INTRODUCTION

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THE UBIQUITOUS ROLE of cellular Ca²⁺ signaling depends on its complexity and variability. Why do changes in the intracellular concentration of one ion, Ca²⁺, selectively trigger different responses in the same cell dependent on the nature of the stimulus, and why does the same stimulus elicit different Ca²⁺-mediated responses in smooth muscle cells located at different sites? The answers to these questions lie in the temporal fluctuations and spatial variations of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), which are dependent on the interactions of ion transport proteins located in the plasma membrane (PM) and membranes of the sarcoplasmic reticulum (SR), nuclear envelope, and mitochondria. These interactions are either transient or steady-state processes that prevent ionic concentrations from reaching equilibrium. In general, a cytoplasmic Ca²⁺ gradient results from the separation of a Ca²⁺ source (channel supplying Ca²⁺ from the extracellular space or organelle) from a Ca²⁺ sink (Ca²⁺ pump or exchanger). As discussed in more detail below, the degree of this separation varies from close alignment causing the two transport molecules to function as a unit to distances of several hundred nanometers. Recent evidence indicates that the cytoskeleton plays an essential role in maintaining the functional alignments among the PM, SR, and mitochondria. The resulting microstructural arrangements of the apposing membranes create diffusional barriers defining different types of junctional spaces within the cytoplasm. As we will discuss throughout this review, these junctional spaces appear to play important roles in both active and resting state Ca²⁺ cycling. The diffusional limitations of these junctional spaces allow for accumulation of ions such as Na⁺ and Ca²⁺ in concentrations greatly exceeding that in the bulk cytoplasm. These cytoplasmic microdomains have important functional implications. For instance, Ca²⁺-sensitive ion channels selective for K⁺, Cl, and Ca²⁺ and Ca²⁺-sensitive enzymes, such as protein kinase C and phospholipase C, which are located in membranes bordering the restricted space between the PM and superficial SR, can be regulated separately from the myofilaments occupying the bulk of the cytoplasm. The high [Ca²⁺] of the junctional space ([Ca²⁺]_js) also enables rapid and selective transport of Ca²⁺ between different organelles and the extracellular space with little disturbance to the bulk cytoplasm. More importantly, we will illustrate how the localization of different channels, transporters, and Ca²⁺-sensitive enzymes to this sheltered microenvironment of the junctional region can lead to functionally distinct junctional complexes that underlie the observed vascular heterogeneity in [Ca²⁺]_i signaling. In addition to spatial variations in [Ca²⁺]_i, temporal variations in the Ca²⁺ signal, such as waves and oscillations, may selectively encode messages for frequency-sensitive processes in the nucleus and other cellular spaces. In principle, it is therefore possible for Ca²⁺ to regulate the processes of contraction, relaxation, growth, migration, and matrix synthesis independently of each other.

The purpose of this review is to examine the evidence for the above general hypotheses and describe in detail how oscillating Ca²⁺ waves are generated and maintained during activation of vascular smooth muscle and how Ca²⁺ cycles through the smooth muscle cell during quiescent periods.

	[CA2+]i OSCILLATIONS

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Agonist-induced [Ca²⁺]_i oscillations constitute a ubiquitous intracellular signaling mechanism in both excitable and nonexcitable cells. Although all smooth muscle [Ca²⁺]_i oscillations depend on PM-SR interactions, there are two fundamentally different types of [Ca²⁺]_i oscillations, depending on their immediate source of Ca²⁺. When Ca²⁺ used to generate each spike is derived directly from intermittent influx across the PM, it typically appears as a uniform rise in [Ca²⁺]_i throughout the cell (55), even though Ca²⁺-induced Ca²⁺ release (CICR) may be involved to amplify the Ca²⁺ signal. In this case, where [Ca²⁺]_i rises more or less evenly across the entire cell, no apparent Ca²⁺ waves are observed. In contrast, when the endoplasmic reticulum (ER)/SR is the immediate Ca²⁺ source for each Ca²⁺ spike, [Ca²⁺]_i initially rises in a specific cellular locus, and this regional elevation in [Ca²⁺]_i propagates in a wavelike fashion throughout the length of the cell (39). In vascular smooth muscle cells, both non-wavelike and wavelike [Ca²⁺]_i oscillations are observed.

	SYNCHRONIZED NON-WAVELIKE [CA2+]i OSCILLATIONS

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The non-wavelike [Ca²⁺]_i oscillations are commonly observed in vascular smooth muscle cells (VSMCs) of small resistance arteries and arterioles and typically display a frequency of ~0.05 and ~0.1 Hz, respectively (3, 42, 55, 75). The Ca²⁺ spikes are driven by periodic depolarizations stimulating the opening of L-type, voltage-gated Ca²⁺ channels (VGCCs). Because the smooth muscle cells are electrically coupled via gap junctions, these non-wavelike oscillations in [Ca²⁺]_i are synchronized between neighboring VSMCs and have the functional role of signaling force oscillations or vasomotion in the small arteries and arterioles. The oscillations in force follow the oscillations in [Ca²⁺]_i with a delay of ~300 ms, the time required for phosphorylation of myosin light chain (MLC) and the initiation of cross-bridge cycling. Unfortunately, the mechanism underlying the generation of the synchronized oscillations of VSMC membrane potential has not been definitively elucidated. Thus far, Ca²⁺-activated K⁺ channels (K_Ca), Ca²⁺-activated Cl channels (Cl_Ca), endothelium, and SR have all been implicated. Recently, Peng et al. (55) proposed the intriguing hypothesis that synchronized [Ca²⁺]_i oscillations may be initiated by asynchronous Ca²⁺ waves, which are discussed below.

