Transient receptor potential channels regulate myogenic tone of resistance arteries

Elevation of intravascular pressure causes depolarization and constriction (myogenic tone) of small arteries and arterioles, and this response is a key element in blood flow regulation. However, the nature of pressure-induced depolarization has remained elusive. In the present study, we provide evidence that a transient receptor potential channel (TRPC6) homologue has a major role in this depolarizing response to pressure. Antisense oligodeoxynucleotides to TRPC6 decreased TRPC6 protein expression and greatly attenuated arterial smooth muscle depolarization and constriction caused by elevated pressure in intact cerebral arteries. Suppressing the expression of this channel protein also reduced the current density of a major cation current in resistance artery smooth muscle cells. We propose that TRPC6 channels play an essential role in regulation of myogenic tone.     

Arteriolar myogenic signalling mechanisms: Implications for local vascular function

Arterioles typically exist in a state of partial constriction that is related to the level of intraluminal pressure. This vasomotor response is a function of the vascular smooth muscle and occurs independently of neurohumoral and endothelial input. The physiological relevance of myogenic constriction relates to the setting of peripheral resistance, provision of a level of tone that vasodilators can access, and a contribution to control of capillary pressure. Despite its importance in the regulation of microvascular haemodynamics the exact cellular mechanisms linking intraluminal pressure to myogenic constriction remain uncertain. Studies using isolated, cannulated arteriole techniques, and freshly dispersed smooth muscle cells, have shown that increased intraluminal pressure/cell stretch leads to smooth muscle cell membrane depolarisation, the opening of L-type voltage-gated Ca2+ channels (VGCC), Ca2+-dependent activation of myosin light chain kinase and actomyosin-based contraction. Questions remain as to how the initial stimulus is detected and how these events lead to membrane depolarisation. A candidate pathway for the mechanosensory events involves the link between extracellular matrix proteins, cell surface integrins and the subsequent activation of intracellular signalling events. Membrane depolarisation may occur through the involvement of various ion channels, including non-selective cation channels (possibly themselves mechanosensitive) that predominantly pass Na+ from the extracellular space. Evidence suggests that this may involve TRP-like channels, possibly TRPM4 or TRPC6 isoforms that are modulated by diacylglycerol and protein kinase C. In addition, the exact roles played by various Ca2+ pools, including those occurring in spatially-restricted domains, and Ca2+ sensitisation, remain uncertain despite the clearly important role of VGCC. Similarly, while a change in intraluminal pressure is associated with the generation of a number of second messengers and the activation of various protein kinases, their roles in myogenic contraction versus long-term adaptive responses, such as tissue remodelling, are still to be defined.     

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.     

The role of thermosensitive TRP (transient receptor potential) channels in insulin secretion

Insulin secretion from pancreatic β-cells is the only efficient means to decrease blood glucose concentrations. Glucose is the principal stimulator of insulin secretion with the ATP-sensitive K+ channel-voltage-gated Ca2+ channel-mediated pathway being the primary one involved in glucose-stimulated insulin secretion. Recently, several reports demonstrated that some transient receptor potential (TRP) channels are expressed in pancreatic β-cells and contribute to pancreatic β-cell functions. Interestingly, six of them (TRPM2, TRPM4, TRPM5, TRPV1, TRPV2 and TRPV4) are thermosensitive TRP channels. Thermosensitive TRP channels in pancreatic β-cells can function as multimodal receptors and cause Ca2+ influx and membrane depolarization at physiological body temperature. TRPM channels (TRPM2, TRPM4 and TRPM5) control insulin secretion levels by sensing intracellular Ca2+ increase, NAD metabolites, or hormone receptor activation. TRPV2 is involved not only in insulin secretion but also cell proliferation, and is regulated by the autocrine effects of insulin. TRPV1 expressed in sensory neurons is involved in β-cell stress and islet inflammation by controlling neuropeptide release levels. It is thus clear that thermosensitive TRP channels play important roles in pancreatic β-cell functions, and future analyses of TRP channel function will lead to better understanding of the complicated mechanisms involved in insulin secretion and diabetes pathogenesis.     

