Mark T. Nelson

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.

Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure

1 The regulation of intracellular [Ca2+] in the smooth muscle cells in the wall of small pressurized cerebral arteries (100‐200 μm) of rat was studied using simultaneous digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2 Elevation of intravascular pressure (from 10 to 100 mmHg) caused a membrane depolarization from ‐63 ± 1 to ‐36 ± 2 mV, increased arterial wall [Ca2+] from 119 ± 10 to 245 ± 9 nM, and constricted the arteries from 208 ± 10 μm (fully dilated, Ca2+ free) to 116 ± 7 μm or by 45 % (‘myogenic tone’). 3 Pressure‐induced increases in arterial wall [Ca2+] and vasoconstriction were blocked by inhibitors of voltage‐dependent Ca2+ channels (diltiazem and nisoldipine) or to the same extent by removal of external Ca2+. 4 At a steady pressure (i.e. under isobaric conditions at 60 mmHg), the membrane potential was stable at ‐45 ± 1 mV, intracellular [Ca2+] was 190 ± 10 nM, and arteries were constricted by 41 % (to 115 ± 7 μm from 196 ± 8 μm fully dilated). Under this condition of ‐45 ± 5 mV at 60 mmHg, the voltage sensitivity of wall [Ca2+] and diameter were 7.5 nM mV−1 and 7.5 μm mV−1, respectively, resulting in a Ca2+ sensitivity of diameter of 1 μm nM−1. 5 Membrane potential depolarization from ‐58 to ‐23 mV caused pressurized arteries (to 60 mmHg) to constrict over their entire working range, i.e. from maximally dilated to constricted. This depolarization was associated with an elevation of arterial wall [Ca2+] from 124 ± 7 to 347 ± 12 nM. These increases in arterial wall [Ca2+] and vasoconstriction were blocked by L‐type voltage‐dependent Ca2+ channel inhibitors. 6 The relationship between arterial wall [Ca2+] and membrane potential was not significantly different under isobaric (60 mmHg) and non‐isobaric conditions (10‐100 mmHg), suggesting that intravascular pressure regulates arterial wall [Ca2+] through changes in membrane potential. 7 The results are consistent with the idea that intravascular pressure causes membrane potential depolarization, which opens voltage‐dependent Ca2+ channels, acting as ‘voltage sensors’, thus increasing Ca2+ entry and arterial wall [Ca2+], which leads to vasoconstriction.

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.

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 4&agr;-phorbol 12,13-didecanoate (4&agr;-PDD) (5 &mgr;mol/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 &mgr;mol/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.

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.

Targeted Disruption of Kir2.1 and Kir2.2 Genes Reveals the Essential Role of the Inwardly Rectifying K + Current in K + -Mediated Vasodilation

The molecular bases of inwardly rectifying K(+) (Kir) currents and K(+)-induced dilations were examined in cerebral arteries of mice that lack the Kir2.1 and Kir2.2 genes. The complete absence of the open reading frame in animals homozygous for the targeted allele was confirmed. Kir2.1(-/-) animals die 8 to 12 hours after birth, apparently due to a complete cleft of the secondary palate. In contrast, Kir2.2(-/-) animals are viable and fertile. Kir currents were observed in cerebral artery myocytes isolated from control neonatal animals but were absent in myocytes from Kir2.1(-/-) animals. Voltage-dependent K(+) currents were similar in cells from neonatal control and Kir2.1(-/-) animals. An increase in the extracellular K(+) concentration from 6 to 15 mmol/L caused Ba(2+)-sensitive dilations in pressurized cerebral arteries from control and Kir2.2 mice. In contrast, arteries from Kir2.1(-/-) animals did not dilate when the extracellular K(+) concentration was increased to 15 mmol/L. In summary, Kir2.1 gene expression in arterial smooth muscle is required for Kir currents and K(+)-induced dilations in cerebral arteries.

EXTRACELLULAR K+-INDUCED HYPERPOLARIZATIONS AND DILATATIONS OF RAT CORONARY AND CEREBRAL ARTERIES INVOLVE INWARD RECTIFIER K+ CHANNELS

1. The hypothesis that inward rectifier K(+) channels are involved in the vasodilatation of small coronary and cerebral arteries (100‐200 microm diameter) in response to elevated [K+]o was tested. The diameters and membrane potentials of pressurized arteries from rat were measured using a video‐imaging system and conventional microelectrodes, respectively. 2. Elevation of [K+]o from 6 to 16 mM caused the membrane potential of pressurized (60 mmHg) arteries to hyperpolarize by 12‐14 mV. Extracellular Ba(2+) (Ba2+(o)) blocked K(+)‐induced membrane potential hyperpolarizations at concentrations (IC(50), 6 microM) that block inward rectifier K(+) currents in smooth muscle cells isolated from these arteries. 3. Elevation of [K+]o from 6 to 16 mM caused sustained dilatations of pressurized coronary and cerebral arteries with diameters increasing from 125 to 192 microm and 110 to 180 microm in coronary and cerebral arteries, respectively. Ba2+(o) blocked K(+)‐induced dilatations of pressurized coronary and cerebral arteries (IC50, 3‐8 microM). 4. Elevated [K+]o‐induced vasodilatation was not prevented by blockers of other types of K(+) channels (1 mM 4‐aminopyridine, 1 mM TEA+, and 10 mu M glibenclamide), and blockers of Na(+)‐K(+)‐ATPase. Elevated [K+]o‐induced vasodilatation was unaffected by removal of the endothelium. 5. These findings suggest that K+(o) dilates small rat coronary and cerebral arteries through activation of inward rectifier K(+) channels. Furthermore, these results support the hypothesis that inward rectifier K(+) channels may be involved in metabolic regulation of coronary and cerebral blood flow in response to changes in [K+]o.

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels

1 The effects of inhibitors of ryanodine‐sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+‐dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100‐200 μm) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2 Ryanodine (10 μM), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nM and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 μm (≈22 %). Iberiotoxin (100 nM), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nM, and constricted by 37 μm (≈19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage‐dependent Ca2+ channels. 3 Caffeine (10 mM), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 ± 9 nM in control arteries and by 158 ± 12 nM in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 ± 5 nM. 4 In the presence of blockers of voltage‐dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. 5 These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L‐type voltage‐dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+].

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.