Contribution of calcium dysregulation to impaired coronary artery contraction in Zucker diabetic fatty rats

Diabetic coronary artery injury is closely associated with Ca²⁺ dysregulation, although the underlying mechanism remains unclear. This study explored the role and mechanism of Ca²⁺ handling in coronary artery dysfunction in type 2 diabetic rats. Zucker diabetic fatty (ZDF) rats were used as the type 2 diabetes mellitus model. The contractility of coronary artery rings induced by KCl, CaCl₂, 5-HT and U46619 was significantly lower in ZDF rats than in Zucker lean rats. Vasoconstriction induced by 5-HT and U46619 was greatly inhibited by nifedipine. However, in the presence of 1 μM nifedipine or in the Ca²⁺-free KH solution containing 1 μM nifedipine, there was no difference in the vasoconstriction between Zucker lean and ZDF rats. Store-operated calcium channels (SOCs) were not involved in coronary vasoconstriction. The downregulation of contractile proteins and the upregulation of synthesized proteins were in coronary artery smooth muscle cells (CASMCs) from ZDF rats. Metformin reversed the reduction of vasoconstriction in ZDF rats. Taken together, L-type calcium channel is important for regulating the excitation–contraction coupling of VSMCs in coronary arteries, and dysregulation of this channel contributes to the decreased contractility of coronary arteries in T2DM.     

The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca channel subunits Orai1 and TRPC1

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca²⁺ sensor STIM1 and Ca²⁺ permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca²⁺ permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca²⁺ channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Store-operated calcium entry: From physiology to tubular aggregate myopathy

Store-Operated Ca²⁺ entry (SOCE) is recognized as a key mechanism in muscle physiology necessary to refill intracellular Ca²⁺ stores during sustained muscle activity. For many years the cell structures expected to mediate SOCE in skeletal muscle fibres remained unknown. Recently, the identification of Ca²⁺ Entry Units (CEUs) in exercised muscle fibres opened new insights into the role of extracellular Ca²⁺ in muscle contraction and, more generally, in intracellular Ca²⁺ homeostasis. Accordingly, intracellular Ca²⁺ unbalance due to alterations in SOCE strictly correlates with muscle disfunction and disease. Mutations in proteins involved in SOCE (STIM1, ORAI1, and CASQ1) have been linked to tubular aggregate myopathy (TAM), a disease that causes muscle weakness and myalgia and is characterized by a typical accumulation of highly ordered and packed membrane tubules originated from the sarcoplasmic reticulum (SR). Achieving a full understanding of the molecular pathways activated by alterations in Ca²⁺ entry mechanisms is a necessary step to design effective therapies for human SOCE-related disorders.     

Polychlorinated biphenyl 19 blocks the most common form of store-operated Ca2+ entry through Orai

PCB19, a 2,2′,6-trichlorinated biphenyl, is one of many non-dioxin-like polychlorinated biphenyls (NDL-PCBs), which are ubiquitous pollutants. NDL-PCBs affect cytosolic Ca²⁺ signaling by promoting Ca²⁺ release from ryanodine receptor-sensitive Ca²⁺ pools and inhibiting store-operated Ca²⁺ entry (SOCE) from the extracellular space. However, NDL-PCB-mediated SOCE inhibition has only been demonstrated in PC12 cells, in which SOCE is thought to be mainly mediated by TRPC family channels. Here, we investigated the effect of PCB19 on SOCE using human embryonic kidney 293 (HEK293) cells, human leukemia T cell line Jurkat-T cells and human promyelocytoma HL-60 cells which are the cell lines that are previously demonstrated to mediate the most common form of SOCE solely by the intrinsic Orai channels. PCB19 reduced thapsigargin-induced Ca²⁺ influx after Ca²⁺ pool depletion in HEK293 cells. SOCEs in HEK293, Jurkat T, HL-60 and PC12 cells showed distinct sensitivities to SOCE inhibitors such as Gd³⁺ and ML-9; however, PCB19 also showed a common effect of inhibiting SOCEs in all cell lines. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current but not the TRPM7 current. These results imply that PCB19 inhibits not only TRPC-mediated SOCE as in PC12 cells but also Orai-mediated SOCE as in many other cells including HEK293, Jurkat T and HL-60 cells.

