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This Article Full Text (PDF) All Versions of this Article: 293/3/H1317    most recent 00613.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jaggar, J. H. Search for Related Content PubMed PubMed Citation Articles by Jaggar, J. H.

EDITORIAL FOCUS

Smooth muscle sparklet Ca_v channels defined: 1.2 is the number

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

SYSTEMIC BLOOD PRESSURE and tissue blood flow are regulated by the contractile status of smooth muscle cells located within the wall of small arteries and arterioles. One important signal that regulates arterial smooth muscle cell contractility is the intracellular calcium ion (Ca²⁺) concentration. Although Ca²⁺ was once considered to act only as a global intracellular signaling element, research performed over the past decade has revealed Ca²⁺ to act in a far more dynamic and versatile manner than previously thought possible.

The ability of Ca²⁺ to regulate a wide variety of cellular functions stems primarily from the diversity of local and global Ca²⁺ signals that can be generated. In arterial smooth muscle cells, local and global Ca²⁺ signals differ with respect to their spatial, temporal, and amplitude properties. In addition, whether proteins detect local or global Ca²⁺ signals depends on several factors, including their Ca²⁺ sensitivity and proximity to the source of a Ca²⁺ signal. In arterial smooth muscle cells, the global intracellular Ca²⁺ concentration ([Ca²⁺]_i) occurs because of Ca²⁺ influx through the plasma membrane and Ca²⁺ release from intracellular stores (8). A nanomolar elevation in global [Ca²⁺]_i activates Ca²⁺/calmodulin-dependent myosin light chain kinase, leading to contraction. In contrast, a reduction in global [Ca²⁺]_i results in relaxation.

Localized [Ca²⁺]_i transients, termed "Ca²⁺ sparks," occur because of the opening of multiple ryanodine-sensitive Ca²⁺ release (RyR) channels on the sarcoplasmic reticulum (SR) (8, 14). When imaged by using fluorescent Ca²⁺ indicators, Ca²⁺ sparks are transient events that last for only 100 ms. Within the vicinity of RyR channels, Ca²⁺ sparks produce a localized micromolar [Ca²⁺]_i elevation that activates several nearby plasma membrane large-conductance Ca²⁺-activated potassium (K_Ca) channels, resulting in a transient K_Ca current. Consistent with Ca²⁺ sparks acting as local signals, large-conductance K_Ca channels are sensitive to micromolar Ca²⁺ concentrations and are relatively insensitive to nanomolar Ca²⁺ concentrations found globally. Asynchronous transient K_Ca currents cause an arterial wall membrane hyperpolarization that reduces voltage-dependent Ca²⁺ channel activity, leading to a decrease in global [Ca²⁺]_i and relaxation. Thus Ca²⁺ sparks induce vasodilation by reducing global [Ca²⁺]_i. In smooth muscle cells, Ca²⁺ sparks occur at 1 event/s and do not directly elevate global Ca²⁺ because of their transient and localized temporal and spatial properties. Although global [Ca²⁺]_i and Ca²⁺ sparks are discrete Ca²⁺ signals with distinct targets, these events are functionally coupled. Ca²⁺ sparks regulate global Ca²⁺, and global Ca²⁺ feeds back to regulate Ca²⁺ sparks by modulating the activity of RyR channels, which are Ca²⁺ sensitive.

Another primary Ca²⁺ signal that occurs in arterial smooth muscle cells is a "Ca²⁺ wave" (6). These propagating Ca²⁺ transients occur due the activation of SR inositol trisphosphate (IP₃)-gated Ca²⁺-release channels and RyR channels. Ca²⁺ waves have been proposed to stimulate contraction, but Ca²⁺ waves and oscillations produced in response to alkaline pH or vasoconstrictors have also been described that do not induce contraction (3–5, 7, 10, 15) Thus the physiological functions of Ca²⁺ waves are less clear.

