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Calcium sparks represent local, rapid, and transient calcium release events from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In arterial smooth muscle cells (SMCs), calcium sparks activate calcium-dependent potassium channels causing decrease in the global intracellular [Ca2+] and oppose vasoconstriction. This is in contrast to cardiac and skeletal muscle, where spatial and temporal summation of calcium sparks leads to global increases in intracellular [Ca2+] and myocyte contraction. We summarize the present data on local RyR calcium signaling in arterial SMCs in comparison to striated muscle and muscle-specific differences in coupling between L-type calcium channels and RyRs. Accordingly, arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux though RyRs. Downregulation of RyR2 up to a certain degree is compensated by increased SR calcium content to normalize calcium sparks. This indirect coupling between Cav1.2 and RyR in arterial SMCs is opposite to striated muscle, where triggering of calcium sparks is controlled by rapid and direct cross-talk between Cav1.1/Cav1.2 L-type channels and RyRs. We discuss the role of RyR isoforms in initiation and formation of calcium sparks in SMCs and their possible molecular binding partners and regulators, which differ compared to striated muscle.

L-type, voltage-dependent calcium (Ca2+) channels, ryanodine-sensitive Ca2+ release (RyR) channels, and large-conductance Ca2+-activated potassium (KCa) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters Ca2+ spark to KCa channel coupling and Ca2+ spark regulation by voltage-dependent Ca2+ channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control (cav-+/+) cells but were not observed in cav-1-deficient (cav-1−/−) cells. Ca2+ spark and transient KCa current frequency were approximately twofold higher in cav-1−/− than in cav-1+/+ cells. Although voltage-dependent Ca2+ current density was similar in cav-1+/+ and cav-1−/− cells, diltiazem and Cd2+, voltage-dependent Ca2+ channel blockers, reduced transient KCa current frequency to ∼55% of control in cav-1+/+ cells but did not alter transient KCa current frequency in cav-1−/− cells. Furthermore, although KCa channel density was elevated in cav-1−/− cells, transient KCa current amplitude was similar to that in cav-1+/+ cells. Higher Ca2+ spark frequency in cav-1−/− cells was not due to elevated intracellular Ca2+ concentration, sarcoplasmic reticulum Ca2+ load, or nitric oxide synthase activity. Similarly, Ca2+ spark amplitude and spread, the percentage of Ca2+ sparks that activated a transient KCa current, the amplitude relationship between sparks and transient KCa currents, and KCa channel conductance and apparent Ca2+ sensitivity were similar in cav-1+/+ and cav-1−/− cells. In summary, cav-1 ablation elevates Ca2+ spark and transient KCa current frequency, attenuates the coupling relationship between voltage-dependent Ca2+ channels and RyR channels that generate Ca2+ sparks, and elevates KCa channel density but does not alter transient KCa current activation by Ca2+ sparks. These findings indicate that cav-1 is required for physiological Ca2+ spark and transient KCa current regulation in cerebral artery smooth muscle cells.

Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.

Objective

To demonstrate that TNFα, via sphingosine-1-phosphate (S1P) signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss.

Methods and Results

TNFα induced a pro-constrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing S1P receptor 2 subtype signaling (1µmol/L JTE013) attenuated the effects of TNFα in all models. TNFα activated Sk1 and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1G82D) eliminated both baseline spiral modiolar artery calcium sensitivity and TNFα effects, while a non-phosphorylatable Sk1 mutant (Sk1S225A) only blocked the effects of TNFα. A small group of etanercept-treated hearing loss patients recovered with a one-phase exponential decay (t½=1.56±0.20 weeks), which matched a kinetic predicted for a vascular origin.

Conclusions

TNFα indeed reduces cochlear blood flow via the activation of vascular S1P signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis and treatment.

Background

Vasospasm of the spiral modiolar artery (SMA) may cause ischemic stroke of the inner ear. Endothelin-1 (ET-1) induces a strong, long-lasting constriction of the SMA by increasing contractile apparatus Ca2+ sensitivity via Rho-kinase. We therefore tested several Rho-kinase inhibitors and a cell-permeable analogue of cAMP (dbcAMP) for their ability to reverse ET-1-induced constriction and Ca2+-sensitization.

Methods

The present study employed SMA isolated from gerbil temporal bones. Ca2+sensitivity was evaluated by correlating vascular diameter and smooth muscle cell [Ca2+]i, measured by fluo-4-microfluorometry and videomicroscopy.

