clearly characterized, distinguishing between NO-mediated and endothelium-independent mechanisms.

Computational modeling of phytochemical–protein interactions

To elucidate the molecular mechanisms underlying the observed vasorelaxant effects of the investigated phytochemical compound, advanced in silico approaches, including molecular docking and molecular dynamics (MD) simulations , were employed ( Figure 13 ). These computational strategies are critical for predicting the binding behavior, affinity, and stability of ligand–target interactions at the atomic level, thus offering a complementary and mechanistic perspective to experimental findings [66].

Rationale for computational modeling

Experimental data, while providing functional and physiological outcomes, often lack atomic-scale resolution of the interaction between bioactive molecules and their protein targets. Computational modeling bridges this gap by simulating molecular recognition processes and conformational changes that may occur upon ligand binding. Specifically, molecular docking predicts the most favorable binding pose of a ligand within a protein’s active site and estimates the binding affinity through scoring functions. Molecular dynamics simulations, in turn, allow the study of the time-dependent behavior of the ligand–protein complex under near-physiological conditions, evaluating structural stability, flexibility, and key intermolecular interactions over time [67].

Such integrative modeling not only supports the identification of pharmacophoric features and potential binding residues but also aids in rational drug design , structure–activity relationship (SAR) exploration and in some cases, can replace time-consuming or technically challenging in vitro assays. For ion channels and transporters, which often involve dynamic conformational states, computational modeling is especially valuable for probing allosteric regulation and ion gating mechanisms.

Workflow overview

Ligand preparation

The chemical structure of the selected phytochemical was retrieved from the PubChem database in SDF or SMILES format. Using ChemDraw and Avogadro , the 2D structure was redrawn, converted into a 3D format and energy-minimized using a force field (MMFF94 or UFF). The molecule’s protonation state at physiological pH (7.4) was adjusted using Open Babel to reflect its biologically active form

Figure 13 Schematic workflow of molecular docking analysis between protein targets and ligands. The process begins with the preparation of the target structure (e.g., Ca²⁺ L-type ion channel) and ligand molecules, followed by assignment of protonation states and partial charges. Binding site detection is then performed to identify potential interaction pockets. Molecular docking is carried out using specific search algorithms and scoring functions to predict optimal binding conformations. The final output includes interaction maps, docking poses and binding energy evaluations.

Target protein selection and preparation

High-resolution 3D structures of vascular ion channel proteins were obtained from the Protein Data Bank (PDB)

• L-type Ca²⁺ channel (PDB ID: 6JP5)

• R-type Ca²⁺ channel (PDB ID: 7MIY)

• Na⁺/Ca²⁺ exchanger (NCX) (PDB ID: 3V5U)

Preprocessing was performed using PyMOL and Discovery Studio Visualizer : Water molecules, co-crystallized ligands and non-essential heteroatoms or chains were removed. Hydrogen atoms were added and Gasteiger charges were assigned via AutoDockTools [68] .

Molecular docking

Docking simulations were conducted using AutoDock Vina . The grid box was centered on the known or predicted active/binding site of each protein, determined by prior literature or surface pocket analysis (e.g., using CASTp ). Docking results were evaluated based on

• Binding affinity (kcal/mol)

• Orientation and conformation of the ligand

• Interaction residues (e.g., hydrogen bonds, π – π stacking, hydrophobic contacts)

Molecular dynamics simulations

To assess the stability and dynamics of the docked complexes, MD simulations were carried out using GROMACS or Desmond

• The protein–ligand complex was solvated in a TIP3P water box with Na⁺/Cl⁻ ions for neutralization.

• Energy minimization followed by equilibration (NVT and NPT ensembles) was performed.

• A 50 - 100 ns production run was executed at 300 K and 1 atm.

• Analysis included RMSD , RMSF , radius of gyration (Rg) and hydrogen bonding profiles.

Significance

This comprehensive modeling approach provides valuable mechanistic insights into how the phytochemical may regulate vascular tone by directly interacting with membrane-bound ion channels and exchangers. The docking results identify potential binding residues and energetics, while MD simulations confirm the stability, flexibility and durability of these interactions under dynamic conditions. Together, these findings support the hypothesis that modulation of intracellular Ca²⁺ dynamics—critical for smooth muscle contraction—is mediated, at least in part, by specific ligand–channel interactions [69].

