Vasodilation by the calcium-mobilizing messenger cyclic ADP-ribose

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.     

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.     

Hypoxic fetoplacental vasoconstriction in humans is mediated by potassium channel inhibition

Fetal to maternal blood flow matching in the placenta, necessary for optimal fetal blood oxygenation, may occur via hypoxic fetoplacental vasoconstriction (HFPV). We hypothesized that HFPV is mediated by K(+) channel inhibition in fetoplacental vascular smooth muscle, as occurs in several other O(2)-sensitive tissues. With the use of an isolated human placental cotyledon perfused at a constant flow rate, we found that hypoxia reversibly increased perfusion pressure by >20%. HFPV was unaffected by cyclooxygenase or nitric oxide synthase inhibition. HFPV and 4-aminopyridine, an inhibitor of voltage-dependent K(+) (K(v)) channels, increased pressure in a nonadditive manner, suggesting they act via a common mechanism. Iberiotoxin, a large conductance Ca(2+)-sensitive K(+) (BK(Ca)) channel inhibitor, had little effect on normoxic pressure. Immunoblotting and RT-PCR showed expression of several putative O(2)-sensitive K(+) channels in peripheral fetoplacental vessels. In patch-clamp experiments with smooth muscle cells isolated from peripheral fetoplacental arteries, hypoxia reversibly inhibited K(v) but not BK(Ca) or ATP-dependent currents. We conclude that human fetoplacental vessels constrict in response to hypoxia. This response is largely mediated by hypoxic inhibition of K(v) channels in the smooth muscle of small fetoplacental arteries.     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.     

Relaxing effects of 5-oxo-ETE on human bronchi involve BK Ca channel activation

The present study investigated the ability of 5-oxo-EicosaTetraEnoic acid (5-oxo-ETE) for modulating airway smooth muscle (ASM) tone in human bronchi. 5-Oxo-ETE induced a concentration-dependent relaxing effect on human bronchi pre-contracted with methacholine (MCh) and arachidonic acid (AA). This relaxing response was highly sensitive to Iberiotoxin (IbTx), a large conducting Ca(2+)-activated K(+) channel (BK(Ca)) inhibitor. Furthermore, microelectrode measurements revealed that 5-oxo-ETE (0.1-10 microM) hyperpolarizes the membrane potential of human bronchial ASM cells. These hyperpolarizing effects were also inhibited in the presence of 10nM IbTx. Lastly, 5-oxo-ETE was shown to directly activate reconstituted BK(Ca) channels derived from human airway smooth muscles. In summary, the 5-oxo-ETE eicosanoid activates a specific K(+) conductance, involved in membrane hyperpolarization, which in turn reduces Ca(2+) entry and facilitates relaxation of smooth muscle cells.     

Theoretical model of metabolic blood flow regulation: roles of ATP release by red blood cells and conducted responses

A proposed mechanism for metabolic flow regulation involves the saturation-dependent release of ATP by red blood cells, which triggers an upstream conducted response signal and arteriolar vasodilation. To analyze this mechanism, a theoretical model is used to simulate the variation of oxygen and ATP levels along a flow pathway of seven representative segments, including two vasoactive arteriolar segments. The conducted response signal is defined by integrating the ATP concentration along the vascular pathway, assuming exponential decay of the signal in the upstream direction with a length constant of approximately 1 cm. Arteriolar tone depends on the conducted metabolic signal and on local wall shear stress and wall tension. Arteriolar diameters are calculated based on vascular smooth muscle mechanics. The model predicts that conducted responses stimulated by ATP release in venules and propagated to arterioles can account for increases in perfusion in response to increased oxygen demand that are consistent with experimental findings at low to moderate oxygen consumption rates. Myogenic and shear-dependent responses are found to act in opposition to this mechanism of metabolic flow regulation.     

Polydatin, a natural polyphenol, protects arterial smooth muscle cells against mitochondrial dysfunction and lysosomal destabilization following hemorrhagic shock

The main objective of this study was to investigate the activity of polydatin on mitochondrial dysfunction and lysosomal stability of arteriolar smooth muscle cells (ASMCs) in severe shock. The experimental animals (rats) were divided into five groups: control, hemorrhagic shock, shock + CsA, shock + Res, and shock + PD (exposed to cyclosporin A, resveratrol, or polydatin following induction of hemorrhagic shock, respectively). The calcein-Co(2+) technique revealed opening of ASMC mitochondrial permeability transition pores (mPTP) after shock with resulting mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm), and reduced intracellular ATP levels. These alterations were all inhibited by exposure to PD, which was significantly more effective than CsA and Res. PD also preserved lysosomal stability, suppressed activation of K(ATP) channels, ASMC hyperpolarization, and reduced vasoresponsiveness to norepinephrine that normally follows severe shock. The results demonstrate that exposure to PD after initiation of severe shock effectively preserves ASMC mitochondrial integrity and has a significant therapeutic effect in severe shock. The effects may partially result from lysosomal stabilization against shock-induced oxidative stress and depressed relocation of hydrolytic enzymes and redox-active lysosomal iron that, in turn, may induce mPTP opening.     