     

Mechanism of asynchronous Ca2+ waves underlying agonist-induced contraction in the rat basilar artery

Background and purpose:

Uridine 5''-triphosphate (UTP) is a potent vasoconstrictor of cerebral arteries and induces Ca²⁺ waves in vascular smooth muscle cells (VSMCs). This study aimed to determine the mechanisms underlying UTP-induced Ca²⁺ waves in VSMCs of the rat basilar artery.

Experimental approach:

Isometric force and intracellular Ca²⁺ ([Ca²⁺]_i) were measured in endothelium-denuded rat basilar artery using wire myography and confocal microscopy respectively.

Key results:

Uridine 5''-triphosphate (0.1–1000 µmol·L⁻¹) concentration-dependently induced tonic contraction (pEC₅₀ = 4.34 ± 0.13), associated with sustained repetitive oscillations in [Ca²⁺]_i propagating along the length of the VSMCs as asynchronized Ca²⁺ waves. Inhibition of Ca²⁺ reuptake in sarcoplasmic reticulum (SR) by cyclopiazonic acid abolished the Ca²⁺ waves and resulted in a dramatic drop in tonic contraction. Nifedipine reduced the frequency of Ca²⁺ waves by 40% and tonic contraction by 52%, and the nifedipine-insensitive component was abolished by SKF-96365, an inhibitor of receptor- and store-operated channels, and KB-R7943, an inhibitor of reverse-mode Na⁺/Ca²⁺ exchange. Ongoing Ca²⁺ waves and tonic contraction were also abolished after blockade of inositol-1,4,5-triphosphate-sensitive receptors by 2-aminoethoxydiphenylborate, but not by high concentrations of ryanodine or tetracaine. However, depletion of ryanodine-sensitive SR Ca²⁺ stores prior to UTP stimulation prevented Ca²⁺ waves.

Conclusions and implications:

Uridine 5''-triphosphate-induced Ca²⁺ waves may underlie tonic contraction and appear to be produced by repetitive cycles of regenerative Ca²⁺ release from the SR through inositol-1,4,5-triphosphate-sensitive receptors. Maintenance of Ca²⁺ waves requires SR Ca²⁺ reuptake from Ca²⁺ entry across the plasma membrane via L-type Ca²⁺ channels, receptor- and store-operated channels, and reverse-mode Na⁺/Ca²⁺ exchange.     

Paracetamol inhibits Ca2+ permeant ion channels and Ca2+ sensitization resulting in relaxation of precontracted airway smooth muscle

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K⁺- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca²⁺ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca²⁺ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca²⁺ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca²⁺ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca²⁺ sensitization. These results suggest that paracetamol might be a new bronchodilator.     

Contraction of the sheep middle cerebral,pulmonary and coronary arteries initiated by release of intracellular calcium

