© 2001 American Heart Association, Inc.
Editorials |
Calcium Sparks Unleashed in Vascular Smooth Muscle
Lessons From the RyR3 Knockout Mouse
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 (Vm) 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 Vm 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 (BKCa) 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 BKCa 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 BKCa 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 BKCa current and should therefore be sensitive to blockers of this channel. Surprisingly, iberiotoxin, a selective and potent blocker of BKCa, 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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