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(Circulation Research. 2001;89:941.)
© 2001 American Heart Association, Inc.

Editorials

Calcium Sparks Unleashed in Vascular Smooth Muscle

Lessons From the RyR3 Knockout Mouse

Harm J. Knot

From the Department of Pharmacology and Therapeutics, University of Florida, and the McKnight Brain Institute, Gainesville, Fla.

Correspondence to Harm J. Knot, University of Florida, Department of Pharmacology, Box 100267, Gainesville, FL 32610. E-mail hknot{at}college.med.ufl.edu

Key Words: calcium • sparks • cerebral arteries • ryanodine receptors • calcium channels

Hypertension is a progressive disease involving an increase in arterial constriction. It constitutes a major risk factor leading to stroke, heart disease, and kidney failure. Understanding the molecular pathways involved in arterial tone regulation is crucial to increasing our understanding of blood pressure regulation. Recently, a novel mechanism involving small-localized bursts of calcium inside vascular smooth muscle cells, termed Ca²⁺ sparks, was identified that acts as a negative feedback mechanism that opposes vasoconstriction.¹

Ca²⁺ Sparks in Muscle

Ca²⁺ sparks are thought to be the elementary functional Ca²⁺ release signals in heart, skeletal, and smooth muscle cells.^1–4 Ca²⁺ sparks are but one example of the increasing complexity in Ca²⁺ signaling in the spatial and temporal domain in a variety of tissues including arterial smooth muscle.⁵ Ca²⁺ sparks result from the opening of several or the coordinated opening of many, tightly clustered ryanodine receptor Ca²⁺ release channels (RyRs) in the sarcoplasmic reticulum (SR) of muscle cells.^6,7 In arterial smooth muscle and intact arteries, Ca²⁺ sparks are observed just under the cell membrane consistent with a predominant subsarcolemmal localization of the RyR Ca²⁺ release channels in the sarcoplasmic reticulum.^3,8

Function, Distribution, and Localization of RyR Isoforms

The mammalian RyRs are encoded by 3 different genes (RYR1, RYR2, and RYR3). Initial DNA cloning studies in mammalian tissues have assigned the names RyR1 to the subtype dominantly expressed in skeletal muscle, RyR2 to the one in cardiac muscle and RyR3 in brain tissue, respectively.^9,10 The functional properties of RyR1 and RyR2 are well studied because of their dominant expression and role in excitation-contraction coupling in skeletal and cardiac muscle, respectively. RyR1 is essential for EC coupling in skeletal muscle and is thought to act as the Ca²⁺ release channel that is mechanically linked to the sarcolemmal voltage-sensor constituted by the L-type voltage-dependent Ca²⁺ channel, often referred to as the dihydropyridine receptor (DHPR). In cardiac muscle, RyR2 is thought to mediate Ca²⁺ release in response to Ca²⁺ entry through DHPRs via a process named Ca²⁺-induced Ca²⁺ release (CICR). Despite the fact that RyR3 has now been shown to exist in neurons, smooth muscle cells, skeletal muscle cells, lymphocytes, and other nonexcitable tissues, the functional role of RyR3 remains elusive.¹¹ RyR3 has been able to substitute for RyR1 and is capable of CICR in skeletal muscle of the RyR1 null mouse.¹²

To further investigate the physiological role of RyR3, RNA silencing approaches have been used and 2 independent strains of RyR3 null mice have been generated.^13–15 In this issue of Circulation Research, Löhn et al¹⁶ use one of the RyR3 null mice to study consequences of RyR3 knockout on cerebral arterial smooth muscle function. To understand the implications of their findings, let me briefly summarize the current hypothesis on the physiological regulation of arterial diameter by Ca²⁺ sparks.

Physiological Role of Ca²⁺ Sparks in Arterial Smooth Muscle

In cerebral resistance arteries, intracellular pressure causes a graded membrane potential (V_m) depolarization to approximately -40 mV at 60 to 80 mm Hg.^17,18 The result is an elevation of arterial wall [Ca²⁺] due to activation of voltage-dependent L-type Ca²⁺ channels leading to a sustained contraction termed myogenic tone.¹⁹ At physiological pressures, the resulting arterial diameter is very sensitive to changes in membrane potential (1 to 2 µm · nmol/L^-1 Ca²⁺ · mV^-1 V_m change).²⁰ Ca²⁺ sparks are thought to be involved in opposing this tonic contraction of small resistance arteries by activating large conductance Ca²⁺-sensitive potassium channels (BK_Ca) in the cell membrane, causing hyperpolarization, and thus limiting the opening of voltage-dependent Ca²⁺ channels.^1,21,22 Ca²⁺ sparks depend on influx of extracellular Ca²⁺ via L-type Ca²⁺ channels and the level of Ca²⁺ in the lumen of the SR.^23–25 The result of these two opposing forces, involving different spatio-temporal Ca²⁺ signals, is a partially contracted resistance artery that can dilate or constrict depending on the demand for blood. Further studies have shown that circulating systemic and/or locally produced metabolic vasoconstrictors and vasodilators can either directly or via second messenger pathways modulate the Ca²⁺ spark pathway to further fine tune arterial tone and thus control local blood flow.^26–28 Recent studies in transgenic mice that lack the ß1 subunit of the BK_Ca channel, the subunit that modulates the Ca²⁺-sensitivity of the channel, indicate that relatively small molecular interventions in the Ca²⁺ spark pathway have significant effects on vascular resistance and blood pressure.^29,30