1 The aim of the study was to compare contraction initiated by intracellular Ca²⁺ release in the middle cerebral, coronary and pulmonary arteries of the sheep. With all three arteries from the sheep, incubation in Ca²⁺-free physiological salt solution (PSS) reduced agonist-induced contraction much more than occurred with the rabbit aorta. The intracellular Ca²⁺ store appeared to be of limited capacity, since contraction was transient in Ca²⁺-free conditions with most agonists. 2 In the middle cerebral artery, contraction in Ca²⁺-free conditions was much reduced if a previous contraction had been obtained (for 5-hydroxytryptamine, 5-HT, from 11 + 4 to 1 + 0.5% of control contraction in 2.5 mm Ca²⁺), suggesting that the previous contraction had partly discharged the intracellular Ca²⁺ store. Contraction was less affected in the pulmonary artery and almost unaffected in the coronary artery (for 5-HT, from 15 + 1 to 11 + 1%) by a previous contraction in Ca²⁺-free conditions. 3 Rings prepared from small branches of the pulmonary and coronary arteries were affected by Ca²⁺ deprivation in a similar manner to large diameter pulmonary and coronary artery rings. 4 In Ca²⁺-free PSS, contraction induced by prostaglandin E₂ was almost eliminated (3 + 1% of control contraction in 2.5 mm Ca²⁺), contractions induced by 5-HT and noradrenaline were reduced, and contraction induced by the thromboxane mimetic U46619 was least affected (up to 73 + 8%). 5 Increasing agonist concentration from EC₅₀ to the maximally effective concentration raised the percentage contraction remaining in the middle cerebral artery (for noradrenaline from 7 + 2% to 12 + 3%) but not in the pulmonary artery (for noradrenaline from 22 + 2% to 24 + 6%). 6 The present study has revealed notable differences, in coupling to intracellular Ca²⁺ release between the three vascular territories studied.     

IL-4 and serum amyloid P inversely regulate fibrocyte differentiation by targeting store-operated Ca2+ channels

Background

Circulating fibrocytes (CFs) have been shown to participate in subepithelial fibrosis of asthma with chronic airflow limitation by acting as an important source of fibroblasts deposited beneath airway epithelia. Serum amyloid P (SAP) is an innate inhibitor of fibrocytes differentiation. Store-operated Ca²⁺ entry (SOCE) is the major Ca²⁺ influx of non-excitable cells. In this study, the role of SOCE in the regulation of fibrocytes differentiation and the effects of Th2 cytokine IL-4 and SAP on SOCE of fibrocytes were investigated.

Increased availability of angiotensin AT 1 receptors leads to sustained arterial constriction to angiotensin II in diabetes - role for Rho-kinase activation

BACKGROUND AND PURPOSE

Antagonists of angiotensin AT₁ receptors elicit beneficial vascular effects in diabetes mellitus. We hypothesized that diabetes induces sustained availability of AT₁ receptors, causing enhanced arterial constriction to angiotensin II.

EXPERIMENTAL APPROACH

To assess functional availability of AT₁ receptors, constrictions to successive applications of angiotensin II were measured in isolated skeletal muscle resistance arteries (∼150 µm) of Zucker diabetic fatty (ZDF) rats and of their controls (+/Fa), exposed acutely to high glucose concentrations (HG, 25 mM, 1 h). AT₁ receptors on cell membrane surface were measured by immunofluorescence.

KEY RESULTS

Angiotensin II-induced constrictions to first applications were greater in arteries of ZDF rats (maximum: 82 ± 3% original diameter) than in those from +/Fa rats (61 ± 5%). Constrictions to repeated angiotensin II administration were decreased in +/Fa arteries (20 ± 6%), but were maintained in ZDF arteries (67 ± 4%) and in +/Fa arteries vessels exposed to HG (65 ± 6%). In ZDF arteries and in HG-exposed +/Fa arteries, Rho-kinase activities were enhanced. The Rho-kinase inhibitor, Y27632 inhibited sustained constrictions to angiotensin II in ZDF arteries and in +/Fa arteries exposed to HG. Levels of surface AT₁ receptors on cultured vascular smooth muscle cells (VSMCs) were decreased by angiotensin II but were maintained in VSMCs exposed to HG. In VSMCs exposed to HG and treated with Y27632, angiotensin II decreased surface AT₁ receptors.

CONCLUSIONS AND IMPLICATIONS

In diabetes, elevated glucose concentrations activate Rho-kinase which inhibits internalization or facilitates recycling of AT₁ receptors, leading to increased functional availability of AT₁ receptors and sustained angiotensin II-induced arterial constriction.     