Junctional Ca²⁺ transients (jCaTs) are localized [Ca²⁺]_i elevations that occur in arterial smooth muscle cells in response to ATP release from nerve fibers (9). jCaTs are caused by Ca²⁺ influx through purinergic P2X receptors and stimulate mesenteric artery contraction. Additional Ca²⁺ signals have also been described in arterial smooth muscle cells, including "Ca²⁺ ripples," which occur because of IP₃-induced Ca²⁺ release, and "Ca²⁺ flashes," which occur in a synchronous manner in small groups of cells at a very low rate (2). Thus the inventory of Ca²⁺ signals generated in arterial smooth muscle cells is diverse, and novel Ca²⁺ signals are still being discovered.

A series of elegant studies (1, 11, 12) by the Santana group at the University of Washington has recently described another intracellular Ca²⁺ signal produced in arterial smooth muscle cells. Termed "Ca²⁺ sparklets," these subsarcolemmal transients are generated by Ca²⁺ influx through one or several tightly clustered voltage-dependent Ca²⁺ channels. Previously, arterial smooth muscle cell voltage-dependent Ca²⁺ channels were studied by using patch-clamp electrophysiology. Electrophysiological techniques generated high-resolution temporal information of currents generated by Ca²⁺ channels but could not provide spatial data concerning channel location and the impact of channel opening on submembrane Ca²⁺ concentrations. A significant advantage to imaging Ca²⁺ sparklets is that important spatial information can be obtained regarding the location of active voltage-dependent Ca²⁺ channels in the membrane and the profiles of the local Ca²⁺ transients that are produced by these channels. Combining imaging and electrophysiological techniques provides an even more powerful approach, because both high-resolution temporal and spatial information about intracellular Ca²⁺ changes that occur in response to voltage-dependent Ca²⁺ channel activation can be obtained.

With the use of these techniques, it has been found that arterial smooth muscle cell Ca²⁺ sparklets occur in two distinct functional modes: low and high activity. Low-activity Ca²⁺ sparklets occur due to the opening of a single voltage-dependent Ca²⁺ channel, at low frequency, and in an apparent stochastic manner throughout the plasma membrane. In contrast, high-activity sparklets occur at recurring sites through the opening of small clusters of voltage-dependent Ca²⁺ channels that exhibit such a high open probability that they produce almost constant Ca²⁺ influx. An important distinguishing feature of these apparently different Ca²⁺ signals is that the high-activity mode results from protein kinase C (PKC)--induced Ca²⁺ channel activation. Protein phosphatases 2A and 2B counter PKC-mediated Ca²⁺ sparklets because phosphatase inhibition stimulates previously silent high-activity-mode sparklet sites. One major discovery is that Ca²⁺ sparklets not only cause a local [Ca²⁺]_i elevation within the vicinity of the Ca²⁺ channel, but these events also increase global [Ca²⁺]_i. A striking finding is that at physiological voltages and external Ca²⁺ concentrations (2 mM), 50% of voltage-dependent Ca²⁺ influx in arterial smooth muscle cells occurs because of the PKC--induced high-activity Ca²⁺ sparklet mode.

