Calcium dynamics in vascular smooth muscle

Abstract

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells. These signals vary with respect to their mechanisms of generation, temporal properties, and spatial distributions. The calcium signals discussed include calcium waves, junctional calcium transients, calcium sparks, calcium puffs, and L-type calcium channel sparklets. For each calcium signal we address underlying mechanisms, general properties, physiological importance, and regulation.

Introduction

The otherwise unassuming inorganic divalent cation calcium (Ca²⁺) is the most important signaling molecule in mammalian cells. Cellular responses to changes in intracellular Ca²⁺ ([Ca²⁺]_i) often underlie our definition of what a given cell “is” (22). For example, following an increase in [Ca²⁺]_i, endocrine cells secrete hormones and neurons release neurotransmitters. Muscle cells, including vascular smooth muscle cells, generally contract when [Ca²⁺]_i rises. Ca²⁺ induces contraction in these cells by complexing with the ubiquitous Ca²⁺ binding protein calmodulin and subsequently increasing the activity of myosin light-chain kinase (MLCK; see Figure 1). Changes in the contractile state of vascular smooth muscle either increases or decreases vascular diameter, which in turn increases or decreases blood flow through the vessel and subsequently the vascularized tissue. Thus, events which influence [Ca²⁺]_i are critical regulators of vascular function with clear physiological (and pathophysiological) ramifications.

Figure 1. Overview of calcium dynamics in vascular smooth muscle.

Ca²⁺ influences the state of vascular smooth muscle contraction directly and indirectly. Direct mechanisms include changes (Δ) in Ca²⁺ influx and release from intracellular Ca²⁺ stores leading to increased Ca²⁺ within the cell, binding of Ca²⁺ to calmodulin (CaM), and activation of myosin light chain kinase (MLCK). Indirect mechanisms include changes in Ca²⁺ influx via alterations in plasma membrane potential (Vm), Ca²⁺ release from intracellular stores, Ca²⁺ sequestration, and Ca²⁺ sensitization of the contractile apparatus.

Due to the importance of Ca²⁺, it is not unexpected that changes in vascular smooth muscle [Ca²⁺]_i (i.e., “Ca²⁺ dynamics”) are governed by a complex array of cellular processes. Indeed, to arrive at the appropriate physiological response, vascular smooth muscle cells must integrate multiple signaling events that influence [Ca²⁺]_i either directly or indirectly (see Figure 1). In this paper we provide an overview of the major Ca²⁺ signals that contribute to changes in vascular smooth muscle [Ca²⁺]_i. Note that many excellent and more extensive reviews on the individual topics discussed here are readily found in the published literature.

Calcium signals in vascular smooth muscle

Sources of Ca²⁺ available for cytoplasmic signaling in vascular smooth muscle cells include Ca²⁺ influx through ion-permeable channels located in the plasma membrane and Ca²⁺ release from intracellular Ca²⁺ stores (e.g., the sarcoplasmic reticulum). Ca²⁺ influx in vascular smooth muscle is mediated primarily by voltage-dependent L-type Ca²⁺ channels (39) with contributions from other channels including but not limited to voltage-dependent T-type Ca²⁺ channels, Ca²⁺-permeable members of the transient receptor potential (TRP) superfamily of cation channels, and Ca²⁺-permeable ligand-gated cation channels. Ca²⁺ release from intracellular stores is mediated by two types of Ca²⁺-permeable ion channels located in sarcoplasmic reticular membranes: 1) Ryanodine receptors and 2) inositol 1,4,5-trisphosphate (IP₃) receptors.

Cytoplasmic signals produced by these Ca²⁺ fluxes can be loosely classified as either more or less localized with respect to total cell volume (see Figure 2). Here we have grouped the “less than global” subcellular Ca²⁺ signals observed in vascular smooth muscle into five categories: 1) Ca²⁺ waves, 2) junctional Ca²⁺ transients, 3) Ca²⁺ sparks, 4) Ca²⁺ puffs, and 5) L-type Ca²⁺ channel sparklets. Note that IP₃-receptor-mediated Ca²⁺ puffs have not been definitively isolated and visualized in vascular smooth muscle cells to the best of our knowledge. This non-arbitrary categorization of subcellular Ca²⁺ signaling events is based on a wealth of experimental evidence obtained by numerous research groups over the past few decades. The general biophysical characteristics (when known) of each of these subcellular Ca²⁺ signals is presented in Table 1.