	ASYNCHRONOUS WAVELIKE [CA2+]i OSCILLATIONS

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Since the first report by Iino and co-workers in 1994 (27), asynchronous wavelike [Ca²⁺]_i oscillations have emerged as a common mode of Ca²⁺ signaling in in situ VSMCs (Table 1). In these isolated blood vessel preparations, confocal microscopy of intracellular Ca²⁺-sensitive dyes reveals recurrent intracellular Ca²⁺ waves traveling through the longitudinal axis of the ribbon-shaped VSMCs. These Ca²⁺ waves, which are usually but not always initiated by agonists, result from SR Ca²⁺ release and do not propagate between cells. Because the mechanism of the repetitive Ca²⁺ waves depends on interaction of the peripheral SR with the PM, we will first examine the ultrastructural evidence.

	ULTRASTRUCTURE OF VSMC

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The SR is composed of an interconnected tubular and sheetlike network, the membranes of which bound the SR lumen. It extends throughout the spindle-shaped VSMC and is contiguous with the nuclear envelope (64). The SR contributes to Ca²⁺-signaling by virtue of active Ca²⁺ transport via sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) from the cytoplasm to the SR lumen and Ca²⁺ release from the SR into the cytoplasm via inositol 1,4,5-trisphosphate (InsP₃)-sensitive SR Ca²⁺ release channels (IP₃R) and ryanodine-sensitive SR Ca²⁺ release channels (RyR). Its capacity for Ca²⁺ storage is greatly enhanced by the high-capacity, low-affinity Ca²⁺-binding proteins calsequestrin (57) and calreticulin (44). The SR has been classified according to its location as superficial or deep, with distinct functions being ascribed to the superficial SR. In domains where the SR apposes the PM, it creates a narrow space that extends on average in two dimensions for about 300-400 nm and has a depth of about 15-20 nm. The structures responsible for this spacing have not yet been identified, although in some instances "feet" similar to those seen in triadic junctions in skeletal muscle have been reported (64), and proteins called "junctophillins" have been isolated from the diads of cardiac muscle (67). The narrow cytoplasmic space between the junctional SR and PM is referred to as the PM-SR junctional space and is thought to present an imperfect barrier to diffusion of small molecules and ions, in particular, Ca²⁺ and Na⁺. As can be seen from the electron micrographs of serial sections of smooth muscle of the inferior vena cava of the rabbit and the schematic three-dimensional interpretations in Fig. 1, the caveolae are able to perforate the junctional SR sheet such that their apexes frequently remain in contact with the bulk cytoplasm. As shown in Fig. 1, E-G, the apexes of the caveolae oftentimes are close to the perpendicular or radial SR sheets that appear to arise from the superficial SR sheets. Because of these varying geometric arrangements, it is plausible that different membrane domains perform specialized functions. For instance, the L-type VGCCs that supply the majority of the Ca²⁺ required for activation of actomyosin in small resistance arteries may be located at the apexes of the caveolae. This would allow entering Ca²⁺ to bypass the PM-SR junction and to access the deeper cytoplasm where the myofilaments are located. In contrast, channels such as the store-operated channels (SOC), which function in refilling the SR, are most probably located in the PM-SR junctional complex. Ca²⁺ influx mediated by the SOC could thus be selectively directed into the PM-SR junctional space from which SERCA on the apposing SR membranes could efficiently take up Ca²⁺ into the SR lumen with minimal influence on the bulk [Ca²⁺]_i. Furthermore, it is important to note that the periphery of the VSMC is typically very low in density of myosin polymers as shown by the electron micrograph picture in Fig. 2. This includes the PM-SR junctional space, which is completely myosin free and a peripheral zone of ~200-300 nm in width here referred to as the myosin-poor space. If we assume that the VSMC is a cylindrical tube with a diameter of 3 µm and between 10 and 15% of its surface is closely apposed by the superficial SR, the PM-SR junctional space constitutes ~0.3% of the overall cellular volume of the VSMC. In comparison, the myosin-poor space would constitute between 25 and 36% of the total cellular volume.

View larger version (90K): [in this window] [in a new window]   Fig. 1.   Ultrastructure of vascular smooth muscle of the rabbit inferior vena cava (IVC) revealed with electron microscopy. Serial cross-sections (electron micrograph) of vascular smooth muscle cells (VSMC) are shown in series 1 (A-D) and series 2 (E-G). Series 1 illustrates the close spatial apposition between the superficial sarcoplasmic reticulum (SR) sheet and the plasmalemma (PM) with the apexes of the caveolae perforating through the superficial SR sheets to come into contact with the bulk cytoplasm. Membranes of the PM (dotted line) and the SR (solid line) in A-D are outlined to the right of the respective panels. Close apposition among the superficial SR sheet, the PM, and the neck region of the caveolae creates a narrow and expansive restricted space. Series 2 (E and F) illustrates the perpendicular sheets of SR, which appear to arise from the superficial SR sheets. Mitochondria also come into close contact with the perpendicular SR sheets. H: a stylized illustration of the close association between the superficial SR sheet, which is continuous with the perpendicular sheet, the perforating caveolae (C), the PM, and a mitochondrion (M). I: calyculin-A (100 nM)-mediated dissociation of the superficial SR sheets from the PM (see arrows). Solid scale bars indicated represents 200 nm of distance. [Adapted from Lee et al. (39)].