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).     

重症休克时平滑肌细胞内的钙动力学变化

使用荧光比率方法研究血管平滑肌细胞钙动力学变化在重症休克血管反应性降低中的作用．复制SD大鼠失血性休克模型,分离肠系膜细动脉血管平滑肌(ASMC)．使用荧光探针Fluo-3／AM、FuraRed双标记比率方法结合激光扫描共聚焦显微成像技术测定单个平滑肌细胞钙动力学变化,观察ATP敏感钾通道(KATP)特异性阻滞剂优降糖对血管反应性和钙动力学影响、实验结果发现休克后2h(失代偿期),血管反应性明显降低,norepinephrine作用带来的平滑肌细胞内钙离子浓度升高的程度明显减弱;加入优降糖可明显提高NE对平滑肌细胞内钙离子的升高作用,改善细动脉对NE的反应性．带来血管反应性部分恢复．     

失血性休克引起大鼠肠系膜动脉平滑肌依钙K^＋通道活动改变

在由股动脉放血制备的失血性休克大鼠模型急性分离的肠系膜动脉平滑肌细胞上,利用膜片箝单通道记录技术观察了血管平滑肌依钙K^ 通道（BKca)的活动,发现在对去甲肾上腺素（NE）反应性增高的休克代偿期,BKca的开放概率（P0)和单位电导都显著较正常动物的低,P0的改变主要是由通道的慢关闭时间常数（τcs)增大引起关闭时间延长所致;而处于对NE反应性降低的休克失代偿期,BKca的P0和单位电导都高于正常动物,P0的变化也主要是τcs减小所致。     

Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation

Airway smooth muscle (ASM) metabolizes arachidonic acid (AA) through various enzymatic pathways, including cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either methacholine or AA. Relaxing effects of 20-HETE on resting tone were prevented by 10 nM iberiotoxin (IbTx), a BK(Ca) channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1-10 microM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentrations. Together, these results indicate that 20-HETE-dependent activation of BK(Ca) channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.     

Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.

Functional hyperemia requires the coordination of smooth muscle cell relaxation along and between branches of the arteriolar network. Vasodilation is conducted from cell to cell along the arteriolar wall through gap junction channels composed of connexin protein subunits. Within skeletal muscle, it is unclear whether arteriolar endothelium, smooth muscle, or both cell layers provide the cellular pathway for conduction. Furthermore, the constitutive profile of connexin expression within the microcirculation is unknown. We tested the hypothesis that conducted vasodilation and connexin expression are intrinsic to the endothelium of arterioles (17 +/- 1 microm diameter) that supply the skeletal muscle fibers in the cremaster of anesthetized C57BL/6 mice. ACh delivered to an arteriole (500 ms, 1-microA pulse; 1-microm micropipette) produced local dilation of 17 +/- 1 microm; conducted vasodilation observed 1 mm upstream was 9 +/- 1 microm (n = 5). After light-dye treatment to selectively disrupt endothelium (250-microm segment centered 500 microm upstream, confirmed by loss of local response to ACh while constriction to phenylephrine and dilation to sodium nitroprusside remained intact), we found that conducted vasodilation was nearly abolished (2 +/- 1 microm; P < 0.05). Whole-mount immunohistochemistry for connexins revealed punctate labeling at borders of arteriolar endothelial cells, with connexin40 and connexin37 in all branches and connexin43 only in the largest branches. Immunoreactivity for connexins was not apparent in smooth muscle or in capillary or venular endothelium, despite robust immunolabeling for alpha-actin and platelet endothelial cell adhesion molecule-1, respectively. We conclude that vasodilation is conducted along the endothelium of mouse skeletal muscle arterioles and that connexin40 and connexin37 are the primary connexins forming gap junction channels between arteriolar endothelial cells.     

Whole-cell voltage clamp and intracellular perfusion technique on single smooth muscle cells

Using freshly isolated single smooth muscle cells prepared by collagenase treatment, membrane currents were recorded by whole-cell voltage clamp. Intracellular constituents were modified by using an intracellular perfusion technique, i.e., pipette solutions were continuously exchanged from control to test solutions during current recording. In smooth muscle cells, intracellular application of ATP, but not cyclic AMP, enhanced the amplitude of Ca2+ currents and prevented current run-down. In addition, with this stabilization of Ca2+ current recording by ATP, introduction of various chemicals into the cell using the intracellular perfusion technique is useful for investigations of regulation of ion channels in smooth muscle cells.     

Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.     

Vasomotion in critically perfused muscle protects adjacent tissues from capillary perfusion failure

We analyzed the incidence and interaction of arteriolar vasomotion and capillary flow motion during critical perfusion conditions in neighboring peripheral tissues using intravital fluorescence microscopy. The gracilis and semitendinosus muscles and adjacent periosteum, subcutis, and skin of the left hindlimb of Sprague-Dawley rats were isolated at the femoral vessels. Critical perfusion conditions, achieved by stepwise reduction of femoral artery blood flow, induced capillary flow motion in muscle, but not in the periosteum, subcutis, and skin. Strikingly, blood flow within individual capillaries was decreased (P < 0.05) in muscle but was not affected in the periosteum, subcutis, and skin. However, despite the flow motion-induced reduction of muscle capillary blood flow during the critical perfusion conditions, functional capillary density remained preserved in all tissues analyzed, including the skeletal muscle. Abrogation of vasomotion in the muscle arterioles by the calcium channel blocker felodipine resulted in a redistribution of blood flow within individual capillaries from cutaneous, subcutaneous, and periosteal tissues toward skeletal muscle. As a consequence, shutdown of perfusion of individual capillaries was observed that resulted in a significant reduction (P < 0.05) of capillary density not only in the neighboring tissues but also in the muscle itself. We conclude that during critical perfusion conditions, vasomotion and flow motion in skeletal muscle preserve nutritive perfusion (functional capillary density) not only in the muscle itself but also in the neighboring tissues, which are not capable of developing this protective regulatory mechanism by themselves.     

Potassium (BK(Ca)) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats

Insulin resistance (IR) syndrome is associated with impaired vascular relaxation; however, the underlying pathophysiology is unknown. Potassium channel activation causes vascular smooth muscle hyperpolarization and relaxation. The present study determined whether a reduction in large conductance calcium- and voltage-activated potassium (BK(Ca)) channel activity contributes to impaired vascular relaxation in IR rats. BK(Ca) channels were characterized in mesenteric microvessels from IR and control rats. Macroscopic current density was reduced in myocytes from IR animals compared with controls. In addition, inhibition of BK(Ca) channels with tetraethylammonium (1 mM) or iberiotoxin (100 nM) was greater in myocytes from control (70%) compared with IR animals (approximately 20%). Furthermore, activation of BK(Ca) channels with NS-1619 was three times more effective at increasing outward current in cells from control versus IR animals. Single channel and Western blot analysis of BK(Ca) channels revealed similar conductance, amplitude, voltage sensitivity, Ca2+ sensitivity, and expression density between the two groups. These data provide the first direct evidence that microvascular potassium currents are reduced in IR and suggest a molecular mechanism that could account for impaired vascular relaxation in IR.     

PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase

Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BK(Ca)-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BK(Ca) channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BK(Ca)-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BK(Ca)-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BK(Ca) channels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BK(Ca)-channel activity was blocked by G?-6983, which selectively blocks PKC-alpha, -delta, -gamma, and -zeta, and by rottlerin, which selectively inhibits PKC-delta. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, G?-6983, or the BK(Ca)-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BK(Ca)-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.     

BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle

The large-conductance, Ca2+-activated K+ (BK) channels are regulators of voltage-dependent Ca2+ entry in many cell types. The BK channel accessory beta1-subunit promotes channel activation in smooth muscle and is required for proper tone in the vasculature and bladder. However, although BK channels have also been implicated in airway smooth muscle function, their regulation by the beta1-subunit has not been investigated. Utilizing the gene-targeted mice for the beta1-subunit gene, we have investigated the role of the beta1-subunit in tracheal smooth muscle. In mice with the beta1-subunit-knockout allele, BK channel activity was significantly reduced in excised tracheal smooth muscle patches and spontaneous BK currents were reduced in whole tracheal smooth muscle cells. Knockout of the beta1-subunit resulted in an increase in resting Ca2+ levels and an increase in the sustained component of Ca2+ influx after cholinergic signaling. Tracheal constriction studies demonstrate that the level of constriction is the same with knockout of the beta1-subunit and BK channel block with paxillin, indicating that BK channels contribute little to airway relaxation in the absence of the beta1-subunit. Utilizing nifedipine, we found that the increased constriction caused by knockout of the beta1-subunit could be accounted for by an increased recruitment of L-type voltage-dependent Ca2+ channels. These results indicate that the beta1-subunit is required in airway smooth muscle for control of voltage-dependent Ca2+ influx during rest and after cholinergic signaling in BK channels.