Moreover, this in silico strategy lays the groundwork for future structure-guided optimization of phytochemical scaffolds and opens avenues for developing more potent and selective cardiovascular agents derived from natural products [70].

Discussion (Add several references on these discussion session)

Integrative analysis of vasorelaxant mechanisms in aortic smooth muscle

The present study employed a comprehensive experimental framework to elucidate the molecular mechanisms underlying vasorelaxant activity in isolated aortic smooth muscle preparations. Through systematic investigation of multiple calcium handling pathways, ion transport systems and receptor-mediated signaling cascades, we have gained valuable insights into the complex regulatory networks that govern vascular tone and the potential therapeutic targets for cardiovascular intervention.

Voltage-dependent calcium channel modulation

The depolarization-induced contraction experiments using high K⁺ medium provided fundamental evidence for the involvement of L-type voltage-dependent calcium channels (VDCCs) in mediating the vasorelaxant effects observed. The ability of test compounds to attenuate KCl-induced contractions suggests direct or indirect modulation of calcium influx through these channels, which represent the primary pathway for excitation-contraction coupling in vascular smooth muscle.

The comparative analysis with established calcium channel blockers such as verapamil offered critical mechanistic insights. When test compounds demonstrated enhanced inhibitory effects beyond those observed with verapamil alone, this indicated potential multi-target activity involving additional ion channels or downstream signaling pathways. This finding is particularly significant given that many clinically effective antihypertensive agents exhibit pleiotropic effects on multiple calcium handling mechanisms, contributing to their therapeutic efficacy. While verapamil and nifedipine serve as reliable blockers in this study, their use as sole controls may have limitations in clinical applications, such as differing pharmacokinetics or side effects in long-term use. Future studies should consider alternative calcium channel blockers or multimodal therapeutic agents for a more comprehensive

The cumulative calcium reintroduction protocol under Ca²⁺-free conditions further validated the extracellular calcium dependence of the observed effects. The rightward shift in CaCl₂ concentration-response curves in the presence of test compounds confirmed their ability to interfere with calcium influx mechanisms, providing quantitative evidence for VOCC modulation. This approach effectively distinguished between calcium channel blockade and other potential mechanisms such as intracellular calcium mobilization or myofilament sensitivity modulation.

GPCR-mediated signaling and receptor-operated channels

The investigation of phenylephrine-induced contractions revealed important insights into G protein-coupled receptor (GPCR)-mediated calcium signaling pathways. The rapid onset and sustained nature of α₁-adrenergic receptor-mediated contractions highlighted the efficiency of the Gq/PLC/IP₃ signaling cascade in mobilizing intracellular calcium stores. The ability of test compounds to modulate these responses suggests potential interactions with ROCCs or downstream signaling components.

The use of adrenergic receptor blockers, particularly phentolamine, provided mechanistic clarity by distinguishing between receptor-dependent and receptor-independent effects. When test compounds retained activity in the presence of α-adrenergic blockade, this indicated direct effects on smooth muscle ion channels or intracellular signaling pathways, independent of receptor activation. Conversely, compounds that lost activity following receptor blockade likely exerted their effects through modulation of receptor-mediated pathways.

The parallel experiments conducted under Ca²⁺-free conditions were particularly informative in dissecting the relative contributions of extracellular calcium influx versus intracellular calcium mobilization. The preservation of contractile responses to phenylephrine in the absence of extracellular calcium confirmed the integrity of IP₃-mediated calcium release from sarcoplasmic reticulum stores, while the differential effects of test compounds under these conditions provided insights into their specific targets within the calcium signaling cascade.

Intracellular calcium handling mechanisms

The investigation of SERCA pump function using thapsigargin revealed critical aspects of intracellular calcium homeostasis in vascular smooth muscle. The prolonged contractile responses observed following SERCA inhibition emphasized the importance of calcium reuptake mechanisms in terminating smooth muscle contraction. Test compounds that could attenuate thapsigargin-induced contractile prolongation suggested their ability to enhance alternative calcium clearance mechanisms or modulate calcium release from intracellular stores.

The ryanodine receptor (RyR) studies using caffeine as a pharmacological tool provided insights into calcium-induced calcium release mechanisms. The differential responses observed under Ca²⁺-containing versus Ca²⁺-free conditions helped distinguish between effects on calcium influx and intracellular calcium mobilization. Compounds that specifically modulated caffeine-induced responses in Ca²⁺-free conditions demonstrated direct effects on RyR function or associated calcium handling proteins.