Preserved BK channel function in vasospastic myocytes from a dog model of subarachnoid hemorrhage

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is due to contraction of smooth muscle cells in the cerebral arteries. The mechanism of this contraction, however, is not well understood. Smooth muscle contraction is regulated in part by membrane potential, which is determined by K+ conductance in smooth muscle. Voltage-gated (Kv) and large-conductance, Ca2+-activated K+ (BK) channels dominate arterial smooth muscle K+ conductance. Vasospastic smooth muscle cells are depolarized relative to normal cells, but whether this is due to altered Kv or BK channel function has not been determined. This study determined if BK channels are altered during vasospasm after SAH in dogs. We first characterized BK channels in basilar-artery smooth muscle using whole-cell patch clamping and single-channel recordings. Next, we compared BK channel function between normal and vasospastic cells. There were no significant differences between normal and vasospastic cells in BK current density, kinetics, Ca2+ and voltage sensitivity, single-channel conductance or apparent Ca2+ affinity. Basilar-artery myocytes had no, small- or intermediate-conductance, Ca2+-activated K+ channels. The lack of difference in BK channels between vasospastic and control cells suggests alteration(s) in other K+ channels or other ionic conductances may underlie the membrane depolarization and vasoconstriction observed during vasospasm after SAH.     

The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction

Growing evidence suggests that mechanisms which regulate the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca2+ sensitivity is suited to partially uncouple intracellular Ca2+ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca2+-dependent processes. Following a brief review of 'classical' Ca2+-dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca2+ sensitivity. For the purpose of this review, Ca2+ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin light-chain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca2+ sensitivity will be discussed. Although Ca2+ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca2+-dependent 'initiation/trigger' phase followed by a Ca2+-independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca2+-dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca2+ sensitivity, as an equal and complimentary partner of Ca2+-dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.     

Viewpoints on the Role of Transient Receptor Potential Melastatin Channels in Cardiovascular System and Disease: A Systematic Review

Transient receptor potential (TRP) family play critical roles in cardiovascular system. TRPM family as largest TRP subfamily is non-voltage Ca2+-activated selective channels which has 8 members. This study aimed to discuss the role of TRPM family in cardiovascular system and diseases. Systematic search was performed covering PubMed, ISI Web of Science, and Google Scholar from inception until June 2021 using related keywords and Mesh terms for English studies with human, animal and in-vitro subjects. Finally 10 studies were selected for data extraction. Reviewing the articles showed that TRPM2, TRPM4, TRPM5, TRPM6 and TRPM7 play important roles in cardiovascular system and diseases. TRPM2 could be activated by reactive oxygen species (ROS) and effects on cardiac injury and cardiac fibrosis. TRPM7 and TRPM6 also have been reported to be associated with cardiac fibrosis and atrial fibrosis development respectively. TRPM4 channels contributed to resting membrane potential of cerebral artery smooth muscle cells and atrial contraction. TRPM5 channels are bitter taste sensors and prevent high salt intake and consequently high blood pressure due to the high salt intake. In conclusion based on the proof of the effectiveness of some members of TRPM family in the cardiovascular system, research on other members of this channel group seems to be useful and necessary to find their possible connection to the cardiovascular system.     

Potassium channels in the peripheral microcirculation

Vascular smooth muscle (VSM) cells, endothelial cells (EC), and pericytes that form the walls of vessels in the microcirculation express a diverse array of ion channels that play an important role in the function of these cells and the microcirculation in both health and disease. This brief review focuses on the K+ channels expressed in smooth muscle and endothelial cells in arterioles. Microvascular VSM cells express at least four different classes of K+ channels, including inward-rectifier K+ channels (Kin), ATP-sensitive K+ channels (KATP), voltage-gated K+ channels (Kv), and large conductance Ca2+-activated K+ channels (BKCa). VSM KIR participate in dilation induced by elevated extracellular K+ and may also be activated by C-type natriuretic peptide, a putative endothelium-derived hyperpolarizing factor (EDHF). Vasodilators acting through cAMP or cGMP signaling pathways in VSM may open KATP, Kv, and BKCa, causing membrane hyperpolarization and vasodilation. VSMBKc. may also be activated by epoxides of arachidonic acid (EETs) identified as EDHF in some systems. Conversely, vasoconstrictors may close KATP, Kv, and BKCa through protein kinase C, Rho-kinase, or c-Src pathways and contribute to VSM depolarization and vasoconstriction. At the same time Kv and BKCa act in a negative feedback manner to limit depolarization and prevent vasospasm. Microvascular EC express at least 5 classes of K+ channels, including small (sKCa) and intermediate(IKCa) conductance Ca2+-activated K+ channels, Kin, KATP, and Kv. Both sK and IK are opened by endothelium-dependent vasodilators that increase EC intracellular Ca2+ to cause membrane hyper-polarization that may be conducted through myoendothelial gap junctions to hyperpolarize and relax arteriolar VSM. KIR may serve to amplify sKCa- and IKCa-induced hyperpolarization and allow active transmission of hyperpolarization along EC through gap junctions. EC KIR channels may also be opened by elevated extracellular K+ and participate in K+-induced vasodilation. EC KATP channels may be activated by vasodilators as in VSM. Kv channels may provide a negative feedback mechanism to limit depolarization in some endothelial cells.     