Results

The Rho-kinase inhibitors Y-27632, fasudil, and hydroxy-fasudil reversed ET-1-induced vasoconstriction with an IC50 of 3, 15, and 111 μmol/L, respectively. DbcAMP stimulated a dose-dependent vasodilation (Ec50 = 1 mmol/L) and a reduction of [Ca2+]i (EC50 = 0.3 μmol/L) of ET-1-preconstricted vessels (1 nmol/L). Fasudil and dbcAMP both reversed the ET-1-induced increase in Ca2+ sensitivity.

Conclusion

Rho-kinase inhibition and dbcAMP reversed ET-1-induced vasoconstriction and Ca2+-sensitization. Therefore, Rho-kinase inhibitors or cAMP modulators could possess promise as pharmacological tools for the treatment of ET-1-induced constriction, ischemic stroke and sudden hearing loss.

Rationale

Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BKCa) channels by local Ca2+ signals (Ca2+ sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known.

Objective

The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BKCa channels in this response.

Methods and Results

Internal diameter and vascular smooth muscle cell (VSMC) Ca2+ signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), VSMCs exhibited primarily RyR-dependent Ca2+ waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca2+ wave activity, and dramatically increased Ca2+ spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BKCa channel or RyR blockers, in a non-additive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BKCa channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of KATP channels or nitric oxide synthase.

Conclusions

These results support the novel concept that acidification, by converting Ca2+ waves to sparks, leads to the activation of BKCa channels to induce dilation of cerebral parenchymal arterioles.

Summary

Background

Ca2+ signaling mechanisms are crucial for proper regulation of vascular smooth muscle contractility and vessel diameter. In cerebral artery myocytes, a rise in global cytosolic Ca2+ concentration ([Ca2+]i) causes contraction while an increase in local Ca2+ release events from the sarcoplasmic reticulum (Ca2+ sparks) leads to increased activity of large-conductance Ca2+-activated (BK) K+ channels, hyperpolarization and relaxation. Here, we examined the impact of SAH on Ca2+ spark activity and [Ca2+]i in cerebral artery myocytes following SAH.

Methods

A rabbit double injection SAH model was used in this study. Five days after the initial intracisternal injection of whole blood, small diameter cerebral arteries were dissected from the brain for study. For simultaneous measurement of arterial wall [Ca2+]i and diameter, vessels were cannulated and loaded with the ratiometric Ca2+ indicator fura-2. For measurement of Ca2+ sparks, individual myocytes were enzymatically isolated from cerebral arteries and loaded with the Ca2+ indicator fluo-4. Sparks were visualized using laser scanning confocal microscopy.

Results

Arterial wall [Ca2+]i was significantly elevated and greater levels of myogenic tone developed in arteries isolated from SAH animals compared with arteries isolated from healthy animals. The L-type voltage-dependent Ca2+ channel (VDCC) blocker nifedipine attenuated increases in [Ca2+]i and tone in both groups suggesting increased VDCC activity following SAH. Membrane potential measurement using intracellular microelectrodes revealed significant depolarization of vascular smooth muscle following SAH. Further, myocytes from SAH animals exhibited significantly reduced Ca2+ spark frequency (~50%).

Conclusions

Our findings suggest decreased Ca2+ spark frequency leads to reduced BK channel activity in cerebral artery myocytes following SAH. This results in membrane potential depolarization, increased VDCC activity, elevated [Ca2+]i and decreased vessel diameter. We propose this mechanism of enhanced cerebral artery myocyte contractility may contribute to decreased cerebral blood flow and development of neurological deficits in SAH patients.

Ca2+ sparks are highly localized cytosolic Ca2+ transients caused by a release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca2+ in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca2+ indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca2+ released into the cytosol, and its rate of rise is proportional to the Ca2+ current flowing through the RyRs during a spark (ICa(spark)). Thus, Ca2+ currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of ICa(spark) in different sparks varies more than fivefold, Ca2+ sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca2+ current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca2+] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca2+ concentration resulting from the measured range of ICa(spark). At the onset of a spark, the Ca2+ concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca2+]EC50 for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca2+] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca2+ action on a variety of molecular targets within cellular microdomains.

Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-NG-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca2+ concentration and/or the Ca2+ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca2+, and Ca2+ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K+ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca2+ concentration in smooth muscle cells but increased the Ca2+ sensitivity. This increase in the Ca2+ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca2+ sensitivity and abolished the LNNA-induced increase in Ca2+ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca2+ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca2+ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.