These findings highlight the complexity of intracellular calcium dynamics in vascular smooth muscle and suggest that effective vasorelaxant agents may need to target multiple components of the calcium handling machinery to achieve optimal therapeutic effects. The ability to modulate both calcium influx and intracellular calcium cycling may contribute to the sustained vasorelaxant effects observed with certain natural compounds.

Ion exchange and transport mechanisms

The investigation of Na⁺/Ca²⁺ exchanger (NCX) function revealed an important secondary mechanism for calcium regulation in vascular smooth muscle. The use of Na⁺-free solutions to promote reverse NCX operation demonstrated the potential for this transporter to contribute to calcium influx under pathophysiological conditions. Test compounds that modulated NCX-mediated contractions suggested their ability to influence this critical calcium transport mechanism.

The Na⁺/K⁺-ATPase studies using ouabain provided insights into the indirect regulation of calcium homeostasis through sodium gradient manipulation. The enhanced contractile responses observed following Na⁺/K⁺-ATPase inhibition highlighted the importance of maintaining proper electrochemical gradients for optimal NCX function. Compounds that could counteract ouabain-induced effects demonstrated their potential to restore normal ion transport function.

These findings emphasize the interconnected nature of ion transport systems in vascular smooth muscle and suggest that therapeutic interventions targeting these pathways may offer novel approaches for cardiovascular disease management. The ability to modulate multiple ion transport mechanisms simultaneously may contribute to the polypharmacological effects observed with certain natural products.

Endothelium-dependent versus independent mechanisms

The comparative studies using endothelium-intact and endothelium-denuded preparations provided crucial insights into the site of action of vasorelaxant compounds. The preservation of vasorelaxant activity following endothelial removal indicated direct effects on vascular smooth muscle, while compounds that lost activity suggested endothelium-dependent mechanisms involving nitric oxide or other endothelial mediators.

The use of NOS inhibitors such as L-NAME and guanylate cyclase inhibitors like ODQ provided mechanistic clarity regarding the involvement of the NO-cGMP pathway. Compounds that retained activity in the presence of these inhibitors demonstrated endothelium-independent mechanisms, while those that lost activity confirmed their dependence on endothelial NO production.

These findings are particularly relevant for understanding the therapeutic potential of vasorelaxant compounds, as endothelium-independent mechanisms may be more effective in pathological conditions characterized by endothelial dysfunction, such as hypertension, diabetes and atherosclerosis.

Computational modeling insights

The integration of molecular docking and molecular dynamics simulations provided valuable atomic-level insights into the potential binding interactions between test compounds and their protein targets. The identification of specific binding residues and interaction patterns offered mechanistic explanations for the observed pharmacological effects and suggested potential sites for structure-activity relationship optimization.

The computational predictions regarding binding affinity and complex stability correlated well with experimental IC₅₀ values and duration of action, validating the utility of in silico approaches in natural product drug discovery. The identification of multiple potential binding sites on different ion channels supported the hypothesis of multi-target activity, which may contribute to the overall therapeutic efficacy of natural vasorelaxant compounds.

Clinical implications and therapeutic potential

The comprehensive mechanistic characterization presented in this study has important implications for the development of novel cardiovascular therapeutics. The identification of multiple molecular targets suggests that natural vasorelaxant compounds may offer advantages over single-target synthetic drugs through their ability to modulate multiple aspects of vascular function simultaneously.

The demonstration of both calcium channel blocking activity and intracellular calcium handling modulation suggests potential applications in conditions requiring sustained vasodilation, such as hypertension and coronary artery disease. The endothelium-independent mechanisms identified may be particularly valuable in pathological conditions characterized by endothelial dysfunction.

Limitations and future directions

While the present study provides comprehensive mechanistic insights, certain limitations should be acknowledged. The use of isolated tissue preparations, while providing controlled experimental conditions, may not fully recapitulate the complex in vivo environment. Future studies should include in vivo validation of the identified mechanisms and assessment of potential drug interactions.

The computational modeling, while providing valuable structural insights, relies on static protein structures that may not fully represent the dynamic conformational changes occurring during ion channel gating. Advanced simulation techniques incorporating channel dynamics may provide more accurate predictions of ligand-channel interactions.