Figure 2. Overview of calcium signaling events in vascular smooth muscle.

Discrete subcellular Ca²⁺ signaling events (junctional Ca²⁺ transients, Ca²⁺ waves, Ca²⁺ sparks, Ca²⁺ puffs, and Ca²⁺ sparklets) can be classified as either more or less localized with respect to total cell volume. * Direct visualization of Ca²⁺ puffs in arterial smooth muscle is lacking; however, abundant indirect evidence supports their existence.

Table 1.

Properties of vascular smooth muscle subcellular Ca²⁺ signals (approximate values). NA = not available.

	Ca2+ waves (31, 61)	Junctional Ca2+ transients (34)	Ca2+ sparks (8, 26)	Ca2+ puffs	Ca2+ sparklets (3, 41)
Area (μm2)	≈ 50 μm (full-width, half maximum)	≈ 25	≈ 15	NA	≈ 1
Duration (t0.5; ms)	NA	≈ 150	≈ 50	NA	τ1 ≈ 35 τ2 ≈150
Amplitude (F/F0)	≈ 2	≈ 3	≈	NA	NA

In contrast to increased global [Ca²⁺]_i, which is invariably associated with contraction (29), the role of specific subcellular Ca²⁺ signals is not always apparent. For instance, while Ca²⁺ events such as junctional Ca²⁺ transients are inherently contractile (32, 63, 65), Ca²⁺ microdomains such as Ca²⁺ sparks may induce vasodilation (see below; (46)). This highlights the complexity of Ca²⁺ dynamics in vascular smooth muscle and the need for further investigation of subcellular Ca²⁺ signaling in these cells.

Calcium waves

As suggested by the moniker, Ca²⁺ waves are propagating [Ca²⁺]_i elevations produced by sequential series of Ca²⁺ release events from the sarcoplasmic reticulum that extend from one end of the cell to the other (23, 38, 66). Ca²⁺ waves have been shown to be produced by Ca²⁺ release via IP₃ and ryanodine receptors located in the sarcoplasmic reticulum (20, 26). By their nature Ca²⁺ waves inherently require a regenerative Ca²⁺ release mechanism that promotes propagation of the wavefront. This is thought to occur by a Ca²⁺-induced Ca²⁺-release (CICR) mechanism between adjacent IP₃ and/or ryanodine receptors (7, 9, 25, 26). A schematic representation of an IP₃ receptor-dependent Ca²⁺ wave is shown in Figure 3. In this scenario, the Ca²⁺ wave is initiated by localized SR Ca²⁺ release via IP₃-dependent opening of IP₃ receptors. A self-perpetuating Ca²⁺ wave arises when the initiating Ca²⁺ induces Ca²⁺-dependent opening of adjacent IP₃ receptors (i.e., Ca²⁺-induced Ca²⁺-release). Ca²⁺ release via ryanodine receptors has also been implicated in Ca²⁺ wave generation via frequent discharge sites in rat and rabbit portal vein (7, 20, 64). In addition, application of the alkaloid ryanodine eliminated Ca²⁺ waves in pressurized rat cerebral arteries (25, 62). This suggests that in some arterial smooth muscle cells Ca²⁺ waves involve SR Ca²⁺ release via ryanodine receptors.

Figure 3. Vascular smooth muscle calcium waves.

Norepinephrine (NE)-dependent stimulation of α₁ adrenergic receptors (α1R) on the vascular smooth muscle cell plasma membrane (VSMC PM) leads to activation of phospholipase C (PLC) and the production of inositol 1,4,5-trisphosphate (IP3). IP3 in turn promotes the opening of IP3 receptors (IP3R) on the sarcoplasmic reticulum (SR) resulting in a propagating series of Ca²⁺ release events (i.e., a Ca²⁺ wave). Ca²⁺ waves increase contraction directly by elevating global intracellular Ca²⁺ ([Ca²⁺]_i). Ca²⁺ waves also modulate contraction indirectly by altering plasma membrane potential (and thus voltage-dependent Ca²⁺ influx through Cav1.2 L-type Ca²⁺ channels) by stimulating plasmalemmal Ca²⁺-activated chloride channels.

