Adrian D. Bonev

Relaxation of Arterial Smooth Muscle by Calcium Sparks

Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca2+-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.

Vasoregulation by the β1 subunit of the calcium-activated potassium channel

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These ‘calcium sparks’ activate calcium-dependent potassium (BK) channels comprised of α and β1 subunits. Here we show that targeted deletion of the gene for the β1 subunit leadsxa0to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The β1 subunit of the BK channel, by tuning the channels calciumxa0sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.

Local potassium signaling couples neuronal activity to vasodilation in the brain

The mechanisms by which active neurons, via astrocytes, rapidly signal intracerebral arterioles to dilate remain obscure. Here we show that modest elevation of extracellular potassium (K+) activated inward rectifier K+ (Kir) channels and caused membrane potential hyperpolarization in smooth muscle cells (SMCs) of intracerebral arterioles and, in cortical brain slices, induced Kir-dependent vasodilation and suppression of SMC intracellular calcium (Ca2+) oscillations. Neuronal activation induced a rapid (<2 s latency) vasodilation that was greatly reduced by Kir channel blockade and completely abrogated by concurrent cyclooxygenase inhibition. Astrocytic endfeet exhibited large-conductance, Ca2+-sensitive K+ (BK) channel currents that could be activated by neuronal stimulation. Blocking BK channels or ablating the gene encoding these channels prevented neuronally induced vasodilation and suppression of arteriolar SMC Ca2+, without affecting the astrocytic Ca2+ elevation. These results support the concept of intercellular K+ channel–to–K+ channel signaling, through which neuronal activity in the form of an astrocytic Ca2+ signal is decoded by astrocytic BK channels, which locally release K+ into the perivascular space to activate SMC Kir channels and cause vasodilation.

Elementary Ca2+ Signals Through Endothelial TRPV4 Channels Regulate Vascular Function

Blood Pressure Gauge Endothelial cells line blood vessels and, by interacting with smooth muscle, can help to control blood flow. Sonkusare et al. (p. 597; see the Perspective by Lederer et al.) describe how signaling in endothelial cells controls contraction of surrounding smooth muscle cells, which provides an important mechanism for control of blood pressure. A calcium-sensitive fluorescent protein was expressed in endothelial cells of mouse arteries to image small changes in calcium concentration that appear to represent opening of single TRPV4 ion channels and consequent influx of calcium into the cell. Clustering of the channels allowed cooperative activation of a handful of channels, which appeared to produce a sufficient calcium signal to open another set of calcium-sensitive potassium channels. The resulting depolarization of the endothelial cells then passes an electrical connection to smooth muscle cells through gap junctions. Imaging reveals single-channel openings of cation channels at the heart of endothelial cell–mediated blood pressure control. Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca2+) signals (“sparklets”) in the vascular endothelium of resistance arteries that represent Ca2+ influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca2+-sensitive potassium (K+) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca2+ influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca2+ sensitivity of IK and SK channels to cause vasodilation.

Gender Differences in Coronary Artery Diameter Involve Estrogen, Nitric Oxide, and Ca2+-Dependent K+ Channels

During their reproductive years, women have a much lower incidence of coronary heart disease compared with men of similar age. Estrogen appears to be largely responsible for this decrease in cardiovascular mortality in women. In the present study, isolated pressurized coronary arteries from rats were used to assess the role of gender and circulating estrogen on coronary vascular function. Pressure-induced constrictions (myogenic tone) were greater (approximately 2-fold) in isolated coronary arteries from estrogen-deficient male or ovariectomized (OVX) rats compared with similar arteries obtained from female rats or OVX rats receiving physiological levels of estrogen replacement (OVX+E group). These differences in coronary artery diameter were abolished by removal of the vascular endothelium or chemical inhibition of NO synthase. The anti-estrogen, tamoxifen, increased pressure-induced constrictions of coronary arteries from female and OVX+E rats. Dilations of pressurized coronary arteries from female and OVX animals to sodium nitroprusside, a nitrovasodilator that generates NO, were reduced by > 50% by iberiotoxin (IBTX), an inhibitor of Ca(2+)-dependent K+ (KCa) channels. Sodium nitroprusside (10 mumol/L) hyperpolarized coronary arteries by 13 +/- 2 mV, an effect that was greatly diminished (approximately 80%) by IBTX. Coronary arteries isolated from female rats produced greater constrictions in response to IBTX and KT 5823, an inhibitor of cGMP-dependent protein kinase, compared with coronary arteries from OVX rats. cGMP-dependent protein kinase increased the activity of KCa channels 16.5 +/- 5-fold in excised membrane patches from smooth muscle cells enzymatically isolated from these small coronary arteries. We propose that physiological levels of circulating 17 beta-estradiol elevate basal NO release from the endothelial cells, which increases the diameter of pressurized coronary arteries. Further, our results suggest that part of the effect of this NO is through activation of KCa channels in the smooth muscle cells of the coronary arteries.