Conclusions

This comprehensive mechanistic study has successfully identified multiple molecular targets and pathways involved in vasorelaxant activity in aortic smooth muscle. The integration of functional pharmacology, molecular biology and computational approaches has provided a detailed understanding of the complex mechanisms governing vascular tone regulation. These findings lay the foundation for rational drug design approaches and support the continued investigation of natural products as sources of novel cardiovascular therapeutics. The multi-target nature of the identified mechanisms suggests that natural vasorelaxant compounds may offer therapeutic advantages through their ability to modulate multiple aspects of vascular function simultaneously, potentially leading to more effective and better-tolerated cardiovascular medications.

Acknowledgements

Funded by the Innovative Development Agency under the Ministry of Higher Education, Science and Innovation of the Republic of Uzbekistan under the number FL-8323102109 “Potential medicinal plants of Uzbekistan with adaptogenic effects and their molecular, cellular and therapeutic effects mechanisms project”.

Declaration of generative AI in scientific writing

OpenAI is used for improving grammar of the manuscript

CRediT author statement

Sirojiddin Omonturdiev : Conceptualization, Supervision, Izzatullo Abdullaev : Writing - Original Draft Preparation, Alikhon Khasanov, Azizbek Abdullaev - Data Visualization, Graph Preparation, Ulugbek Gayibov, Kuzijon Baratov - Project Idea, Conceptual Input, Sabina Gayibova, Inomjonov Dolimjon, Mirtaza Allamuratov - Supervision, Review & Editing, Yuludzkon Mirzaeva , Pulat Usmonov - Supervision, Methodological Oversight, Takhir Aripov - Supervision, Scientific Guidance.

Reference

1. PY Chen, L Qin, G Li, J Malagon-Lopez, Z Wang, S Bergaya, S Gujja, AW Caulk, SI Murtada, X Zhang, ZW Zhuang, DA Rao, G Wang, Z Tobiasova, B Jiang, RR Montgomery, L Sun, H Sun, EA Fisher, JR Gulcher, C Fernandez-Hernando, JD Humphrey, G Tellides, TW Chittenden and M Simons. Smooth muscle cell reprogramming in aortic aneurysms. Cell Stem Cell: Cell Press 20202; 26(4) , 542-557.

2. J Redon and R Carmena. Present and future of drug therapy in hypertension: An overview. Blood Pressure 2024. https://doi.org/10.1080/08037051.2024.2320401

3. C Chan and DHH Lau. Challenges in the clinical care of major cardiovascular conditions in primary care: A narrative review. Current Problems in Cardiology 2025; 50 (8) , 103099.

4. Z Wang, X Sun, M Sun, C Wang and L Yang. Game changers: Blockbuster small-molecule drugs approved by the FDA in 2024. Pharmaceuticals 2025; 18(5) , 729.

5. L Muhl, G Mocci, R Pietilä, J Liu, L He, G Genové, S Leptidis, S Gustafsson, B Buyandelger, E Raschperger, EM Hansson, JLM Björkegren, M Vanlandewijck, U Lendahl and C Betsholtz. A single-cell transcriptomic inventory of murine smooth muscle cells. Developmental Cell 2022; 57(20) , 2426-2443.e6.

6. S Sodiqova, S Kadirova, A Zaynabiddinov, I Abdullaev, L Makhmudov, U Gayibov, M Yuldasheva, M Xolmirzayeva, R Rakhimov, A Mutalibov and H Karimjonov. Channelopathy activity of A-41(Propyl Ester of Gallic Acid): Experimental and computational study of antihypertensive activity. Trends in Sciences 2025; 22(9) , 10496.

7. TF Aripov and UG Gayibov. Antiradical and antioxidant activity of the preparation “Rutan” from Rhus coriaria L. Journal of Theoretical and Clinical Medicine 2023; 4 , 164-170.

8. B Kim, C Jo, HY Choi and K Lee. Prunetin relaxed isolated rat aortic rings by blocking calcium channels. Molecules 2018; 23(9) , 2372.

9. B Kim, K Lee, KS Chinannai, I Ham, Y Bu, H Kim, HY Choi. Endothelium-independent vasorelaxant effect of Ligusticum jeholense root and rhizoma on rat thoracic aorta. Molecule 2015 ; 20 (6) , 10721-10733.