View larger version (130K): [in this window] [in a new window]   Fig. 2.   Myosin-poor space and myosin-rich myoplasm in VSMC of the rabbit inferior vena cava. Electron micrograph image of the VSMC shows the position of myosin filaments (indicated by arrows) in relation to the PM. It reveals a peripheral zone ~200-300 nm in width that possesses nearly no myosin filaments. This is referred to as the myosin-poor space, which separates the PM and the myosin-rich myoplasm. Solid scale bar indicated represents 200 nm of distance.

As mentioned above, the junctional SR is connected to sheets of radial SR (Fig. 1, E-G), which extend from the PM through the myosin-poor space into the deeper myoplasm. In theory, these perpendicular/radial SR sheets may provide an effective conduit for Ca²⁺ to access the myosin-rich myosplasm for efficient activation of the myofilaments. In this context it is important to note that calmodulin is bound to the MLC kinase (MLCK), which is in turn tethered to the thin filaments and therefore in the appropriate place for direct activation by Ca²⁺ (72).

In addition to forming close contacts with the PM, the SR network also comes into close contact with the mitochondria (MT) (53, 58), forming yet another diffusionally restricted space, referred to as the MT-SR junctional space (Fig. 1, E-G). This <80-nm wide space, sandwiched between the SR and mitochondrial membranes, also appears to be functionally important. As the SR network penetrates deeper into the cell, it inserts into the nuclear membrane such that the lumen of the perinuclear SR network is continuous with the lumen of the nuclear envelope (64).

	CA2+ WAVES AND VASOCONSTRICTION

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The main function of vascular smooth muscle is to distribute blood flow through selective vasoconstriction and vasomotion. The latter is clearly associated with oscillations in [Ca²⁺]_i, but it was long thought that tonic contraction was initiated by SR Ca²⁺ release and maintained by elevated Ca²⁺ influx. However, confocal microscopy of intact blood vessels loaded with Ca²⁺-sensitive dyes has shown that in many blood vessels agonist-induced contractions are maintained by asynchronous wavelike [Ca²⁺]_i oscillations in single smooth muscle cells (Fig. 3, A and B), which summate to give a steady-state elevation in [Ca²⁺]_i for the whole tissue (60). Figure 3C relates the concentration of the -adrenergic agonist phenylephrine (PE) to force developed by the inferior vena cava (IVC) of the rabbit and to various parameters of the [Ca²⁺]_i oscillations. It is clear that at the lower agonist concentrations, force depends on the number of cells recruited to generate fixed amplitude [Ca²⁺]_i oscillations, whereas at higher concentrations, force is regulated by the frequency of the oscillations and perhaps also by the velocity of the recurring Ca²⁺ waves. In addition to causing tonic contraction, low-frequency (<0.05 Hz) asynchronous wavelike [Ca²⁺]_i oscillations, which themselves are associated with only minimal development of tone, appear to be instrumental in the initiation of vasomotion in the rat mesenteric artery (55). In this proposed scenario, asynchronous Ca²⁺ waves can activate an unknown depolarizing current through the plasma membrane of each VSMC. The activation of such depolarizing current can by chance occur synchronously in a sufficiently high fraction of VSMCs within the artery to entrain cycles of membrane depolarization and repolarization in electrically coupled VSMCs. The resulting synchronized, but intermittent activation, of L-type VGCC in all VSMCs subsequently produces synchronized non-wavelike [Ca²⁺]_i oscillations that underlie vasomotion.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Phenylephrine (PE)-mediated asynchronous wavelike cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations and contraction in the rabbit IVC. A: [Ca2+]i oscillations recorded in neighboring smooth muscle cells within the intact vessel are not synchronized between cells as they each display different frequency of oscillations. B: individual Ca2+ spikes in PE-mediated [Ca2+]i oscillations are wavelike as different regions (1, 2, and 3) in the same ribbon-shaped VSMC experience sequential rises of [Ca2+] in time. C: [PE] dependence in force generation compared with the [PE] dependence in the percent recruitment of cells, the amplitude of the [Ca2+]i oscillations, the frequency of the [Ca2+]i oscillations, and the apparent velocity of the recurring Ca2+ waves. [Adapted from Lee et al. (38) and Ruehlmann et al. (60)].

	CA2+ WAVES AND VASODILATATION

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Whereas Ca²⁺ waves can stimulate smooth muscle contraction in large vessels such as the rabbit IVC, similar Ca²⁺ waves have also been associated with the induction of dilatation of cerebral resistance arteries. In this case, the wavelike Ca²⁺ release is thought to stimulate K_Ca on the PM, such that the relaxing effect of the resulting hyperpolarization-induced closing of VGCC outweighs the local stimulation of contraction (29). In accordance with this finding, activation of K_Ca has been reported following Ca²⁺ waves in smooth muscle cells (76). Mechanistically, it is also important to point out that these recurrent Ca²⁺ waves are triggered by elevated [Ca²⁺]_i that results from the maintained opening of the L-type VGCC. The higher surrounding [Ca²⁺]_i sensitizes the RyR to CICR, which initiates the Ca²⁺ waves repetitively (46). This is why diltiazem can completely abolish the occurrence of the recurring Ca²⁺ waves in the rat cerebral resistance artery (Table 1), even though the Ca²⁺ waves are initiated and produced by SR Ca²⁺ release. In rat cerebral resistance vessel, the stimulus sensitizing the SR Ca²⁺ release channels to initiate Ca²⁺ waves is Ca²⁺ rather than InsP₃ as in the case of PE-stimulated rabbit IVC.