The occurrence of Ca²⁺ waves in excised vessel preparations is variable to moderate under resting conditions. For example, smooth muscle cells in mouse cremaster feed arteries (62) and rat retinal arterioles exhibit moderate basal Ca²⁺ wave activity (56) while mouse and rat mesenteric arterial smooth muscle cells often possess minimal Ca²⁺ wave activity in the absence of stimulation (38, 65). The occurrence of basal Ca²⁺ waves appears to be dependent on phospholipase C (PLC) activity (62). Consistent with a role of PLC in regulating Ca²⁺ waves, increased production of IP₃ by PLC following stimulation of α₁ adrenergic receptors by norepinephrine in mesenteric arteries (31) and ET_A receptors by endothelin-1 in retinal arterioles (56) increases Ca²⁺ wave frequency.

Ca²⁺ waves are postulated to contract vascular smooth muscle by at least two distinct mechanisms (21). Ca²⁺ released into the cytosol during the wave event can contribute to global Ca²⁺ thus increasing MLCK activity and promoting contraction (33). This is perhaps the most common interpretation of Ca²⁺ wave function. However, in contrast to this direct mechanism, Ca²⁺ waves can also influence contraction indirectly by interacting with and changing the activity of plasmalemmal Ca²⁺-activated ion channels. Ca²⁺ waves in rat retinal arteriole smooth muscle cells elicit Ca²⁺-activated chloride (Cl_Ca) currents (56). Stimulation of Ca²⁺-activated chloride channels results in a depolarization of the plasma membrane via Cl⁻ efflux (an inward current by convention; see Figure 3), thus enhancing voltage-dependent L-type Ca²⁺ channel activity, and consequently, Ca²⁺ influx, global [Ca²⁺]_i, and ultimately contraction.

As noted above, Ca²⁺ waves are associated with increased vascular smooth muscle contraction in excised vessels. For example, in rat mesenteric arteries, asynchronous Ca²⁺ waves (with respect to adjacent smooth muscle cells) following neurogenic activation of α₁ receptors by norepinephrine contribute to gradually-developing contractions (31). Contractile responses to bath applied α1 agonists (e.g., phenylephrine) also include a rapid initial component due to synchronous Ca²⁺ wave activity. This is likely an experimental artifact resulting from strong uniform (i.e., non-physiological) activation of α1 receptors on a large population of smooth muscle cells (31, 67). Despite a substantial body of experimental data, the physiological significance of Ca²⁺ waves is poorly understood. For example, Ca²⁺ waves in mouse mesenteric arteries appear to cease following the development of tone (65) and evidence suggests that Ca²⁺ waves, which are readily observed in numerous excised vessels (as described above), are not apparent in corresponding in vivo experiments (36). Thus, much work is required to understand the physiological role of Ca²⁺ waves in the vasculature.

Junctional Calcium Transients

Short-lived Ca²⁺ influx events evoked by neural (i.e., sympathetic) stimulation of vascular smooth muscle cells are defined as junctional Ca²⁺ transients (34, 63). In contrast to Ca²⁺ waves, junctional Ca²⁺ transients remain localized, as they do not possess a regenerating or propagating mechanism. Action potentials arriving at perivascular sympathetic nerve terminals culminate in release of neurotransmitters, including ATP and norepinephrine. ATP activates postjunctional vascular smooth muscle P2X₁ purinergic receptors and norepinephrine stimulates α₁ adrenergic receptors giving rise to Ca²⁺ waves as described above (31, 32, 35) (see Figure 4).

Figure 4. Vascular smooth muscle junctional calcium transients.

Neuronal action potentials (AP) promote the release of ATP and norepinephrine (NE) from perivascular nerve terminals. Norepinephrine stimulates α₁ adrenergic receptors (α1R) giving rise to Ca²⁺ waves as described in Figure 3 while ATP opens Ca²⁺-permeable purinergic receptors (P2XR) on the vascular smooth muscle cell plasma membrane (VSMC PM) producing localized junctional Ca²⁺ transients. Purinergic receptor activation also leads to plasma membrane depolarization (+ Vm) and increased opening of voltage-dependent Cav1.2 L-type Ca²⁺ channels (L). Ca²⁺ influx from junctional Ca²⁺ transients and L-type Ca²⁺ channels summate to increase contraction.