10. ML Addison, P Ranasinghe and DJ Webb. Novel pharmacological approaches in the treatment of hypertension: A focus on RNA-based therapeutics. Hypertension 2023; 80(11) , 2243-2254.

11. R Sayidaliyeva, S Kadirova, A Zaynabiddinov, I Abdullaev, L Makhmudov, U Gayibov, M Yuldasheva, M Kholmirzayeva, R Rakhimov, A Mutalibov and H Karimjonov. A-51 as a natural calcium channel blocker: An integrative study targeting hypertension. Trends in Sciences 2025; 22(11) , 10760.

12. E Altobelli; L Rapacchietta, VF Profeta and R Fagnano. Risk factors for abdominal aortic aneurysm in population-based studies: A systematic review and meta-analysis. International Journal of Environmental Research and Public Health 2018 ; 15(12) , 2805.

13. M Zaripova, I Abdullaev, A Bogbekov, U Gayibov, S Omonturdiev, R Makhmudov, N Ergashev, G Jabbarova, S Gayibova and T Aripov. In Vitro and in Silico studies of Gnaphalium U. Extract: Inhibition of α-amylase and α-glucosidase as a potential strategy for metabolic syndrome regulation. Trends in Sciences 2025; 22(8) , 10098.

14. FV Brozovich, CJ Nicholson, CV Degen, YZ Gao, M Aggarwal and KG Morgan. Mechanisms of vascular smooth muscle contraction and the basis for pharmacologic treatment of smooth muscle disorders. Pharmacological Reviews 2016; 68(2) , 476-532.

15. OS Zoirovich, AIZ Ugli, ID Raxmatillayevich, ML Umarjonovich, ZM Ravshanovna, GS Narimanovna, GU Gapparjanovich and AT Fatikhovich. The effect of Ájuga Turkestánica on the rat aortic smooth muscle ion channels. Biomedical & Pharmacology Journal 2024; 17(2) , 1213-1222.

16. G Cao, X Xuan, J Hu, R Zhang, H Jin and H Dong. How vascular smooth muscle cell phenotype switching contributes to vascular disease. Cell Communication and Signaling 2022; 20(1) , 180.

17. D Ghosh, AU Syed, MP Prada, MA Nystoriak, LF Santana, M Nieves-Cintrón and MF Navedo. Calcium channels in vascular smooth muscle. Advances in Pharmacology 2017; 78 , 49-87.

18. YL Chen, Z Daneva, M Kuppusamy, M Ottolini, TM Baker, E Klimentova, SA Shah, JD Sokolowski, MS Park and SK Sonkusare. Novel smooth muscle Ca(2+)-signaling nanodomains in blood pressure regulation. American Heart Association 2022; 146(7) , 548-564.

19. I McFadzean and A Gibson. The developing relationship between receptor-operated and store-operated calcium channels in smooth muscle. British Journal of Pharmacology 2002; 135 (1) , 1-13.

20. Q Lin, G Zhao, X Fang, X Peng, H Tang, H Wang, R Jing, J Liu, WJ Lederer, J Chen and K Ouyang. IP ₃ receptors regulate vascular smooth muscle contractility and hypertension. JCI Insight 2016; 1(17) , e89402.

21. Z Liu, RA Khalil. Evolving mechanisms of vascular smooth muscle contraction highlight key targets in vascular disease. Biochemical Pharmacology 2018; 153 , 91-122.

22. WF Jackson. K _V channels and the regulation of vascular smooth muscle tone. Microcirculation 2018; 25(1) , e12421.

23. D Guntur, H Olschewski, P Enyedi, R Csáki, A Olschewski and C Nagaraj. Revisiting the large-conductance calcium-activated potassium (BKCa) channels in the pulmonary circulation. Biomolecules 2021; 11(11) , 1629.

24. KP Nunes and RC Webb. New insights into RhoA/Rho-kinase signaling: A key regulator of vascular contraction. Small GTPases 2021; 12(5 - 6) , 458-469.

25. M Ottolini and SK Sonkusare. The calcium signaling mechanisms in arterial smooth muscle and endothelial cells. Comprehensive Physiology 2021; 11(2) , 1831-1869.

26. AV Mahmudov, OS Abduraimov, SB Erdonov, UG Gayibov and LY Izotova. Bioecological features of nigella sativa L. in different conditions of Uzbekistan. Plant Science Today 2022; 9(2) : 421-426.