This dual function of Ca²⁺ waves provides an intriguing example of vascular heterogeneity. How does apparently the same Ca²⁺ wave elicit contraction in one VSMC type and induce relaxation in another? The answer to this question may reside in the molecular makeup of the PM-SR junctional complex. We have shown previously (51) that blockade of K_Ca did not affect contractions of the rabbit inferior vena cava. This could be due to a lack of expression of these channels in this tissue; however, unpublished data from our laboratory indicate active large conductance K_Ca (BK_Ca) mRNA expression by the VSMC of the rabbit IVC. Alternatively, given that the activation of BK_Ca requires high [Ca²⁺]_i that can be achieved in the PM-SR junctional space, it is also possible that the BK_Ca in the rabbit IVC is not located in the PM-SR junctional complex and therefore not activated by each passing Ca²⁺ wave. In contrast, the cerebral resistance artery relies heavily on BK_Ca activation for the maintenance of membrane potential and regulation of pressure-induced myogenic tone (22, 32, 52). Because of the low Ca²⁺ affinity of BK_Ca, their open probability is regulated by the spontaneous opening of clusters of RyR releasing Ca²⁺ sparks near the PM (5, 31). Recent calculations place the BK_Ca within 20 nm of the RyR, making it likely that in the resistance arteries, both types of channels are localized within the PM-SR junctional complex (56). With this particular PM-SR junctional complex in the cerebral resistance artery containing BK_Ca, each Ca²⁺ wave would induce membrane hyperpolarization, which in turn would inhibit the opening of the L-type VGCC and induce relaxation. We speculate that L-type VGCC must be located in the apexes of the caveolae away from the PM-SR junction, such that the activating Ca²⁺ current would be delivered to the myoplasm. As Ca²⁺ originating from VGCC diffuses from the PM to the deeper myoplasm, the signal could be attenuated by SERCA located in the peripheral SR, but outside the junctional complex, or amplified by CICR. Evidence suggests that in vascular smooth muscle the peripheral SR functions as a superficial buffer barrier rather than amplifying through CICR. Interestingly, in bladder myocytes, where there is direct evidence for CICR (14), the coupling between VGCC and RyR has been found to be of the "loose" type in support of the idea that the VGCC are not part of the PM-SR junctional complexes (6). This is further supported by the structural finding that the L-type VGCC is localized on the caveolin-rich portion of the PM (8). The concept of two different PM-SR junctional complexes having opposite effects on smooth muscle contractility is illustrated in Fig. 5. It is important to note that the interaction illustrated between the RyR and the BK_Ca represents only one aspect of the regulation of the membrane potential by the SR. Given the recent findings that revealed an inhibitory regulation by type 3 RyR on Ca release by other RyR isoforms (41), it is likely that there are more complex interactions between different isoforms of RyR and BK_Ca in these junctional regions.

	MECHANISM OF WAVELIKE [CA2+]i OSCILLATIONS

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Although rhythmic events have been observed in VSM for many decades, their molecular mechanisms remain to be fully elucidated. One possible reason for our limited understanding is that blood vessels display different types of rhythmic activity, which impedes consensus between different laboratories. At the present time, the molecular basis for agonist-induced [Ca²⁺]_i oscillations has been resolved in some detail for the smooth muscle of the inferior vena cava of the rabbit (39, 61), which may therefore serve as a basis for comparison with other types of blood vessels. As shown in Table 1 and Fig. 4A, SR store depletion by SERCA blockade can inhibit the wavelike [Ca²⁺]_i oscillations, indicating the importance of intermittent SR-mediated Ca²⁺ release. In the case of the rabbit IVC, waves of SR-mediated Ca²⁺ release begin with the opening of IP₃R because they are prevented or instantly blocked by concentrations of 2-aminoethoxydiphenyl borate (2-APB), which block IP₃R (Fig. 4B). As some of the released Ca²⁺ is extruded to the extracellular space, recurrence of the Ca²⁺ waves depends on replenishment of the SR by stimulated Ca²⁺ influx. This occurs through three different pathways: L-type VGCC, nonselective cation channels (NSCC), and the Na⁺/Ca²⁺ exchanger (NCX) in its reverse mode. In this large vein, the VGCCs play a relatively minor role because nifedipine will only decrease the frequency of the oscillations (Fig. 4C) and inhibit contraction by 27%. Blockade of the NCX eliminates the oscillations (Fig. 4D), leaving a slightly elevated [Ca²⁺]_i, which can be reduced to baseline value by the receptor-operated channel/SOC (ROC/SOC) blocker SKF-96365 (Fig. 4D) and also by 2-APB (Fig. 4B). These observations led to postulation of the following sequence of events, illustrated in Fig. 5A: 1) PE activates phospholipase C, which catalyzes the synthesis of InsP₃; 2) activation of IP₃R and Ca²⁺ release from the SR near calmodulin tethered to the thin filaments; 3) opening of NSCC in the plasma membrane and influx of mainly Na⁺ and some Ca²⁺ into the PM-SR junctional space; 4) depolarization, opening of the L-type VGCC, and reversal of NCX resulting in Ca²⁺ influx; and 5) Ca²⁺ uptake into the SR by SERCA. The identity and the mode of activation of the NSCC are not yet resolved. The PM-SR junction complexes are likely the sites for interactions among NSCC, NCX, and SERCA during SR refilling and are thus crucial for the occurrence of the recurring Ca²⁺ waves. In addition, the low Na⁺-affinity Na-K-ATPase isoforms ₂ and ₃ have been localized to the junctional PM (33), which would promote elevated junctional [Na⁺] and reversal of the NCX (1). The importance of the PM-SR junctional complex in refilling the SR was confirmed by the finding that dissociation of the superficial SR sheets from the PM by calyculin-A inhibits maintenance of the agonist-induced wavelike [Ca²⁺]_i oscillations (Fig. 1I, arrows) (38). In addition, the rabbit IVC does express Trp1 (unpublished results), which has been shown to constitute a NSCC and to be activated by SR Ca²⁺ release (74, 78), but direct evidence for its participation in the above events requires further experimentation with knockout or antisense techniques. It is of interest to note that oscillatory inward nonselective cationic current has been described in endothelin-stimulated rat aorta, a large vessel that exhibits asynchronous wavelike [Ca²⁺]_i oscillations as well (61).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Mechanism of PE-mediated wavelike [Ca2+]i oscillations in the rabbit inferior vena cava. A: PE-mediated [Ca2+]i oscillations are completely inhibited by 10 µM cyclopiazonic acid (CPA), but the average [Ca2+]i remains elevated. B: PE-mediated [Ca2+]i oscillations are abolished by 75 µM 2-aminoethoxydiphenyl borate (2-APB). C: application of 10 µM nifedipine (Nif) reduced the frequency of PE-mediated [Ca2+]i oscillations while additional application of SKF-96365 (SKF) completely abolished the remaining [Ca2+]i oscillations. D: application of 100 µM 2,4-dichlorobenzamil (2,4-DCB) completely inhibited Nif-resistant, PE-induced [Ca2+]i oscillations and lowered the [Ca2+]i to a level that is slightly higher than baseline. Additional application of SKF-96365 returned the [Ca2+]i level to baseline. [Adapted from Lee et al. (38).]