P2X₁ receptors are ligand-gated Ca²⁺-permeable nonselective cation channels (16). In vascular beds innervated by the sympathetic nervous system (e.g., mesenteric arteries), stimulation of vascular smooth muscle P2X₁ receptors by ATP generates localized junctional Ca²⁺ transients on account of the relatively high Ca²⁺ permeability of these channels (32, 34). Temporally mirroring the kinetics of the junctional Ca²⁺ transients, the Ca²⁺ influx associated with these events induces a rapid but brief increase in contraction (31, 32). Non-neuronal stimulation of P2X₁ receptors has also been described. In mouse renal afferent arterioles, increasing intraluminal pressure promotes local release of ATP, stimulation of smooth muscle P2X₁ receptors, and contraction (24). This paracrine signaling mechanism is thought to contribute to autoregulation of blood flow in the kidney.

In addition to evoking local junctional Ca²⁺ transients, activation of P2X₁ receptors by ATP induces depolarizing excitatory junctional potentials as a result of Na⁺ and (to a lesser extent) Ca²⁺ influx (16, 67). Depolarization increases the opening of L-type Ca²⁺ channels resulting in increased Ca²⁺ influx and elevated global [Ca²⁺]_i. Thus, stimulation of P2X₁ receptors with ATP increases vascular smooth muscle Ca²⁺ influx by two independent mechanisms: 1) Local junctional Ca²⁺ transients via Ca²⁺ entry through the P2X₁ receptors and 2) non-localized Ca²⁺ influx through L-type Ca²⁺ channels following depolarizing excitatory junction potentials. These two complimentary Ca²⁺ influx mechanisms summate to underlie the initial component of sympathetic-mediated contraction of rat mesenteric arteries (67).

Calcium Sparks

Ca²⁺ sparks are localized Ca²⁺ microdomains produced specifically by the opening of the ryanodine receptor class of intracellular Ca²⁺ release channels located in sarcoplasmic reticular membranes (12). In vascular smooth muscle Ca²⁺ sparks have minimal direct impact on global [Ca²⁺]_i and contraction as a consequence of their limited spatial spread (46). However, Ca²⁺ sparks can have substantial influence on global [Ca²⁺]_i and contraction through indirect mechanisms. Accordingly, regions of sarcoplasmic reticulum are often found in close juxtaposition to the plasma membrane of vascular smooth muscle cells (≈10–20 nm; (14, 28, 57)). As noted above, Ca²⁺-activated plasmalemmal ion channels (e.g., K_Ca and Cl_Ca) are expressed in various vascular smooth muscle cells. Ca²⁺ sparks occurring in close proximity to these channels can increase local [Ca²⁺]_i to levels sufficient (i.e., μM; (48)) for channel activation (see Figure 5). Thus, the contractile response of vascular smooth muscle to Ca²⁺ sparks depends on the expression and coupling of the sparks to Ca²⁺-activated plasmalemmal ion channels.

Figure 5. Vascular smooth muscle calcium sparks.

Ca²⁺-permeable ryanodine receptors (RR) on the sarcoplasmic reticulum (SR) produce highly localized sites of elevated Ca²⁺ (i.e., Ca²⁺ sparks). Ca²⁺ sparks near the vascular smooth muscle cell plasma membrane (VSMC PM) increase the activity of plasmalemmal ion channels including Ca²⁺ activated K⁺ (K) and Ca²⁺-activated Cl⁻ (Cl) channels. Ca²⁺ spark-dependent activation of K⁺ channels generates hyperpolarizing outward currents (via K⁺ efflux) resulting in decreased opening of voltage-dependent Cav1.2 L-type Ca²⁺ channels (L) and decreased contraction. Conversely, Ca²⁺ spark-dependent activation of Cl⁻ channels generates depolarizing (+ Vm) inward currents (via Cl⁻ efflux) resulting in increased opening of L-type Ca²⁺ channels and increased contraction.