Dexamethasone restrains ongoing expression of interleukin-23p19 in peripheral blood-derived human macrophages

Background

Store-Operated Calcium Entry (SOCE) is the major Ca²⁺ ion entry pathway in lymphocytes and is responsible of a severe combined immunodeficiency (SCID) when deficient. It has recently been observed or highlighted in other cell types such as myoblasts and neurons, suggesting a wider physiological role of this pathway. Whereas Orai1 protein is considered to be the channel allowing the SOCE in T cells, it is hypothesized that other proteins like TRPC could associate with Orai1 to form SOCE with different pharmacology and kinetics in other cell types. Unraveling SOCE cell functions requires specific effectors to be identified, just as dihydropyridines were crucial for the study of Ca²⁺ voltage-gated channels, or spider/snake toxins for other ion channel classes. To identify novel SOCE effectors, we analyzed the effects of 2-aminoethyl diphenylborinate (2-APB) and its analogues. 2-APB is a molecule known to both potentiate and inhibit T cell SOCE, but it is also an effector of TRP channels and endoplasmic reticulum Ca²⁺-ATPase.

Results

A structure-function analysis allowed to discover that the boron-oxygen core present in 2-APB and in the borinate ester analogues is absolutely required for the dual effects on SOCE. Indeed, a 2-APB analogue where the boron-oxygen core is replaced by a carbon-phosphorus core is devoid of potentiating capacity (while retaining inhibition capacity), highlighting the key role of the boron-oxygen core present in borinate esters for the potentiation function. However, dimesityl borinate ester, a 2-APB analogue with a terminal B-OH group showed an efficient inhibitory ability, without any potentiating capacity. The removal or addition of phenyl groups respectively decrease or increase the efficiency of the borinate esters to potentiate and inhibit the SOCE. mRNA expression revealed that Jurkat T cells mainly expressed Orai1, and were the more sensitive to 2-APB modulation of SOCE.

Conclusions

This study allows the discovery of new boron-oxygen core containing compounds with the same ability as 2-APB to both potentiate and inhibit the SOCE of different leukocyte cell lines. These compounds could represent new tools to characterize the different types of SOCE and the first step in the development of new immunomodulators.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats

Background and purpose:

The endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in mesenteric small arteries from 21 week old Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats was investigated using (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309), a potent activator of small-conductance, calcium-activated potassium channel (SK_Ca) and intermediate-conductance, calcium-activated potassium channel (IK_Ca).

Experimental approach:

In the presence of inhibitors of cyclooxygenase and nitric oxide synthase [indomethacin and N^ω-nitro-L-arginine methyl ester (l-NAME), respectively], acetylcholine (ACh)-induced hyperpolarization and EDHF-type relaxation were investigated under isometric conditions in the wire myograph using 0.5 and 1 µM NS309 and/or selective blockers of SK_Ca and IK_Ca channels. Membrane potential was recorded with glass microelectrodes, and changes in the intracellular calcium concentration of endothelial cells were visualized by confocal microscopy. SK_Ca expression was assessed by Western blotting.

Key results:

In arteries from ZDF rats, ACh-induced relaxation and membrane hyperpolarization were attenuated and, compared with arteries from ZL rats, NS309 was less potent at causing relaxation. Incubation with 0.5 µM NS309 did not increase ACh-induced relaxation in arteries from ZDF rats significantly. However, 1 µM NS309 restored it (both in the absence and in the presence of indomethacin and l-NAME) without changing endothelial intracellular calcium concentration. The restored EDHF-type relaxation was more sensitive to TRAM-34 (1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole) (1 µM) than to apamin. Expression of the SK_Ca channel was unaltered.

Conclusions and implications:

The attenuated EDHF-type relaxation in mesenteric small arteries from ZDF rats can be restored by NS309 without changes in the intracellular calcium concentration of endothelial cells. These results may have clinical implications for the treatment of endothelial dysfunction in overweight type 2 diabetic patients.     

Ryanodine receptor 2 contributes to hemorrhagic shock-induced bi-phasic vascular reactivity in rats

Aim:

Ryanodine receptor 2 (RyR2) is a critical component of intracellular Ca²⁺ signaling in vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the role of RyR2 in abnormal vascular reactivity after hemorrhagic shock in rats.