Putative Role of RyR3 in the Arterial Smooth Muscle Ca²⁺ Spark Pathway

In this issue, Löhn et al study the potential physiological role of the RyR3 isoform in the regulation of cerebral arterial smooth muscle function by comparing Ca²⁺ sparks and spontaneous transient outward currents or STOCs resulting from clusters of activated BK_Ca channels, in freshly isolated smooth muscle cells and intact pressurized arteries from the brain of wild-type (WT) and RyR3 null mice.¹⁶ They provide experimental evidence for a proposed inhibitory role for RyR3 on Ca²⁺ spark generation in mouse cerebral arteries. Using quantitative reverse transcriptase–polymerase chain reaction on freshly isolated intact basilar cerebral arteries the authors demonstrate that all three RyR isoforms are expressed at the RNA level. Although this approach includes possible contamination of neurons and endothelial cells, the relative RNA levels indicate high levels of RyR3 and RyR1 and, to a lesser extent, RyR2 in this these arteries. The authors first studied the spatio-temporal properties and kinetics of Ca²⁺ sparks in isolated cells from cerebral arteries and found no significant differences between the RyR3 null and WT mouse. They do note more repetitive spark sites in the RyR3 null myocytes. This is an interesting observation in other studies as well that we will revisit in relation to the sarcolemmal Ca²⁺ channel.²⁷ In the next set of experiments, the authors present the main finding of this study. There is a remarkable shift in the voltage-dependence of Ca²⁺ spark generation, measured as spontaneous transient outward currents (STOCs), in isolated cerebral myocytes under voltage-clamp. To further illustrate this fact, the authors show that in isolated cells from the RyR3 null mouse at a membrane potential of -40 mV, STOC frequency was nearly 10-fold higher compared with cells from WT mice. The voltage-dependence of Ca²⁺ sparks and STOCs in cerebral arteries has been approximated. A 4.3-fold increase in Ca²⁺ spark frequency was observed by raising potassium from 6 to 30 mmol/L K⁺ (the equivalent of a membrane depolarization from -60 to -40 mV) in the bath of intact pressurized posterior cerebral arteries from rat.³¹ Another recent study showed a 6-fold increase in STOC frequency between -50 and -20 mV in voltage-clamped cerebral myocytes (this would encompass the entire physiological range of membrane potential²⁰). In comparing these studies with the current study by Löhn et al, the increase in STOC frequency should result in hyperpolarization of the smooth muscle cells in situ.¹⁶ Consistent with this expectation is the finding that myogenic tone in isolated arteries from these animals is greatly blunted. A larger component of arterial tone should now depend on the BK_Ca current and should therefore be sensitive to blockers of this channel. Surprisingly, iberiotoxin, a selective and potent blocker of BK_Ca, causes only a few microns more constriction in arteries from the RyR3 null mouse. Moreover, the overall effect of iberiotoxin in both types of mouse arteries is similar than that observed in cerebral arteries from rat at the same pressure but with significant more myogenic tone.²¹ It is unclear whether this difference reflects species differences and/or compensatory regulatory mechanisms in the RyR3 null mouse. Future studies should therefore include blood pressure measurements in these knockout animals to extend the present observations to the impact in vivo.

Future Directions

Testing an interesting hypothesis has a tendency to generate more new intriguing questions, and this study by Löhn et al is no exception.¹⁶ The key question that arises from this study is how the RyR3 channel interacts with the Ca²⁺-spark release cluster that is presumably formed by the RyR2/1 isoforms.³² The authors propose 3 possible mechanisms based on the fact that RyR3 channels are thought to be less sensitive to Ca²⁺-dependent inactivation.³³ First, they hypothesize that prolonged Ca²⁺ release through RyR3 may inactivate or desensitize the RyR2/1 cluster, presumably by maintaining elevated levels of Ca²⁺ in the vicinity of the functional spark release unit. The fluorescence data thus far do not reveal this extra Ca²⁺ from RyR3 as part of the observed Ca²⁺ sparks, and this hypothesis seems at first glance at odds with the observation of repetitive firing of the same RyR2/1 clusters. Second, prolonged Ca²⁺ release may contribute to Ca²⁺ dependent inactivation of the sarcolemmal Ca²⁺ channel. This is an interesting hypothesis that requires further thought. A clear and consistent finding among previous studies is that extracellular Ca²⁺ and Ca²⁺ channel openers such as BayK8644 are among the most potent inducers of Ca²⁺ sparks. Recent studies on single Ca²⁺ channels in cerebral vascular myocytes, at physiological membrane potentials and using physiological concentrations of Ca²⁺ as the charge carrier, have revealed multiple inactivation processes that regulate Ca²⁺ channel activity during steady depolarization.³⁴ The same study estimates the number of active Ca²⁺ channels open at any one time in the physiological range of membrane potentials from -50 to -20 mV to be 1 to 10 channels. These numbers are in the same order of magnitude as the observed number of Ca²⁺ sparks in these cells at these potentials. In this context, could repetitive firing reflect a RyR2/1 Ca²⁺ spark release cluster in the vicinity of an active Ca²⁺ channel? Finally, a prolonged open RyR3 might act as a Ca²⁺ leak channel, partially or locally emptying the SR of Ca²⁺ and thus decrease the probability of the RyR2/1 cluster to reopen. As the authors point out, the unaltered STOC and Ca²⁺ spark amplitudes do not suggest such a mechanism, and the latter hypothesis also seems at odds with the repetitive firing observation.

In conclusion, Löhn et al show that knockout of the RyR3 isoform unleashes the full impact of the Ca²⁺ spark pathway in controlling cerebral arterial smooth muscle membrane potential and contractile function.¹⁶ They provide evidence for a novel and surprising role for RyR3 in smooth muscle, thus sparking a wealth of new ideas for future studies to further elucidate this remarkable signaling system controlling arterial tone.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

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