View larger version (62K): [in this window] [in a new window]   Fig. 5.   Model for Ca2+ movements during wavelike [Ca2+]i oscillations and during resting state in VSMC. A: SR is composed of double-membrane sheets separating its continuous lumen from the cytoplasm. The portion of the SR that is closely apposed to the PM is called junctional SR (jSR). The jSR is separated from the PM by 15-20 nm, which creates an irregular narrow PM-SR junctional space with a "diameter" of ~300 nm. Caveolae tend to perforate the jSR, such that the PM-SR junctional space includes the cytoplasm between the necks of the caveolae and the jSR and the tips of the caveolae face the peripheral cytoplasm. The peripheral cytoplasm has a low density of myosin filaments with average density reached ~200-300 nm from the PM. This myosin-poor space, which does not support contractile force development, comprises about 25~36% of the total cellular space. PM-SR junctional space is much smaller, comprising 0.3% of the cell volume (see text for calculations). Data obtained in the IVC of the rabbit support the model where during -adrenergic stimulation Ca2+ is transiently released from the radial SR through inositol 1,4,5 trisphosphate (InsP3)-sensitive SR Ca2+ release channel (IP3R) near the calmodulins tethered to the myofilaments. Subsequent depletion of the SR, which may be augmented by mitochondrial Ca2+ uptake across the mitochondria (Mito)-SR junctional space, opens store-operated channels (SOC) in the PM-SR junction, allowing thousands of Na+ and some Ca2+ to enter the PM-SR junctional space. Na+ entry depolarizes the membrane to activate voltage-gated Ca2+ channels (VGCC) and drives the Na-Ca exchanger (NCX) backward to supply Ca2+ from the extracellular space to junctional sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) for refilling of the SR. As the SR is replenished, the InsP3-sensitized IP3R become once again activated by the locally raised [Ca2+]i to start the next wave of regenerative Ca2+ release through IP3R. [Na+] of junctional space ([Na+]js) is elevated at least transiently because diffusion from the PM-SR junctional space is restricted, and the Na+-K+-ATPase isoform localized to the PM-SR junction is of the low Na+ affinity 2- or 3-type. The jSR release channels would be inactivated by the high [Ca2+] of junctional space ([Ca2+]js) seen during activation. Such a junctional complex composed of the SOC, the NCX, and SERCA serves to facilitate smooth muscle contraction in the rabbit IVC. NCSS, nonselective cationic channel; VGCC, L-type voltage-gated Ca2+ channel. B: in contrast, a different junctional complex composed of the ryanodine-sensitive SR Ca2+ release channel (RyR), the SERCA, and the large conductance Ca2+-activated K+ channel (BKCa) functions to relax VSMC in the rat cerebral resistance artery. Recurring Ca2+ waves mediated by the RyR can elevate the [Ca2+] in the PM-SR junctional space sufficiently high to activate KCa, leading to hyperpolarization of the membrane potential and inhibition of the L-type VGCC.