In rat and mouse cerebral arteries, large-conductance, Ca²⁺-activated K⁺ (BK; maxi K) channels are a primary K_Ca target for Ca²⁺ sparks (27, 46). Ca²⁺ spark activation of BK channels results in a stereotypical temporal pattern of hyperpolarizing K⁺ currents called spontaneous transient outward currents (STOCs) (6, 46). Simultaneous recording of intracellular Ca²⁺ and membrane potential has provided definitive evidence that Ca²⁺ sparks give rise to STOCs as the two events correlate not only in time but also in magnitude (1, 10, 46, 48). In rat cerebral arteries elimination of Ca²⁺ sparks eliminates contractile responses to BK channel inhibition (30, 46) thus indicating that Ca²⁺ sparks are the physiological activators of BK channels in these vascular smooth muscle cells.

In rabbit portal vein, Ca²⁺ sparks are coupled to Cl_Ca channels (59). In contrast to Ca²⁺ spark activation of BK channels, which results in hyperpolarization, Ca²⁺ spark activation of Ca²⁺-activated Cl⁻ channels causes plasma membrane depolarizing outward currents (via Cl⁻ efflux; see Figure 5). Consequently, similar to Ca²⁺-activated K⁺ channels, activation of Ca²⁺-activated Cl⁻ channels by Ca²⁺ sparks produces a signature pattern of inward current called spontaneous transient inward currents (STICs) that correlate in time and magnitude with each other. Depolarization induced by Ca²⁺-activated Cl⁻ channel activity results in activation of voltage dependent L-type Ca²⁺ channels and increased contraction (see Figure 5) (51).

The dependence of Ca²⁺-activated ion channels on underlying Ca²⁺ sparks provides an opportunity for regulation of vascular smooth muscle function by vasodilators and vasoconstrictors. In rat cerebral arteries, vasodilators increase the hyperpolarizing influence of BK channels by increasing the sensitivity of the BK channels to Ca²⁺ and by increasing Ca²⁺ spark (thus STOC) frequency (49, 54). These effects are mediated by activation of adenosine 3′, 5′-cyclic monophosphate (cAMP)/protein kinase A (PKA) and guanosine 3′,5′-cyclic monophosphate (cGMP)/protein kinase G (PKG) signaling cascades. Thus, vasodilators can increase BK channel activity by increasing the coupling strength between the channel and the Ca²⁺ sparks (increasing STOC amplitude) and increasing the occurrence of Ca²⁺ sparks (increasing STOC frequency). Conversely, vasoconstrictors associated with protein kinase C (PKC) decrease the hyperpolarizing influence of BK channels by decreasing BK channel sensitivity to Ca²⁺ (reducing STOC amplitude) and by reducing the occurrence of Ca²⁺ sparks (reducing STOC frequency) (8, 54).

The coupling strength between Ca²⁺ sparks and BK channels is also associated with vascular dysfunction during diseases such as hypertension. The sensitivity of vascular smooth muscle BK channels to Ca²⁺ is greatly dependent on expression of BK channel β1 subunits (10). Genetic ablation of the β1 subunit decreases the sensitivity of cerebral arterial smooth muscle BK channels to Ca²⁺. As a consequence, Ca²⁺ sparks in these cells do not efficiently generate corresponding STOCs (10). This results in increased contraction of excised cerebral arterial segments and increased mean arterial blood pressure. Similarly, in rat models of hypertension, the function and expression of BK channel β1 subunits in cerebral arteries is decreased and contributes to vascular dysfunction (1, 4).

Calcium Puffs

Ca²⁺ puffs are localized Ca²⁺ microdomains produced by the opening of sarcoplasmic reticulum IP₃ receptors (58). Although Ca²⁺ puffs have been observed in colonic smooth muscle cells (5), they have not been visualized in vascular smooth muscle cells. However, indirect experimental evidence in rat cerebral arterial smooth muscle cells indicates that physiologically-relevant localized IP3 receptor-dependent Ca²⁺ release does occur (17, 18). Indeed, in these smooth muscle cells, IP3-dependent Ca²⁺ release has been shown to promote the opening of transient receptor potential melastatin 4 (TRPM4) channels. Opening of these Ca²⁺-activated, Na⁺-permeable cation channels causes arterial smooth muscle membrane depolarization, opening of voltage-dependent L-type Ca²⁺ channels, Ca²⁺ influx, and ultimately contraction (see Figure 6) (17–19). A more detailed explanation of these findings can be found in the accompany paper by Gonzales and Earley in this special issue of Microcirculation.