Modulation of the molecular composition of large conductance, Ca 2+ activated K + channels in vascular smooth muscle during hypertension

Hypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca2+ release events through ryanodine receptors (Ca2+ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca2+-activated K+ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca2+ spark-BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the beta1 subunit was decreased relative to the pore-forming alpha subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca2+ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the beta1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.

Calcium Dynamics in Cortical Astrocytes and Arterioles During Neurovascular Coupling

Neuronal activity in the brain is thought to be coupled to cerebral arterioles (functional hyperemia) through Ca2+ signals in astrocytes. Although functional hyperemia occurs rapidly, within seconds, such rapid signaling has not been demonstrated in situ, and Ca2+ measurements in parenchymal arterioles are still lacking. Using a laser scanning confocal microscope and fluorescence Ca2+ indicators, we provide the first evidence that in a brain slice preparation, increased neuronal activity by electrical stimulation (ES) is rapidly signaled, within seconds, to cerebral arterioles and is associated with astrocytic Ca2+ waves. Smooth muscle cells in parenchymal arterioles exhibited Ca2+ and diameter oscillations (“vasomotion”) that were rapidly suppressed by ES. The neuronal-mediated Ca2+ rise in cortical astrocytes was dependent on intracellular (inositol trisphosphate [IP3]) and extracellular voltage-dependent Ca2+ channel sources. The Na+ channel blocker tetrodotoxin prevented the rise in astrocytic [Ca2+]i and the suppression of Ca2+ oscillations in parenchymal arterioles to ES, indicating that neuronal activity was necessary for both events. Activation of metabotropic glutamate receptors in astrocytes significantly decreased the frequency of Ca2+ oscillations in parenchymal arterioles. This study supports the concept that astrocytic Ca2+ changes signal the cerebral microvasculature and indicate the novel concept that this communication occurs through the suppression of arteriolar [Ca2+]i oscillations and corresponding vasomotion. The full text of this article is available online at http://circres.ahajournals.org.

Calcitonin gene‐related peptide activated ATP‐sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.

1. Whole‐cell K+ currents activated by calcitonin gene‐related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole‐cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP‐sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide‐sensitive currents. CGRP increased the glibenclamide‐sensitive currents by 3‐ to 6‐fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide‐sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current‐voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp‐cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide‐sensitive K+ currents 2.1‐, 3.3‐ and 8.2‐fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide‐sensitive K+ currents. 5. Dialysis of the cells interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H‐8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP‐sensitive K+ channels.

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections

Calcium (Ca2+) release through inositol 1,4,5-trisphosphate receptors (IP3Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca2+ events (“sparks”) that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP3Rs. Here, we have identified spatially fixed, local Ca2+ release events (“pulsars”) in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca2+ pulsars are mediated by IP3Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca2+ pulsars, whereas blunting IP3 production, blocking IP3Rs, or depleting endoplasmic reticulum Ca2+ inhibited these events. The elementary properties of Ca2+ pulsars were distinct from ryanodine-receptor-mediated Ca2+ sparks in smooth muscle and from IP3-mediated Ca2+ puffs in Xenopus oocytes. The intermediate conductance, Ca2+-sensitive potassium (KCa3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca2+ pulsars also depolarized to a similar extent, and blocking KCa3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP3 signaling in which Ca2+ release is spatially restricted to transmit intercellular signals.

Frequency modulation of Ca2+sparks is involved in regulation of arterial diameter by cyclic nucleotides

Forskolin, which elevates cAMP levels, and sodium nitroprusside (SNP) and nicorandil, which elevate cGMP levels, increased, by two- to threefold, the frequency of subcellular Ca2+ release (Ca2+ sparks) through ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolated from cerebral and coronary arteries of rats. Forskolin, SNP, nicorandil, dibutyryl-cAMP, and adenosine increased the frequency of Ca2+-sensitive K+(KCa) currents [spontaneous transient outward currents (STOCs)] by two- to threefold, consistent with Ca2+ sparks activating STOCs. These agents also increased the mean amplitude of STOCs by 1.3-fold, an effect that could be explained by activation of KCa channels, independent of effects on Ca2+ sparks. To test the hypothesis that cAMP could act to dilate arteries through activation of the Ca2+spark→KCa channel pathway, the effects of blockers of KCachannels (iberiotoxin) and of Ca2+sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating external K+. Forskolin lowered average Ca2+ in pressurized arteries while increasing ryanodine-sensitive, caffeine-induced Ca2+ transients. These experiments suggest a new mechanism for cyclic nucleotide-mediated dilations through an increase in Ca2+ spark frequency, caused by effects on SR Ca2+ load and possibly on the RyR channel, which leads to increased STOC frequency, membrane potential hyperpolarization, closure of voltage-dependent Ca2+ channels, decrease in arterial wall Ca2+, and, ultimately, vasodilation.