Emergence of a R-type Ca2+ channel (CaV 2.3) contributes to cerebral artery constriction after subarachnoid hemorrhage

Cerebral aneurysm rupture and subarachnoid hemorrhage (SAH) inflict disability and death on thousands of individuals each year. In addition to vasospasm in large diameter arteries, enhanced constriction of resistance arteries within the cerebral vasculature may contribute to decreased cerebral blood flow and the development of delayed neurological deficits after SAH. In this study, we provide novel evidence that SAH leads to enhanced Ca2+ entry in myocytes of small diameter cerebral arteries through the emergence of R-type voltage-dependent Ca2+ channels (VDCCs) encoded by the gene CaV 2.3. Using in vitro diameter measurements and patch clamp electrophysiology, we have found that L-type VDCC antagonists abolish cerebral artery constriction and block VDCC currents in cerebral artery myocytes from healthy animals. However, 5 days after the intracisternal injection of blood into rabbits to mimic SAH, cerebral artery constriction and VDCC currents were enhanced and partially resistant to L-type VDCC blockers. Further, SNX-482, a blocker of R-type Ca2+ channels, reduced constriction and membrane currents in cerebral arteries from SAH animals, but was without effect on cerebral arteries of healthy animals. Consistent with our biophysical and functional data, cerebral arteries from healthy animals were found to express only L-type VDCCs (CaV 1.2), whereas after SAH, cerebral arteries were found to express both CaV 1.2 and CaV 2.3. We propose that R-type VDCCs may contribute to enhanced cerebral artery constriction after SAH and may represent a novel therapeutic target in the treatment of neurological deficits after SAH.     

TRPM5 is a transient Ca2+-activated cation channel responding to rapid changes in [Ca2+]i

Transient receptor potential (TRP) proteins are a diverse family of proteins with structural features typical of ion channels. TRPM5, a member of the TRPM subfamily, plays an important role in taste receptors, although its activation mechanism remains controversial and its function in signal transduction is unknown. Here we characterize the functional properties of heterologously expressed human TRPM5 in HEK-293 cells. TRPM5 displays characteristics of a calcium-activated, nonselective cation channel with a unitary conductance of 25 pS. TRPM5 is a monovalent-specific, nonselective cation channel that carries Na+, K+, and Cs+ ions equally well, but not Ca2+ ions. It is directly activated by [Ca2+]i at concentrations of 0.3-1 microM, whereas higher concentrations are inhibitory, resulting in a bell-shaped dose-response curve. It activates and deactivates rapidly even during sustained elevations in [Ca2+]i, thereby inducing a transient membrane depolarization. TRPM5 does not simply mirror levels of [Ca2+]i, but instead responds to the rate of change in [Ca2+]i in that it requires rapid changes in [Ca2+]i to generate significant whole-cell currents, whereas slow elevations in [Ca2+]i to equivalent levels are ineffective. Moreover, we demonstrate that TRPM5 is not limited to taste signal transduction, because we detect the presence of TRPM5 in a variety of tissues and we identify endogenous TRPM5-like currents in a pancreatic beta cell line. TRPM5 can be activated physiologically by inositol 1,4,5-trisphosphate-producing receptor agonists, and it may therefore couple intracellular Ca2+ release to electrical activity and subsequent cellular responses.     

