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.

Abstract-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␣-phorbol 12,13-didecanoate (4␣-PDD) (5 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 Ca 2ϩ release events (Ca 2ϩ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca 2ϩ sparks activated nearby sarcolemmal large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels, measured as an increase in the frequency of transient K ϩ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca 2ϩ spark and STOC frequency were inhibited by lowering external Ca 2ϩ from 2 mmol/L to 10 mol/L but not by voltage-dependent Ca 2ϩ channel inhibitors, suggesting that these responses require extracellular Ca 2ϩ influx via channels other than voltage-dependent Ca 2ϩ 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 Ca 2ϩ signaling complex with ryanodine receptors and BK Ca channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca 2ϩ -induced Ca 2ϩ release in response to an endothelial-derived factor. (Circ Res. 2005;97:1270-1279.)Key Words: Ca 2ϩ sparks Ⅲ Ca 2ϩ transients Ⅲ eicosanoids Ⅲ ion channels Ⅲ ryanodine receptor Ⅲ vascular smooth muscle Ⅲ vasodilation D iffusible factors produced by the vascular endothelium are vital for the regulation of smooth muscle membrane potential, arterial tone, blood pressure, and blood flow. Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase products of arachidonic acid, 1 cause vasodilation and account for endothelium-derived hyperpolarizing factor (EDHF) activity in some vascular beds. 2,3 Critical components of signaling pathways activated by these compounds are not fully understood. 1 Although evidence supportive of a receptor-dependent smooth muscle hyperpolarization mechanism has been presented, 4 the molecular identity of the "EET receptor" has not been reported. Vanilloid transient receptor potential (TRPV) channels are important sensors of biochemical and physiological stimuli. 5 Materials and MethodsCerebral and cerebellar arteries used for these studies were isolated from male Sprague-Dawley rats (250 to 350 g; Charles River Laboratories, ...

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.

The goal of this study was to examine the role of large conductance Ca(2+)-activated K+ channels in the regulation of cell excitability in urinary bladder smooth muscle from the guinea pig. Ca(2+)-activated K+ channels were studied with single-channel recording techniques and found to be intracellular Ca2+ and voltage dependent and sensitive to external tetraethylammonium and blocked by nanomolar concentrations of iberiotoxin (apparent dissociation constant of 4 nM). Spontaneous action potentials recorded from intact tissue strips depended on external Ca2+ and were inhibited by Ca2+ channel blockers. Iberiotoxin (100 nM) significantly altered the configuration of the action potential by increasing the duration and peak amplitude of the action potential and decreasing the rate of decay. Iberiotoxin also increased the action potential frequency from 0.11 to 0.29 Hz. This study suggests that Ca(2+)-activated K+ channels play a significant role in the repolarization of the action potential and in the maintenance of the resting membrane potential of the urinary bladder smooth muscle.

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.

The large‐conductance calcium‐activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore‐forming α‐subunit and an accessory, smooth muscle‐specific, β1‐subunit. Our aim was to determine the functional role of the β1‐subunit of the BK channel in controlling the contractions of UBSM by using BK channel β1‐subunit ‘knock‐out’ (KO) mice. The β‐galactosidase reporter (lacZ gene) was targeted to the β1 locus, which provided the opportunity to examine the expression of the β1‐subunit in UBSM. Based on this approach, the β1‐subunit is highly expressed in UBSM. BK channels lacking β1‐subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. The effects of the β1‐subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from β1‐subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from β1‐subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the β1‐subunit. The results indicate that the β1‐subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.

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.

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