Methods:

SD rats were hemorrhaged and maintained mean arterial pressure (MAP) at 40 mmHg for 30 min or 2 h, and then superior mesenteric arteries (SMA) rings were prepared to measure the vascular reactivity. In other experiments, SMA rings of normal rats and rat VSMCs were exposed to a hypoxic medium for 10 min or 3 h. SMA rings of normal rats and VSMCs were transfected with siRNA against RyR2. Intracellular Ca²⁺ release in VSMCs was assessed using Fura-2/AM.

Results:

The vascular reactivity of the SMA rings from hemorrhagic rats was significantly increased in the early stage (30 min), but decreased in the late stage (2 h) of hemorrhagic shock. Similar results were observed in the SMA rings exposed to hypoxia for 10 min or 3 h. The enhanced vascular reactivity of the SMA rings exposed to hypoxia for 10 min was partly attenuated by transfection with RyR2 siRNA, whereas the blunted vascular reactivity of the SMA rings exposed to hypoxia for 3 h was partly restored by transfection with RyR2 siRNA. Treatment with the RyR agonist caffeine (1 mmol/L) significantly increased Ca²⁺ release in VSMCs exposed to hypoxia for 10 min or 3 h, which was partially antagonized by transfection with RyR2 siRNA.

Conclusion:

RyR2-mediated Ca²⁺ release contributes to the development of bi-phasic vascular reactivity induced by hemorrhagic shock or hypoxia.     

Comparative Study of Calcium Ion Dynamics and Contractile Response in Rat Middle Cerebral and Basilar Arteries

The objective of this study was to compare intracellular calcium concentration ([Ca²⁺]_i) and contractile responses in isolated rat middle cerebral artery (MCA) with those in basilar artery (BA) employing real-time confocal laser microscopy. KCl elicited transient [Ca²⁺]_i elevation and sustained contraction in both arteries; moreover, nearly equal responses were evident in both arteries. Application of 5-hydroxytryptamine (5-HT), vasopressin (VP), and α,β-methylene adenosine 5’-triphosphate (α,β-me ATP) also induced elevation of [Ca²⁺]_i and contraction in both arteries. The maximum response of 5-HT and VP necessary to increase [Ca²⁺]_i and to constrict the MCA was less in comparison to the BA; however, a linear relationship emerged between the maximum response of [Ca²⁺]_i and that of contraction. Additionally, the slope of the correlation regression line of MCA was nearly identical to that of BA. On the other hand, cyclopiazonic acid (CPA)-induced Ca²⁺ release from store sites following contraction of MCA was distinct from that of BA. In MCA, velocity of [Ca²⁺]_i elevation in smooth muscle cells and Ca²⁺-wave propagation along smooth muscle cells induced by 5-HT were slower than those in BA. These observations revealed that different regions of arteries along the same cerebral tissue may display distinct [Ca²⁺]_i response; moreover, this difference may be one reason for the distinct contractile response.     

Adenine attenuates the Ca<Superscript>2+</Superscript> contraction-signaling pathway via adenine receptor-mediated signaling in rat vascular smooth muscle cells

Our previous study demonstrated that adenine (6-amino-6H-purine) relaxed contracted rat aorta rings in an endothelial-independent manner. Although adenine receptors (AdeRs) are expressed in diverse tissues, aortic AdeR expression has not been ascertained. Thus, the aims of this study were to clarify the expression of AdeR in rat vascular smooth muscle cells (VSMCs) and to investigate the adenine-induced vasorelaxation mechanism(s). VSMCs were isolated from 8-week-old male Wistar-Kyoto rats and used in this study. Phosphorylation of myosin light chain (p-MLC) was measured by western blot. AdeR mRNA was detected by RT-PCR. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by using Fura-2/AM. Vasorelaxant adenine (10–100 μM) significantly reduced p-MLC by angiotensin II (Ang II, 10 μM) in VSMCs (P < 0.05). We confirmed the expression of aortic AdeR mRNA and the activation of PKA in VSMCs through stimulation of AdeR by adenine by ELISA. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurement demonstrated that adenine inhibits Ang II- and m-3M3FBS (PLC agonist)-induced [Ca²⁺]_i elevation. In AdeR-knockdown VSMCs, PKA activation and p-MLC reduction by adenine were completely abolished. These results firstly demonstrated that vasorelaxant adenine can suppress Ca²⁺ contraction signaling pathways via aortic AdeR/PKA activation in VSMCs.     