The relationship between Ca2+ release (“Ca2+ sparks”) through ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and KCa channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca2+ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca2+ indicator, fluo-3. Virtually all (96%) detectable Ca2+ sparks were associated with the activation of a spontaneous transient outward current (STOC) through KCa channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca2+ spark. The amplitudes of the Ca2+ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca2+ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca2+ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in KCa channel open probability (>104-fold) during a Ca2+ spark is consistent with local Ca2+ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/Fo) measured during a Ca2+ spark (mean 2.04 F/Fo or ∼310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates KCa channels. These results indicate that the majority of ryanodine receptors that cause Ca2+ sparks are functionally coupled to KCa channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs.

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 β1 subunit was decreased relative to the pore-forming α 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 β1 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-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca2+ channels. In heart cells, a tight coupling between the gating of single L-type Ca2+ channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca2+ channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca2+ sparks and propagated Ca2+ waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca2+ channels. L-type Ca2+ channels can open without triggering Ca2+ sparks and triggered Ca2+ sparks are often observed after channel closure. CICR is a function of the net flux of Ca2+ ions into the cytosol, rather than the single channel amplitude of L-type Ca2+ channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca2+ channels are loosely coupled to RYR through an increase in global [Ca2+] due to an increase in the effective distance between L-type Ca2+ channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca2+ releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca2+ and Mg2+ ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg2+. Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg4RYR and CaMg3RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca2Mg2RYR and Ca3MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg2+ unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.

Arterial smooth muscle cell large-conductance Ca2+-activated potassium (KCa) channels have been implicated in modulating hypoxic dilation of systemic arteries, although this is controversial. KCa channel activity in arterial smooth muscle cells is controlled by localized intracellular Ca2+ transients, termed Ca2+ sparks, but hypoxic regulation of Ca2+ sparks and KCa channel activation by Ca2+ sparks has not been investigated. We report here that in voltage-clamped (−40 mV) cerebral artery smooth muscle cells, a reduction in dissolved O2 partial pressure from 150 to 15 mmHg reversibly decreased Ca2+ spark-induced transient KCa current frequency and amplitude to 61% and 76% of control, respectively. In contrast, hypoxia did not alter Ca2+ spark frequency, amplitude, global intracellular Ca2+ concentration, or sarcoplasmic reticulum Ca2+ load. Hypoxia reduced transient KCa current frequency by decreasing the percentage of Ca2+ sparks that activated a transient KCa current from 89% to 63%. Hypoxia reduced transient KCa current amplitude by attenuating the amplitude relationship between Ca2+ sparks that remained coupled and the evoked transient KCa currents. Consistent with these data, in inside-out patches at −40 mV hypoxia reduced KCa channel apparent Ca2+ sensitivity and increased the Kd for Ca2+ from ∼17 to 32 μM, but did not alter single-channel amplitude. In summary, data indicate that hypoxia reduces KCa channel apparent Ca2+ sensitivity via a mechanism that is independent of cytosolic signaling messengers, and this leads to uncoupling of KCa channels from Ca2+ sparks. Transient KCa current inhibition due to uncoupling would oppose hypoxic cerebrovascular dilation.

Expression of Tumor Necrosis Factor alpha (TNF-α), a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF-α regulates arterial diameter, although mechanisms mediating this effect are unclear. Here, we tested the hypothesis that TNF-α regulates the diameter of resistance-size (∼150 μm diameter) cerebral arteries by modulating local and global intracellular calcium (Ca2+) signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF-α increased Ca2+ spark and Ca2+ wave frequency, but reduced global Ca2+ concentration ([Ca2+]i) in smooth muscle cells of intact arteries. TNF-α elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries and this was prevented by apocynin or diphenyleneiodonium (DPI), NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS generating enzymes. In voltage-clamped (-40 mV) cells, TNF-α increased the frequency and amplitude of Ca2+ spark-induced large-conductance Ca2+-activated potassium (KCa) channel transients ∼1.7-fold and ∼1.4-fold, respectively. TNF-α-induced transient KCa current activation was reversed by apocynin or MnTMPyP, a membrane-permeant antioxidant, and prevented by intracellular dialysis of catalase. TNF-α induced reversible and similar amplitude dilations in either endothelium-intact or-denuded pressurized (60 mm Hg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca2+ ATP-ase blocker that inhibits Ca2+ sparks, and iberiotoxin, a KCa channel blocker, reduced TNF-α-induced vasodilations to between 15 and 33% of control. In summary, data indicate that TNF-α activates NAD(P)H oxidase, resulting in an increase in intracellular H2O2 that stimulates Ca2+ sparks and transient KCa currents, leading to a reduction in global [Ca2+]i, and vasodilation.