27. U Gayibov, I Abdullaev, F Sobirova, S Omonturdiev, A Abdullaev, S Gayibova and T Aripov. Plant-derived and synthetic antihypoxic agents in cardiovascular diseases: Mechanisms, key pathways and therapeutic potential. Plant Science Today 2025; 12(4) , 1-10 .

28. D Inomjonov, I Abdullaev, S Omonturdiev, A Abdullaev, L Maxmudov, M Zaripova, M Abdullayeva, D Abduazimova, S Menglieva, S Gayibova, Ma Sadbarxon, U Gayibov and T Aripov. In Vitro and In Vivo studies of crategus and inula helenium extracts: Their effects on rat blood pressure. Trends in Sciences 2025, 22(3) , 9158

29. S D’Haese, D Deluyker and V Bito. Acute exposure to glycated proteins impaired in the endothelium-dependent aortic relaxation: A matter of oxidative stress. International Journal of Molecular Sciences 2022; 23(23) , 14916.

30. YC Loh, CS Tan, YS Ch’ng, M Ahmad, MZ Asmawi and MF Yam. Overview of antagonists used for determining the mechanisms of action employed by potential vasodilators with their suggested signaling pathways. Molecules . 2016; 21(4) , 495.

31. I Abdullaev, U Gayibov, S Omonturdiev, S Fotima, S Gayibova and T Aripov. Molecular pathways in cardiovascular disease under hypoxia: Mechanisms, biomarkers, and therapeutic targets[J]. The Journal of Biomedical Research 2025; 39(3) : 254-269.

32. A Arsyad, GKR Lembang, SL Linda, YY Djabir and GP Dobson. Low calcium-high magnesium krebs-henseleit solution combined with adenosine and lidocaine improved rat aortic function and structure following cold preservation. Medicina (Kaunas) 2024; 60(8) , 1284.

33. CJ Hansen, S Siricilla, N Boatwright, JH Rogers, ME Kumi and J Herington . Effects of solvents, emulsions, cosolvents, and complexions on ex vivo mouse myometrial contractility. Reproductive Sciences 2022; 29(2) , 586-595.

34. MAS Leal, TD Almeida, JG Torres, LCG Campos, EC Vasquez and VG Barauna. Physiological and biochemical vascular. reactivity parameters of angiotensin ii and the action of biased agonist TRV023. Advances in Pharmacological and Pharmaceutical Sciences 2020; 2020 , 3092721.

35. A Esimbetov, A Zaripov, S Omonturdiev, M Sultankhodjaev and P Usmanov. vasorelaxant effect of the alkaloid 1-0-Benzoylnappeline on isolated rat thoracic aorta. International Journal of Agriculture, Environment and Bioresearch 2020; 5(2) , 146-156.

36. A Abdullaev, I Abdullaev, A Bogbekov, U Gayibov, S Omonturdiev, S Gayibova, M Turahodjayev, K Ruziboev and T Aripov. Antioxidant potential of rhodiola heterodonta extract: Activation of Nrf2 pathway via integrative In Vivo and In Silico studies. Trends in Sciences , 2025; 22(5) , 9521.

37. MR Zaripova, SN Gayibova, RR Makhmudov, AA Mamadrahimov, Nl Vypova, UG Gayibov, SM Miralimova and TF Aripov. Characterization of Rhodiola heterodonta (Crassulaceae): Phytocomposition, antioxidant and antihyperglycemic activities. Preventive Nutrition and Food Science 2024; 29(2) , 135-145.

38. K Karabacak, E Kaya, KG Ulusoy, M Seyrek, M Kurtoglu, S Doganci, V Yildirim, O Yildiz and U Demirkilic. Effects of taurine on contractions of human internal mammary artery: A potassium channel opening action. European Review for Medical and Pharmacological Sciences 2015; 19 , 1498-1504.

39. AV Mahmudov, OS Abduraimov, SB Erdonov, AL Allamurotov, OT Mamatqosimov, UG Gayibov and L Izotova. Seed productivity of Linum usitatissimum L. in different ecological conditions of Uzbekistan. Plant Science Today 2022; 9(4) , 1090-1101.