The constant amplitude of the propagated Ca²⁺ wave indicates regenerative transient Ca²⁺ release from the SR. The regenerative nature depends on the positive feedback of increasing [Ca²⁺]_i on the InsP₃ sensitivity of IP₃R and possibly recruitment of Ca²⁺-sensitive RyR. The propagation is due to elevation of [Ca²⁺]_i to the threshold for activation of clusters of release channels in adjacent portions of the SR. In some smooth muscle, the threshold value has been observed as the inflection point between a "foot" segment and the steep portion of the upstroke of [Ca²⁺]_i elevation. The relative involvement of IP₃R and RyR appears to vary between different smooth muscle preparations. The delayed negative feedback on release, which is essential for oscillatory behavior, has been ascribed to a number of mechanisms: 1) inhibition of IP₃R type 1 isoform by high [Ca²⁺]_i (28, 62) or inhibition of RyR by adaptation/inactivation mechanisms (37, 63), 2) inhibition of IP₃R by low luminal SR Ca²⁺ (47), and 3) time-dependent inactivation of both IP₃R and RyR (21). The latter mechanism would imply a maximal limit to the frequency of the wavelike [Ca²⁺]_i oscillations because it requires time for the channels in the inactivated state to return to the closed resting state. This is supported by the observation that [Ca²⁺]_i oscillations in the rabbit IVC appear to peak at a frequency of ~0.5 Hz regardless of further increases in the agonist concentration (60).

As the SR takes up Ca²⁺ from the surrounding cytoplasm, the store-operated NSCC close, and, as a result of repolarization, the VGCC also close. Stimulation of SERCA, PM-Ca²⁺-ATPase, and possibly forward NCX by elevated [Ca²⁺]_i while release terminates may be responsible for the downstroke of the PE-induced [Ca²⁺]_i oscillation (50). In this context, it would be interesting to obtain evidence for NCX reversal during each cycle. The next Ca²⁺ wave may start at the frequent discharge sites (16, 17) when the SR luminal Ca²⁺ has been recharged and spillover from the SR raises the local [Ca²⁺]_i to threshold once again. The observation that waves tend to originate from the same area within the cell has been explained by the observation that such sites posses a higher local density of SR near PM Ca²⁺ channels and are devoid of mitochondria (17).

A conceptually simpler model of [Ca²⁺]_i oscillations is one where the intracellular InsP₃ concentration ([InsP₃]_i) oscillates (25). The delayed negative feedback in this case is Ca²⁺ activation of protein kinase C, which then inhibits phospholipase C. However, it is doubtful that the lower frequency [InsP₃]_i oscillations can be responsible for the high-frequency (~0.5 Hz) [Ca²⁺]_i oscillations observed in these VSMCs.

The apparent complexity of the mechanism of repetitive Ca²⁺ waves leaves many questions unanswered. For example, if the [InsP₃]_i sets the Ca²⁺ sensitivity of the IP₃R, and if this is one of the determinants of the threshold for Ca²⁺ release, then why are the amplitudes constant with increasing PE concentration? Perhaps RyR also plays a role in wave propagation (4). Many aspects of RyR regulation during [Ca²⁺]_i oscillations are as yet unknown. For example, Ca²⁺ is not the only stimulus because cADP-ribose and nicotinic acid-adenine dinucleotide phosphate have also been shown to activate these channels (40, 77). Thus far we have no information on whether the concentrations of these mediators are elevated or oscillate.

	POSSIBLE ADVANTAGE OF WAVELIKE [CA2+]i OSCILLATIONS

TOP ABSTRACT INTRODUCTION [CA2+]i OSCILLATIONS SYNCHRONIZED NON-WAVELIKE [CA2+... ASYNCHRONOUS WAVELIKE [CA2+]i... ULTRASTRUCTURE OF VSMC CA2+ WAVES AND VASOCONSTRICTION CA2+ WAVES AND VASODILATATION MECHANISM OF WAVELIKE [CA2+]i... POSSIBLE ADVANTAGE OF WAVELIKE... CA2+ CYCLING DURING RESTING... MITOCHONDRIAL CA2+ TRANSPORT... NUCLEAR CA2+ SIGNALING CONCLUSION REFERENCES

It is clear that the additive effect of asynchronous Ca²⁺ waves is a steady-state rise in the average tissue [Ca²⁺]_i, but is there a specific functional advantage to the oscillatory pattern of Ca²⁺ release? Several functions have been proposed: 1) several enzymes have been shown to respond to frequency of Ca²⁺ oscillations (see below); 2) maintenance of high [Ca²⁺]_i may be damaging for the cell; 3) if the Ca²⁺ sensor, in this case calmodulin, has a relatively fast Ca²⁺ on rate and a slow off rate, then the peak of the oscillation is the effective signal and signaling can be achieved at a lower average [Ca²⁺]_i; and 4) SR Ca²⁺ release may be more effective in activating the contractile elements. The latter hypothesis was mentioned in the section on smooth muscle ultrastructure. It is clear that each wave depends on SR Ca²⁺ release into the myoplasm, whereas Ca²⁺ influx mainly serves to refill the SR. If the release occurs largely from the radial SR into the myosin-rich space, where the calmodulin involved in activation of MLCK is localized (72), the flow of Ca²⁺ would then bypass the myosin-poor or noncontractile space via the lumen of the radial SR and be more effective in local activation of the myofilaments. Consistent with this postulate is the observation that the entire ER is one continuous Ca²⁺ pool and Ca²⁺ can rapidly move and distribute inside the lumen of the ER (54). Blocking SERCA would disrupt this process, allowing accumulation of Ca²⁺ in the myosin-poor space and decrease the effectiveness of increase in average [Ca²⁺]_i in stimulating contraction. This conclusion is supported by numerous reports that SERCA blockade decreases the ratio of force to [Ca²⁺]_i (34, 68).