In this issue of the American Journal of Physiology-Heart and Circulatory Physiology, Navedo et al. (13) investigated the molecular identity of voltage-dependent Ca²⁺ channels that generate Ca²⁺ sparklets in murine arterial smooth muscle cells. Previous studies using pharmacological approaches had suggested that sparklets occur due to the opening of Ca_V1.2 L-type voltage-dependent Ca²⁺ channels (1, 11, 12). However, because Ca²⁺ sparklets are observed at hyperpolarized voltages where Ca_V1.2 channel activity would be extremely low, additional Ca²⁺ channels with more negative voltage sensitivities than Ca_V1.2 may also generate sparklets. Because Ca_V1.3 channels have been detected in basilar artery and because the voltage sensitivity of Ca_V1.3 is more negative than for Ca_V1.2, the authors tested the hypothesis that Ca_V1.3 channels may also produce Ca²⁺ sparklets in cerebral artery smooth muscle cells. The authors demonstrate that Ca_V1.3 channels expressed in tsA201 cells have a similar conductance to Ca_V1.2 channels. Similarly, when coexpressed with PKC-, Ca_V1.3 channels generate Ca²⁺ sparklets that function in low- and high-activity modes. Ca_V1.3 sparklets were also similar in amplitude and dihydropyridine sensitivity to those generated by Ca_V1.2 channels. However, several important findings support the conclusion that Ca²⁺ sparklets in cerebral artery smooth muscle cells arise due to the opening of Ca_V1.2 channels. These include the observations that in arterial smooth muscle cells, Ca²⁺-current voltage dependence and steady-state inactivation were similar to Ca_V1.2 but not Ca_V1.3 currents and that RT-PCR and immunofluorescence detected only Ca_V1.2 in isolated myocytes. In addition, the authors found that nifedipine did not alter Ca²⁺ sparklets in arterial smooth muscle cells isolated from mice that express dihydropyridine-insensitive Ca_V1.2 channels. If Ca²⁺ sparklets also occurred due to the activation of dihydropyridine-sensitive Ca²⁺ channels other than Ca_V1.2, nifedipine would have reduced Ca²⁺ sparklet frequency in these cells. Together, these data indicate that Ca_V channels other than Ca_V1.2 can generate Ca²⁺ sparklets but that Ca_V1.2 channels produce sparklets in cerebral artery smooth muscle cells.

Although the present study and earlier studies provide some answers as to the source, regulation, and physiological functions of Ca²⁺ sparklets in arterial smooth muscle cells, many questions remain. A major question is what, if any, are local targets for Ca²⁺ sparklets? Many different Ca²⁺-sensitive proteins could be located in close proximity to voltage-dependent Ca²⁺ channels and may be exposed to Ca²⁺ sparklets, including kinases, ion channels, and transporters. Whether Ca²⁺ sparklets regulate local Ca²⁺-sensitive proteins will depend on several factors, including the Ca²⁺ sensitivity of the protein and how close the protein is to the Ca²⁺-channel pore, which would determine the Ca²⁺ concentration to which it is exposed. Any Ca²⁺-permeant channel should generate Ca²⁺ sparklets, with the spatial and temporal properties of the Ca²⁺ transients produced being contingent on the properties of the channel, such as conductance. Arterial smooth muscle cells located in vascular beds other than those in the brain may express voltage-dependent Ca²⁺ channels other than Ca_V1.2. As such, the properties and physiological functions of individual Ca²⁺ sparklets would be defined by those of the Ca²⁺-channel isoforms that generate the signals.

A more specific question that arises from the current study is what is different molecularly between voltage-dependent Ca²⁺ channels that generate low- and high-activity-mode Ca²⁺ sparklets. Although the importance of PKC has been established, it is unclear what predisposes only some Ca_V1.2 channels to PKC activation and thus the high-activity mode. Several possibilities exist, including that PKC is located within the vicinity of only some channels or within certain channel complexes, that Ca_V1.2 splice variants with differing sensitivities to PKC are present, or that protein phosphatases which limit channel phosphorylation are associated with only some Ca_V1.2 channels.

In summary, the excellent study by Navedo et al. (13) demonstrates that although Ca_V1.2 and Ca_V1.3 channels generate quantitatively similar Ca²⁺ sparklets, Ca_V1.2 channels generate sparklets in cerebral artery smooth muscle cells. Data also indicate that Ca_V1.2 channels are responsible for voltage-dependent Ca²⁺ influx in cerebral artery smooth muscle cells. When considering the importance of voltage-dependent Ca²⁺ channels in arterial smooth muscle physiology, future studies are likely to uncover additional functions of Ca²⁺ sparklets in these cells.

FOOTNOTES

Address for reprint requests and other correspondence: J. H. Jaggar, Dept. of Physiology, Univ. of Tennessee Health Science Center, Memphis, TN 38163 (e-mail: jjaggar{at}physio1.utmem.edu)

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This Article Full Text (PDF) All Versions of this Article: 293/3/H1317    most recent 00613.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jaggar, J. H. Search for Related Content PubMed PubMed Citation Articles by Jaggar, J. H.