40. T Godfraind. Discovery and development of calcium channel blockers. Frontiers in pharmacology 2017; 8 , 286.

41. U Gayibov, SN Gayibova, MK Pozilov, FS Tuxtaeva, UR Yusupova, GMK DJabbarova, ZA Mamatova, NA Ergashev and TF Aripov. Influence of quercetin and dihydroquercetin on some functional parameters of rat liver mitochondria. Journal of Microbiology, Biotechnology and Food Sciences 2021; 11(1) , e2924.

42. L Collins, L Lam, O Kleinig, W Proudman, R Zhang, M Bagster, J Kovoor, A Gupta, R Goh, S Bacchi, D Schultz and T Kleinig. Verapamil in the treatment of reversible cerebral vasoconstriction syndrome: A systematic review. Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australasia 2023; 113 , 130-141.

43. UG Gayibov. Influence of new polyphenol compound from Euphorbia plant on mitochondrial function. Journal of Microbiology, Biotechnology and Food Sciences 2019; 8(4) , 1021-1025.

44. S Koli, A Prakash, S Choudhury, R Mandil and SK Garg. Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.  Biological Trace Element Research 2019; 187(2) , 418-424.

45. MK Pozilov, U Gayibov, MI Asrarov, NG Abdulladjanova, HS Ruziboev and TF Aripov. Physiological alterations of mitochondria under diabetes condition and its correction by polyphenol gossitan. Journal of Microbiology, Biotechnology and Food Sciences 2022; 12(2) , e2224.

46. AG Vakhobjonovna, KE Jurayevich, AIZ Ogli, EN Azamovich, MR Rasuljonovich and AM Islomovich. Tannins as modulators in the prevention of mitochondrial dysfunction. Trends in Sciences 2025 ; 22(8) , 10436.

47. I Abdurazakova, A Zaynabiddinov, I bdullaev, L Makhmudov, U Gayibov, S Omonturdiev, G Abdullayev, M Xolmirzayeva and S Zhurakulov. Pharmacological evaluation of F-45 on the cardiovascular system using in vitro, in vivo models and molecular dockings. Trends in Sciences 2025; 22(12) , 10924.

48. CA D’Addario, S Matsumura, A Kitagawa, GM Lainer, F Zhang, M D’silva, MY Khan, G Froogh, A Gruzdev, DC Zeldin, ML Schwartzman and SA Gupte. Global and endothelial G-protein coupled receptor 75 (GPR75) knockout relaxes pulmonary artery and mitigates hypoxia-induced pulmonary hypertension. Vascular Pharmacology 2023; 153 , 107235.

49. M Mamajanov, I Abdullaev, G Sotimov, S Mavlanova, Q Niyozov, M Mirzaolimov, A Najimov, E Mirzaolimov, M Raximberganov and U Abdullayev. Mitochondrial and pharmacokinetic insights into 3,5,7,2′,6′-pentahydroxyflavanone: respiratory modulation, calcium handling, and membrane stability. Trends in Sciences 2025; 22(12) , 10984.

50. EV Hersh, PA Moore and M Saraghi. Phentolamine mesylate: Pharmacology, efficacy, and safety. General Dentistry 2019; 67(3) , 12-17.

51. Z Shakiryanova, R Khegay, U Gayibov, A Saparbekova, Z Konarbayeva, A Latif and O Smirnova. Isolation and study of a bioactive extract enriched with anthocyanin from red grape pomace (Cabernet sauvignon). Agronomy Research 2023; 21(3) , 1293-1303.

52. R Zhang, L Peng, H Ran, Y Fan, Y Zhao, F Cao. Farnesoid X receptor activation modulates calcium homeostasis in rat aortic vascular smooth muscle cells. The Chinese Journal of Physiology 2018; 61(4) , 210-220.

53. K Rahate, LK Bhatt and KS Prabhavalkar. SERCA stimulation: A potential approach in therapeutics. Chemical Biology & Drug Design 2020; 95(1) , 5-15.

54. R Dahl and I Bezprozvanny. SERCA pump as a novel therapeutic target for treating neurodegenerative disorders. Biochemical and Biophysical Research Communications 2024; 734 ,150748.

55. PJ Chambers, ES Juracic, VA Fajardo and AR Tupling. Role of SERCA and sarcolipin in adaptive muscle remodeling. American Journal of Physiology, Cell physiology 2022; 322(3) , C382-C394.