Figure 6. Localized IP₃ receptor-dependent SR Ca²⁺ release in vascular smooth muscle.

Ca²⁺-permeable inositol 1,4,5-trisphosphate receptors (IP3R) on the sarcoplasmic reticulum (SR) can produce non-propagating localized sites of elevated Ca²⁺. IP₃ receptor Ca²⁺ release events near the vascular smooth muscle cell plasma membrane (VSMC PM) increase the activity of plasmalemmal ion channels such as transient receptor potential melastatin 4 (TRPM4). Ca²⁺ puff-dependent activation of TRPM4 channels generates depolarizing (+ Vm) inward currents (via Na⁺ influx) resulting in increased opening of voltage-dependent Cav1.2 L-type Ca²⁺ channels (L) and increased contraction.

Calcium Sparklets

Ca²⁺ sparklets are distinct Ca²⁺ microdomains produced by plasmalemmal Ca²⁺-permeable channels. Despite the name, Ca²⁺ sparklets are not small Ca²⁺ sparks. Ca²⁺ sparklets arise from Ca²⁺ influx through plasmalemmal ion channels (see below) and (as discussed above) Ca²⁺ sparks arise from the release of Ca²⁺ from the sarcoplasmic reticulum through ryanodine receptors (see Figure 7).

Figure 7. Vascular smooth muscle L-type calcium channel calcium sparklets.

Cav1.2 L-type Ca²⁺ channels (L) in the vascular smooth muscle cell plasma membrane (VSMC PM) produce highly localized Ca²⁺ influx events (i.e., Ca²⁺ sparklets). Brief stochastic opening of single L-type Ca²⁺ channels produce low activity Ca²⁺ sparklet sites while prolonged opening of one or more clustered L-type Ca²⁺ channels produce high activity Ca²⁺ sparklet sites. Ca²⁺ influx from low and high activity Ca²⁺ sparklet sites contribute to global intracellular Ca²⁺ ([Ca²⁺]_i) resulting in contraction. Elevated Ca²⁺ concentrations at high activity Ca²⁺ sparklet sites (and perhaps global intracellular Ca²⁺ as well) contribute to changes in gene expression associated with increased contraction.

The original definition of a “Ca²⁺ sparklet” was a visualized Ca²⁺ influx event produced by a voltage-dependent L-type Ca²⁺ channel (60). Since that time, Ca²⁺ influx events produced by other Ca²⁺-permeable channels have been visualized and referred to as “Ca²⁺ sparklets” (e.g., (55). To remove ambiguity we suggest that that the term “Ca²⁺ sparklet” be refined to specifically designate the visualization of a Ca²⁺ influx event through a Ca²⁺-permeable plasmalemmal ion channel. The need for different terms denoting Ca²⁺ influx through different channels can then be eliminated by preceding “sparklets” with the name of the underlying channel (e.g., “L-type Ca²⁺ channel sparklets”, “TRPV4 sparklets”, etc…).

L-Type Calcium Channel Sparklets

Cav1.2 L-type Ca²⁺ channels are the main source of Ca²⁺ influx in vascular smooth muscle cells (39). Conventional electrophysiological recordings of steady-state L-type Ca²⁺ channel activity (50) provide no information with regard to potential spatial heterogeneity of L-type Ca²⁺ channel activity throughout the smooth muscle plasma membrane. However, when conventional electrophysiology and advanced Ca²⁺ imaging techniques (e.g., total internal reflection fluorescence (TIRF) microscopy) are used together this experimental limitation is overcome (41,60).