Nanomolar potency and selectivity of a Ca²⁺ release-activated Ca²⁺ channel inhibitor against store-operated Ca²⁺ entry and migration of vascular smooth muscle cells

BACKGROUND AND PURPOSE

The aim was to advance the understanding of Orai proteins and identify a specific inhibitor of the associated calcium entry mechanism in vascular smooth muscle cells (VSMCs).

EXPERIMENTAL APPROACH

Proliferating VSMCs were cultured from human saphenous veins. Intracellular calcium was measured using fura-2, whole-cell current was recorded using patch-clamp and cell migration quantified in modified Boyden chambers. Subcellular protein localization was determined by microscopy. Isometric tension was recorded from mouse aortic rings.

KEY RESULTS

Molecular disruption and rescue experiments indicated the importance of Orai1 in calcium entry caused by store depletion evoked passively or by platelet-derived growth factor (PDGF), suggesting the presence of Ca²⁺ release-activated Ca²⁺ (CRAC) channels like those of the immune system. The CRAC channel blocker, S66, was a potent inhibitor of the VSMC signals, IC₅₀ 26 nM, which was almost two orders of magnitude greater than with leucocytes. S66 had no effect on PDGF- and ATP-evoked calcium release, overexpressed transient receptor potential canonical (TRPC)5 channels, native TRPC1/5-containing channels, stromal interaction molecule 1 clustering, non-selective cationic current evoked by store depletion and phenylephrine-evoked aortic contraction. S66 reduced PDGF-evoked VSMC migration while having only modest effects on cell proliferation and no effect on cell viability.

CONCLUSIONS AND IMPLICATIONS

The data suggest that Orai1 has a role in human VSMC migration, and that a CRAC channel inhibitor has high potency and selectivity for the associated calcium entry, suggesting a distinct characteristic of vascular CRAC channels and the potential for selective chemical suppression of vascular remodelling.     

Store‐operated Ca2+ entry is not required for store refilling in skeletal muscle

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Augmented inositol phosphate production in mesenteric arteries from diabetic rats

The effects of noradrenaline (NA) on contraction and phosphoinositide metabolism were compared in mesenteric arteries from rats with chronic streptozotocin-induced diabetes and from age-matched control rats. Maximum contractile responses of mesenteric arteries from diabetic rats to NA (30 μM) were significantly greater than control in both the presence and absence of extracellular Ca²⁺. Basal incorporation of [³H]myoinositol into total [³H]inositol phosphates was greater in diabetic than control mesenteric arteries. NA (30 μM) resulted in a time-dependent increase in total [³H]inositol phosphate production, which was also significantly greater in diabetic than in control preparations. The increase in total [³H]inositol phosphates produced by NA in both control and diabetic arterie was blocked by the α₁-adrenoceptor antagonists, prazosin. Absolute levels of inositol 1,4,5-trisphosphate (I(1,4,5)P₃), measured by protein binding assay, were also increased in response to 30 μM NA in both control and diabetic arteries. Although basal I(1,4,5)P₃ levels were not significantly different, NA-induced increases in I(1,4,5)P₃ were significantly greater in diabetic than in control arteries at each time-point measured. These data indicate that phosphoinositide metabolism is enhanced in mesenteric arteries from rats with chronic streptozotocin-induced diabetes in response to a maximum concentration of NA. Augmented production of the second messengers I(1,4,5)P₃ and presumably, 1,2-diacylgylcerol may contribute to the enhanced maximum contractile responses of the diabetic arteries to NA.