	CA2+ CYCLING DURING RESTING STATE

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Even in the resting state, Ca²⁺ enters the smooth muscle cells and cycles through intracellular organelles. The mechanisms involved in the resting Ca²⁺ cycle differ from those seen during stimulated Ca²⁺ oscillations because their function is to sustain low [Ca²⁺]_i essential for cellular homeostasis. The basal rate of Ca²⁺ influx into smooth muscle has been estimated to be ~32 µmol of Ca²⁺ per liter per minute, which is equivalent to >300 times the resting [Ca²⁺]_i per minute (43). This raises the following questions: 1) how does so much Ca²⁺ enter unstimulated cells, 2) how is this Ca²⁺ distributed within the cell, and 3) how is it extruded to maintain resting levels of [Ca²⁺]_i and vascular tone? It is also unknown whether basal Ca²⁺ influx via the leak pathway exhibits variations in magnitude in different regions of the PM. It is becoming clear that the PM is made up of a patchwork of domains, some covered by dense bodies where the myofilaments are attached, some apposed by the superficial SR, some densily covered by caveolaes, and the remainder simply facing the bulk cytoplasm. It would be interesting to determine the relative sizes of these domains for various types of smooth muscle and relate them to specific functional properties.

Resting Ca²⁺ influx is attributed partly to a certain level of the open probability of excitable channels, including ROC, SOC, and VGCC. Some SOC may stay open due to the basal level of InsP₃, whereas the resting membrane potential of vascular smooth muscle of resistance arteries allows for a certain degree of activation of the Ca²⁺ window current (15, 52, 73). Additionally, a nonspecific influx of Ca²⁺, referred to as the calcium leak, substantially contributes to basal influx; however, its precise mechanism remains elusive (71).

The resting [Ca²⁺]_i is ~80 nM and is maintained at that low level despite the fact that every half second the equivalent of the total free cytosolic Ca²⁺ enters the cells. Two mechanisms protect the cell from drastic changes in [Ca²⁺]_i as a result of Ca²⁺ entry: 1) the presence of fixed and diffusible Ca²⁺-binding sites in the cytoplasm and 2) sequestration by SERCA in the SR. It has been shown in several types of vascular smooth muscle that stimulated influx is more effective in raising Ca²⁺ concentration near the myofilaments when Ca²⁺ uptake in the SR is inhibited by either blockade of SERCA or opening of release channels (69). Thus, in the resting smooth muscle, the peripheral SR takes up Ca²⁺ entering the cells before it can equilibrate with the deeper myoplasm (Fig. 6). In the rabbit IVC, continuous unloading of the superficial SR to the extracellular space, involving coupling of SR release channels to the forward mode NCX, permits buffering of Ca²⁺ influx to continue (51). Such spontaneous and preferential unloading of the SR toward the PM has also been observed in the bovine coronary artery (65). In support of this postulate, Fig. 6 shows that Na⁺ removal from the extracellular space inhibits the loss of Ca²⁺ from the SR, whereas returning Na⁺ leads to a rapid transfer of Ca²⁺ from the SR to the extracellular space. This cyclical process of Ca²⁺ buffering and unloading has been referred to as the "superficial buffer barrier" (69, 70). Both the superficial SR and the perpendicular/radial SR traversing through the peripheral myosin-poor space function as the superficial buffer barrier, sequestering Ca²⁺ before it reaches the deep myosin-rich myoplasm. The definition of the superficial SR thus includes the junctional SR and sheets of SR within ~200-300 nm of the PM.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Maintenance of the "superficial buffer barrier" depends on NCX-assisted Ca2+ transport from the SR lumen to the extracellular space. A: rate of loss of SR Ca2+ content, measured as a caffeine transient, into Ca2+-free perfusate at room temperature in the rabbit inferior vena cava. B: rate of decline in [Ca2+]i from an elevated level, measured as the fura 2 fluorescence ratio, into Ca2+-free superfusate, which is either Na+ free or contains 10 µM CPA or is Na+ free and contains CPA. C: representation of a model for maintained buffering by the superficial SR of Ca2+ entry. Structural arrangement of the superficial SR depicted in the figure also allows it to function as the superficial buffer barrier during rest or moderate Ca2+ entry through excitable Ca2+ channels during depolarization. Ca2+ entering the myosin-poor space is partially removed by SERCA of the superficial SR and subsequently unloaded through the PM-SR junctions by vectorial Ca2+ release coupled to the forward mode NCX. [Adapted from Nazer and van Breemen (50, 51)].

	MITOCHONDRIAL CA2+ TRANSPORT AND SIGNALING

TOP ABSTRACT INTRODUCTION [CA2+]i OSCILLATIONS SYNCHRONIZED NON-WAVELIKE [CA2+... ASYNCHRONOUS WAVELIKE [CA2+]i... ULTRASTRUCTURE OF VSMC CA2+ WAVES AND VASOCONSTRICTION CA2+ WAVES AND VASODILATATION MECHANISM OF WAVELIKE [CA2+]i... POSSIBLE ADVANTAGE OF WAVELIKE... CA2+ CYCLING DURING RESTING... MITOCHONDRIAL CA2+ TRANSPORT... NUCLEAR CA2+ SIGNALING CONCLUSION REFERENCES