急性缺氧抑制大鼠肺动脉平滑肌细胞钙ATP钾通道

目的研究急性缺氧对大鼠肺动脉平滑肌细胞钾通道活性的影响，以探讨钾通道活性改变在急性低氧性肺血管收缩（ＨＰＶ）反应中所起的作用。方法应用膜片钳单通道技术，在对称性高钾溶液中，于急性酶分离的大鼠单个肺动脉平滑肌细胞的内面向外式膜片（ｉｎｓｉｄｅｏｕｔｐａｔｃｈ）上，记录外向性钾通道电流，并用常氧和低氧的细胞浴液持续灌流肺动脉平滑肌细胞，以观察其对外向性钾通道电流的影响。结果在记录的外向性钾电流中，证实了一种电流为钙、ＡＴＰ激活性钾通道（Ｋ＋ＣａＡＴＰ）；用低氧的细胞浴液灌流肺动脉平滑肌细胞可明显抑制这种钙、ＡＴＰ激活性钾通道的活性（Ｐ＜０．０１）。而钾通道开放剂卡吗克啉（ｃｒｏｍａｋａｌｉｍ）对低氧所抑制的肺动脉平滑肌细胞钙、ＡＴＰ激活性钾通道具有明显的激活作用（Ｐ＜０．０１）。结论急性低氧可通过对钙、ＡＴＰ激活性钾通道的抑制作用，使肺动脉平滑肌细胞膜发生去极化，肺动脉收缩而导致急性肺血管阻力增加，进而产生肺动脉高压。肺动脉平滑肌细胞钙、ＡＴＰ激活性钾通道活性的降低，可能在低氧性肺血管收缩反应中起着重要的作用。Ｃｒｏｍａｋａｌｉｍ可作为拮抗低氧性肺血管收缩的有效药物之一。     

Comparison of sphingosine 1-phosphate-induced intracellular signaling pathways in vascular smooth muscles: differential role in vasoconstriction

Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160(ROCK) (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate-dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44(mapk) in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P(1)/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.     

Membrane cholesterol depletion with beta-cyclodextrin impairs pressure-induced contraction and calcium signalling in isolated skeletal muscle arterioles

OBJECTIVE: Given evidence for clustering of signalling molecules and ion channels in cholesterol-rich membrane domains, the involvement of such structures in arteriolar smooth muscle mechanotransduction was examined. METHOD: To determine the contribution of smooth muscle cholesterol-rich membrane domains to the myogenic response, isolated arterioles were exposed to the cholesterol-depleting agent beta-cyclodextrin (1-10 mM) in the absence and presence of excess exogenous cholesterol. RESULTS: beta-Cyclodextrin significantly impaired pressure-induced vasoconstriction, while excess cholesterol attenuated this effect. Impaired myogenic constriction was evident in de-endothelialized vessels, indicating an action at the level of smooth muscle. beta-Cyclodextrin treatment uncoupled increases in intracellular Ca(2+) from myogenic constriction and depleted intracellular Ca(2+) stores consistent with a loss of connectivity between plasma membrane and sarcoplasmic reticulum signalling. However, beta-cyclodextrin-treated arterioles showed unaltered constrictor responses to KCl and phenylephrine. Electron microscopy verified that beta-cyclodextrin caused a decrease in caveolae, while confirmation of smooth muscle containing caveolae was obtained by immunostaining for caveolin-1. Viability of beta-cyclodextrin-treated arterioles was confirmed by agonist sensitivity and propidium iodide nuclear staining. CONCLUSION: The data suggest that smooth muscle cholesterol-rich membrane domains contribute to the myogenic response. Further studies are required to determine whether this relates to specific mechanosensory events or generalized alterations in membrane function.     

Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca2+ sparks

Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (-15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (-84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.     

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.     

Oxyhemoglobin-induced suppression of voltage-dependent K+ channels in cerebral arteries by enhanced tyrosine kinase activity

Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences. Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca(2+)-selective and K(+)-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K(+) channel (K(v)) currents by approximately 30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca(2+) channels or large conductance Ca(2+)-activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of K(v) channel currents and oxyhb-induced constriction of isolated cerebral arteries. The K(v) channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in K(v) channel activity could represent either channel block, or a decrease in K(v) channel density on the plasma membrane. To explore whether oxyhb altered trafficking of K(v) channels to the plasma membrane, we used an antibody generated against an extracellular epitope of K(v)1.5 channels. In the presence of oxyhb, staining of K(v)1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with K(v)1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of K(v) currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.     

Essential role for smooth muscle BK channels in alcohol-induced cerebrovascular constriction

Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca(2+) concentration and caused a reversible constriction of cerebral arteries (EC(50) = 27 mM; E(max) = 100 mM) that depended on voltage-gated Ca(2+) entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca(2+) currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (-53%) and amplitude (-32%) of transient Ca(2+)-activated K(+) (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca(2+) spark frequency (-49%), a subsarcolemmal Ca(2+) signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (-44%). In contrast, ethanol failed to modify Ca(2+) waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca(2+) spark/BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.