Using a combinatorial approach of electrophysiology and TIRF microscopy, Ca²⁺ influx through single L-type Ca²⁺ channels (i.e., L-type Ca²⁺ channel sparklets) is readily observed in smooth muscle cells isolated from rat and mouse cerebral and mesenteric arteries (2, 3, 40–42). Data from experiments using this approach have confirmed the importance of Cav1.2 channels in vascular smooth muscle and yielded unexpected information with regard to spatial organization and regulation of L-type Ca²⁺ channels (40, 41)). Previous conventional electrophysiological data suggested that, under steady-state conditions, Ca²⁺ influx in vascular smooth muscle cells was the end result of stochastic opening of L-type Ca²⁺ channels dispersed broadly throughout the plasma membrane (50). However, the exceptional temporal and spatial resolution provided by TIRF microscopy revealed that Ca²⁺ influx in vascular smooth muscle cells through L-type Ca²⁺ channels is not stochastic but rather segregates strikingly into sites of low and high activity (see Figure 7).

Consistent with conventional electrophysiological data, sites of low activity L-type Ca²⁺ channel influx arise from stochastic opening of randomly dispersed L-type Ca²⁺ channels. In contrast, sites of high activity L-type Ca²⁺ channel function result from non-stochastic, apparently coordinated opening of clustered channels (15, 43). High activity L-type Ca²⁺ channel sparklet sites require AKAP150-targeted kinase activity (i.e., PKC and PKA), account for approximately 50 % of the steady-state Ca²⁺ entry through L-type Ca²⁺ channels in isolated rat and mouse cerebral arterial smooth muscle cells. In addition, High activity L-type Ca²⁺ channel sparklets contribute to myogenic tone in mouse mesenteric arteries and are necessary for contractile responses of mouse mesenteric and rat cerebral arteries to angiotensin II (3, 11, 44, 45, 47). Microdomains of elevated Ca²⁺ generated by high-activity L-type Ca²⁺ channel sparklet sites also induce changes in gene expression via specific activation of the Ca²⁺/calcineurin/NFAT signaling pathway (47, 52, 53). A more detailed explanation of these concepts can be found in the accompany paper by Navedo and Amberg in this special issue of Microcirculation.

Other Considerations and Future Directions

The Ca²⁺ signals described above are defined by the molecular mechanisms underlying their initiation and are classified as different “less than global” Ca²⁺ events with unique properties. While mechanisms underlying Ca²⁺ entry into vascular smooth muscle cytosol clearly distinguish one Ca²⁺ signal from another, termination of these events is also critical. Ca²⁺ signals in vascular smooth muscle are terminated by no less than five distinct mechanisms: 1) Diffusion into the surrounding cytosol, 2) extrusion via the Na⁺/Ca²⁺ exchanger, 3) extrusion via the plasma membrane Ca²⁺-ATPase, 4) sequestration via the sarcoplasmic reticular Ca²⁺-ATPase (SERCA), and 5) sequestration and redistribution via mitochondria. Detailed discussion of these important mechanisms in regulating Ca²⁺ signaling in vascular smooth muscle is beyond the scope of this review. However, these mechanisms clearly influence the vascular smooth muscle Ca²⁺ dynamics. For example, mitochondria are known to be important modulators of IP₃-dependent Ca²⁺ signaling (37), the Na⁺/Ca²⁺ exchanger regulates L-type Ca²⁺ channel function (68), and sarcoplasmic reticular Ca²⁺-ATPase function influences ryanodine receptor-dependent Ca²⁺ sparks (13, 61).

Three major areas of future research are necessary to further our understanding of Ca²⁺ dynamics in vascular smooth muscle. First, cryptic Ca²⁺ signals such as Ca²⁺ puffs (see above) need to be visualized directly. Similarly, novel, discrete Ca²⁺ signals associated with store operated Ca²⁺ entry (SOCE) are in need of identification and characterization. Second, the relationship between local Ca²⁺ signals (e.g., Ca²⁺ waves and Ca²⁺ sparklets) and global Ca²⁺ needs to be clarified. Third and finally, future investigations should explore Ca²⁺ dynamics with respect to mechanisms regulating intracellular and intercellular signaling in the intact vascular syncytium. Experiments along these lines are necessary not only to assess the validity of experiments performed on isolated smooth muscle cells, but also to examine the influence of other cell types (i.e., endothelial cells) on Ca²⁺ signaling in vascular smooth muscle. Advances in Ca²⁺ imaging techniques such as high-speed confocal microscopy and availability of cell-specific Ca²⁺ indicators are already and will continue to be critical in advancing these areas of research.