In addition to modulating cellular contractility, the recurring Ca²⁺ waves are likely to affect the regulation of numerous other cellular functions in the vascular smooth muscle. Mitochondrial metabolism is one such function that may be regulated by the recurring Ca²⁺ waves. The mitochondria contain several Ca²⁺-sensitive dehydrogenases, and a rise in mitochondrial [Ca²⁺] can result in increased ATP synthesis (20, 59). Mitochondria also possess low-affinity (dissociation constant for Ca²⁺ is ~10-20 µM), high-capacity Ca²⁺ uptake that is minimally active in the submicromolar range of [Ca²⁺] (18, 36). Given that physiological global [Ca²⁺]_i fluctuations are typically in the submicromolar range, it is not immediately clear how they could affect mitochondrial Ca²⁺ signaling of ATP synthesis. However, there is emerging evidence in both vascular smooth muscle as well as many other cell types to suggest that Ca²⁺ release from the ER-SR through either IP₃R or RyrR can raise mitochondrial [Ca²⁺] to near 100 µM, even though the corresponding elevation in global [Ca²⁺]_i is considerably lower (7, 11, 12, 19, 48, 49, 59, 66). This apparent contradiction can be resolved if one considers the possibility of a high [Ca²⁺] microdomain generated by localized ER-SR Ca²⁺ release via the IP₃R/RyrR near the surface of the mitochondria. Such microdomains are the result of many areas of close contact between SR and mitochondria defining the MT-SR junctional space (58). Although this space may be wider (<80 nm) than the PM-SR junctional space, diffusion from it appears to be sufficiently restricted to support increases of local [Ca²⁺] to 30 µM (66). This would be sufficient to activate mitochondrial Ca²⁺ uptake and raise mitochondrial [Ca²⁺]. Accordingly, [Ca²⁺]_i oscillations in VSMCs have been found to cause oscillations in mitochondrial [Ca²⁺] (12). Furthermore, it has been shown in hepatocytes that ER-mediated oscillatory Ca²⁺ signals can efficiently activate certain Ca²⁺-sensitive dehydrogenase in the mitochondria (20). Thus the evidence strongly suggests that the SR-mediated asynchronous wavelike [Ca²⁺]_i oscillations seen in vascular smooth muscle can raise mitochondrial [Ca²⁺] and ultimately increase ATP generation to match the increased energy demand of contracting vascular smooth muscle. In addition, mitochondrial uptake of Ca²⁺ released by the SR during wavelike [Ca²⁺]_i oscillations may also serve to ensure an adequate level of store depletion for activation of the SOC.

	NUCLEAR CA2+ SIGNALING

TOP ABSTRACT INTRODUCTION [CA2+]i OSCILLATIONS SYNCHRONIZED NON-WAVELIKE [CA2+... ASYNCHRONOUS WAVELIKE [CA2+]i... ULTRASTRUCTURE OF VSMC CA2+ WAVES AND VASOCONSTRICTION CA2+ WAVES AND VASODILATATION MECHANISM OF WAVELIKE [CA2+]i... POSSIBLE ADVANTAGE OF WAVELIKE... CA2+ CYCLING DURING RESTING... MITOCHONDRIAL CA2+ TRANSPORT... NUCLEAR CA2+ SIGNALING CONCLUSION REFERENCES

Because the nucleus is completely surrounded by the nuclear envelope, its Ca²⁺ concentration ([Ca²⁺]_n) has been found to be regulated differently than the [Ca²⁺]_i (24). In particular, [Ca²⁺]_n is lower than [Ca²⁺]_i during rest and higher during activation. Blockade of SERCA also causes the [Ca²⁺]_n to rise in excess of [Ca²⁺]_i. Thus far no specific study has been made of [Ca²⁺]_n during [Ca²⁺]_i oscillations. It is possible that the recurring Ca²⁺ waves or [Ca²⁺]_i oscillations may activate cellular functions other than contraction, such as smooth muscle hypertrophy, proliferation, and synthesis of extracellular matrix indirectly by regulating at the level of gene expression. Experiments in other cell types have revealed a number of Ca²⁺-sensitive cytosolic/nuclear enzymes whose activity is modulated by the frequency domain of fixed-amplitude [Ca²⁺]_i oscillations (9, 10, 13, 26). This includes multipurpose enzymes like calmodulin kinase II and transcription factors such as nuclear factor (NF)-B, NF-AT, and Oct/OAP. There likely exist more Ca²⁺-sensitive enzymes whose activity can either be efficiently and/or selectively activated by [Ca²⁺]_i oscillations. Thus, even though not yet demonstrated, it is highly plausible that the wavelike [Ca²⁺]_i oscillations observed in vascular smooth muscle can convey information in its frequency domain, which can be "decoded" by these Ca²⁺ oscillation-sensitive enzymes to activate other cellular functions (23). Physiologically, this may represents an important adaptive mechanism for blood vessels.

	CONCLUSION

TOP ABSTRACT INTRODUCTION [CA2+]i OSCILLATIONS SYNCHRONIZED NON-WAVELIKE [CA2+... ASYNCHRONOUS WAVELIKE [CA2+]i... ULTRASTRUCTURE OF VSMC CA2+ WAVES AND VASOCONSTRICTION CA2+ WAVES AND VASODILATATION MECHANISM OF WAVELIKE [CA2+]i... POSSIBLE ADVANTAGE OF WAVELIKE... CA2+ CYCLING DURING RESTING... MITOCHONDRIAL CA2+ TRANSPORT... NUCLEAR CA2+ SIGNALING CONCLUSION REFERENCES

Microscopic analysis of Ca²⁺ signaling in vascular smooth muscle has revealed complex and tissue-specific spatial and temporal patterns, generated by interactions of Ca²⁺ transport and Ca²⁺-sensing molecules located in different organelles and bordering different regions of the cytoplasm. The manner in which localized Ca²⁺ signals are coupled to either contraction or relaxation is to a large extent determined by the specific ion pumps and channels contained within the PM-SR junctional complexes. The ultrastructure of these junctional complexes may therefore be responsible for the specific control of different types of blood vessels.