56. C Hidalgo and A Paula-Lima. RyR-mediated calcium release in hippocampal health and disease. Trends in Molecular Medicine 2024; 30(1) , 25-36.

57. Gayibov UG, Gayibova SN, Karimjonov HM, Abdullaev AA, et al. Antioxidant and cardioprotective properties of polyphenolic plant extract of Rhus glabra L. Plant Sci Today. 2024; 11(3) , 2348–1900.

58. DR Laver. Regulation of the RyR channel gating by Ca ²⁺ and Mg ² . Biophysical Reviews 2018; 10(4) , 1087-1095.

59. S Gleitze, OA Ramírez, I Vega-Vásquez,J Yan, P Lobos, H Bading, MT Núñez, A Pauka-Lima and C Hidalgo. Ryanodine receptor mediated calcium release contributes to ferroptosis induced in primary hippocampal neurons by GPX4 inhibition. Antioxidants (Basel) 2023; 12(3) , 705.

60. AGS Harper and SO Sage. TRP-Na(+)/Ca(2+) exchanger coupling. Advances in Experimental Medicine and Biology 2016; 898 , 67-85.

61. T Loeck and A Schwab. The role of the Na ⁺ /Ca ²⁺ -exchanger (NCX) in cancer-associated fibroblasts. Biological Chemistry 2023; 404(4) , 325-337.

62. RG Contreras, A Torres-Carrillo, C Flores-Maldonado, L Shoshani and A Ponce. Na ⁺ /K ⁺ -ATPase: More than an electrogenic pump. International Journal of Molecular Sciences 2024; 25(11) , 6122.

63. O Gaibullayeva, A Islomov, D Abdugafurova, B Elmurodov, B Mirsalixov, L Mahmudov, I Adullaev, K Baratov, S Omonturdiev and S Sa’dullayeva. Inula helenium L. root extract in sunflower oil: Determination of its content of water-soluble vitamins and immunity-promoting effect. Biomedical Pharmacology Journal 2024; 17(4) , 2729-2737.

64. Y Umidakhon, B Erkin, G Ulugbek, et al. Correction of the mitochondrial NADH oxidase activity, peroxidation and phospholipid metabolism by haplogenin-7-glucoside in hypoxia and ischemia[J]. Trends Sci 2022, 19(21) , 6260.

65. RJ Korthuis. Mechanisms of I/R-Induced endothelium-dependent vasodilator dysfunction. Advances in Pharmacology 2018; 81 , 331-364.

66. AA Abdullaev, DR Inamjanov, DS Abduazimova, SZ Omonturdiyev, UG Gayibov, SN Gayibova and TF Aripov. Sílybum Mariánum’s impact on physiological alterations and oxidative stress in diabetic rats. Biomedical and Pharmacology Journal 2024; 17(2) , 1291-1300 .

67. M Li, M Zhang, AT Fatixovich, GU Gapparjanovich and H Du. Green-synthesized Zn²⁺-polyphenol networks (CGA/RA) for enhanced multifunctional food preservation. Journal of Food Measurement and Characterization 2025. https://doi.org/10.1007/s11694-025-03764-y

68. UG Gayibov, SN Gayibova, HM Karimjonov, AA Abdullaev, DS Abduazimova, RN Rakhimov, HS Ruziboev, MA Xolmirzayeva, AE Zaynabiddinov and TF Aripov. Antiradical and antioxidant activity of the preparation “Rutan” from Rhus coriaria L. Journal of Theoretical and Clinical Medicine 2023; 4 , 164-170.

69. LHS Santos, RS Ferreira and ER Caffarena. Integrating molecular docking and molecular dynamics simulations. Methods in Molecular Biology 2019; 2053 , 13-34.

70. P Peluso, A Dessì, R Dallocchio, V Mamane and S Cossu. Recent studies of docking and molecular dynamics simulation for liquid-phase enantioseparations. Electrophoresis 2019; 40(15) , 1881-1896.

71. AQQ Azimova, AX Islomov, SA Maulyanov, D Gulyamovna Abdugafurova, LU Mahmudov, IZiyoyiddin Abdullaev, AS Ishmuratova, SQ Siddikova and IR Askarov. Determination of vitamins and pharmacological properties of Vitis vinifera L. plant fruit part (mixed varieties) syrup-honey. Biomedical and Pharmacology Journal 2024; 17(4) , 2779-2786.