Protein kinase C (PKC) is known to regulate ryanodine receptor (RyR)–mediated local Ca2+ signaling (Ca2+ spark) in airway and vascular smooth muscle cells (SMCs), but its specific molecular mechanisms and functions still remain elusive. In this study, we reveal that, in airway SMCs, specific PKCɛ peptide inhibitor and gene deletion significantly increased the frequency of Ca2+ sparks, and decreased the amplitude of Ca2+ sparks in the presence of xestospogin-C to eliminate functional inositol 1,4,5-triphosphate receptors. PKCɛ activation with phorbol-12-myristate-13-acetate significantly decreased Ca2+ spark frequency and increased Ca2+ spark amplitude. The effect of PKCɛ inhibition or activation on Ca2+ sparks was completely lost in PKCɛ−/− cells. PKCɛ inhibition or PKCɛ activation was unable to affect Ca2+ sparks in RyR1−/− and RyR1+/− cells. Modification of RyR2 activity by FK506-binding protein 12.6 homozygous or RyR2 heterozygous gene deletion did not prevent the effect of PKCɛ inhibition or activation. RyR3 homogenous gene deletion did not block the effect of PKCɛ inhibition and activation, either. PKCɛ inhibition promotes agonist-induced airway muscle contraction, whereas PKCɛ activation produces an opposite effect. Taken together, these results indicate that PKCɛ regulates Ca2+ sparks by specifically interacting with RyR1, which plays an important role in the control of contractile responses in airway SMCs.

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

Objective

Hypercholesterolemia and alcohol drinking constitute independent risk factors for cerebrovascular disease. Alcohol constricts cerebral arteries in several species, including humans. This action results from inhibition of voltage- and calcium-gated potassium channels (BK) in vascular smooth muscle cells (VSMC). BK activity is also modulated by membrane cholesterol. We investigated whether VSMC cholesterol regulates ethanol actions on BK and cerebral arteries.

Methods and Results

After myogenic tone development, cholesterol depletion of rat, resistance-size cerebral arteries ablated ethanol-induced constriction, a result that was identical in intact and endothelium-free vessels. Cholesterol depletion reduced ethanol inhibition of BK whether the channel was studied in VSMC or after rat cerebral artery myocyte subunit (cbv1+β1) reconstitution into phospholipid bilayers. Homomeric cbv1 channels reconstituted into bilayers and VSMC BK from β1 KO mice were both resistant to ethanol-induced inhibition. Moreover, arteries from β1 KO mice failed to respond to ethanol even when VSMC cholesterol was kept unmodified. Remarkably, ethanol inhibition of cbv1+β1 in bilayers and wt mouse VSMC BK were drastically blunted by cholesterol depletion. Consistently, cholesterol depletion suppressed ethanol constriction of wt mouse arteries.

Conclusion

VSMC cholesterol and BK β1 are both required for ethanol inhibition of BK and the resulting cerebral artery constriction, with health-related implications for manipulating cholesterol levels in alcohol-induced cerebrovascular disease.

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.

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

Ca2+ sparks are highly localized, transient releases of Ca2+ from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca2+ sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca2+-activated K+ channels, and also gate Ca2+-activated Cl− (Cl(Ca)) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca2+ sparks is well understood, little information is available on how Ca2+ sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl(Ca) channels in spark sites in airway myocytes from mouse. Ca2+ sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca2+ current underlying a Ca2+ spark (ICa(spark)), with an appropriate correction for endogenous fixed Ca2+ buffer, which was characterized by flash photolysis of NPEGTA. We found that ICa(spark) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca2+ sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the ICa(spark) by less than 3 ms, and its rising phase matches the duration of the ICa(spark). We further determined that Cl(Ca) channels on average are exposed to a [Ca2+] of 2.4 μM or greater during Ca2+ sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca2+] produced by a reaction-diffusion simulation with measured ICa(spark). Finally we estimated that the number of Cl(Ca) channels localized in Ca2+ spark sites could account for all the Cl(Ca) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl(Ca) channels in Ca2+ spark sites localize near to each other, and, moreover, Cl(Ca) channels concentrate in an area with a radius of ∼600 nm, where their density reaches as high as 300 channels/μm2. This model reveals that Cl(Ca) channels are tightly controlled by Ca2+ sparks via local Ca2+ signaling.

In smooth muscle cells, localized intracellular Ca2+ transients, termed “Ca2+ sparks,” activate several large-conductance Ca2+-activated K+ (KCa) channels, resulting in a transient KCa current. In some smooth muscle cell types, a significant proportion of Ca2+ sparks do not activate KCa channels. The goal of this study was to explore mechanisms that underlie fractional Ca2+ spark-KCa channel coupling. We investigated whether membrane depolarization or ryanodine-sensitive Ca2+ release (RyR) channel activation modulates coupling in newborn (1- to 3-day-old) porcine cerebral artery myocytes. At steady membrane potentials of −40, 0, and +40 mV, mean transient KCa current frequency was ∼0.18, 0.43, and 0.26 Hz and KCa channel activity [number of KCa channels activated by Ca2+ sparks × open probability of KCa channels at peak of Ca2+ sparks (NPo)] at the transient KCa current peak was ∼4, 12, and 24, respectively. Depolarization between −40 and +40 mV increased KCa channel sensitivity to Ca2+ sparks and elevated the percentage of Ca2+ sparks that activated a transient KCa current from 59 to 86%. In a Ca2+-free bath solution or in diltiazem, a voltage-dependent Ca2+ channel blocker, steady membrane depolarization between −40 and +40 mV increased transient KCa current frequency up to ∼1.6-fold. In contrast, caffeine (10 μM), an RyR channel activator, increased mean transient KCa current frequency but did not alter Ca2+ spark-KCa channel coupling. These data indicate that coupling is increased by mechanisms that elevate KCa channel sensitivity to Ca2+ sparks, but not by RyR channel activation. Overall, KCa channel insensitivity to Ca2+ sparks is a prominent factor underlying fractional Ca2+ spark uncoupling in newborn cerebral artery myocytes.

We determined the possible role of large-conductance Ca2+-activated K+ (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel α- and β1-subunits were co-expressed, unitary BK currents were detected, and single channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca2+ sparks and iberiotoxin (IBTX)-sensitive spontaneous transient outward currents (STOCs). Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-, KCl (20 mM) and BayK 8644-induced contraction. Acute inhibition of BK channels with IBTX in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel α-subunits and L-type Ca2+ channel α1-C subunits are expressed in murine MV. However, these channels are not functional as murine MV lacked nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl and BayK8644, consistent with our previous studies showing that murine MV do not have BK β1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone, but also that BK channels are required for rhythmic oscillations in venous tone.

Et1-dependent Ca2+-signaling was investigated at the cellular level in retinal vascular smooth muscle. ET1 increased Ca2+-sparks and asynchronous phasic Ca2+-oscillations. This “digital,” frequency-modulated signaling was not previously apparent in microfluorometry recordings of average [Ca2+]i.

Purpose.

To investigate endothelin 1 (Et1)–dependent Ca2+-signaling at the cellular and subcellular levels in retinal arteriolar myocytes.

Methods.

Et1 responses were imaged from Fluo-4–loaded smooth muscle in isolated segments of rat retinal arteriole using confocal laser microscopy.

Results.

Basal [Ca2+]i, subcellular Ca2+-sparks, and cellular Ca2+-oscillations were all increased during exposure to Et1 (10 nM). Ca2+-spark frequency was also increased by 90% by 10 nM Et1. The increase in oscillation frequency was concentration dependent and was inhibited by the EtA receptor (EtAR) blocker BQ123 but not by the EtB receptor antagonist BQ788. Stimulation of Ca2+-oscillations by Et1 was inhibited by a phospholipase C blocker (U73122; 10 μM), two inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs), xestospongin C (10 μM), 2-aminoethoxydiphenyl borate (100 μM), and tetracaine (100 μM), a blocker of ryanodine receptors (RyRs).

Conclusions.

Et1 stimulates Ca2+-sparks and oscillations through EtARs. The underlying mechanism involves the activation of phospholipase C and both IP3Rs and RyRs, suggesting crosstalk between these Ca2+-release channels. These findings suggest that phasic Ca2+-oscillations play an important role in the smooth muscle response to Et1 within the retinal microvasculature and support an excitatory, proconstrictor role for Ca2+-sparks in these vessels.

Pregnancy is a physiological state that involves a significant decrease in uterine vascular tone and an increase in uterine blood flow, which is mediated in part by steroid hormones, including estrogen, progesterone, and cortisol. Previous studies have demonstrated the involvement of these hormones in the regulation of uterine artery contractility through signaling pathways specific to the endothelium and the vascular smooth muscle. Alterations in endothelial nitric oxide synthase expression and activity, nitric oxide production, and expression of enzymes involved in PGI2 production contribute to the uterine artery endothelium-specific responses. Steroid hormones also have an effect on calcium-activated potassium channel activity, PKC signaling pathway and myogenic tone, and alterations in pharmacomechanical coupling in the uterine artery smooth muscle. This review addresses current understanding of the molecular mechanisms by which steroid hormones including estrogen, progesterone, and cortisol modulate uterine artery contractility to alter uterine blood flow during pregnancy with an emphasis on the pregnant ewe model.