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Keywords:

  • Ca2+ entry;
  • Ca2+ waves;
  • capacitative;
  • caveolae;
  • caveolin;
  • eNOS;
  • signal transduction;
  • store-operated Ca2+ entry

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ca2+-Regulated Signal Transduction from Caveolae
  5. Conclusion
  6. Acknowledgments
  7. References

The correct spatial and temporal control of Ca2+ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca2+ concentration. In this review we provide an updated view of how caveolae may regulate both Ca2+ entry into cells and Ca2+-dependent signal transduction


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ca2+-Regulated Signal Transduction from Caveolae
  5. Conclusion
  6. Acknowledgments
  7. References

Some of the earliest experiments probing the function of caveolae centered on their possible function in regulating Ca2+ entry. In 1974, Popescu used potassium oxalate to visualize precipitates of Ca2+ with the electron microscope on the surface of smooth muscle cells. These precipitates were highly localized to invaginated caveolae, subsurface vesicles with the morphology of caveolae vesicles, the sarcoplasmic reticulum and mitochondria (1). On the basis of these studies, he proposed that caveolae were involved in the translocation of Ca2+ from the extracellular space to the cytoplasm during the contraction-relaxation cycle of smooth muscle cells (2). Subsequently, Sugi et al. (3) used pyroantimonate to localize Ca2+ in contracting smooth muscle and found that Ca2+ was highly concentrated in caveolae of relaxed cells but was dramatically reduced in cells that had been stimulated to contract. In contracted cells the Ca2+ precipitate appeared to be diffusely distributed in the cytoplasm of the cell. These results supported Popescu's hypothesis by providing the first evidence that Ca2+ sequestration by caveolae precedes its discharge across caveolae membrane in response to muscle contraction. It is as if caveolae in smooth muscle cells are the equivalent of the T-tubule in skeletal muscle cells.

Ca2+ regulatory molecules in caveolae

If caveolae have anything to do with Ca2+ homeostasis, they should be enriched in molecules that regulate Ca2+ flux across membranes. Two key Ca2+ regulatory molecules are the IP3 receptor (IP3R) and the Ca2+-ATPase. An IP3 receptor-like protein that is recognized by antibodies against a type I IP3 receptor has been localized by immunogold methods to caveolae of endothelial cells, smooth muscle cells, and keratinocytes (4). Most IP3 receptors are thought to exist as homo- or heterotetramers in the ER, where they function to release stored Ca2+ (5). Although the molecular properties and function of the IP3 receptor-like protein in caveolae have not been determined, the presence of this protein is consistent with caveolae being sites where Ca2+ is discharged into the cytosol. Caveolae are also enriched in a Ca2+-ATPase (6), which is concentrated 18- to 25-fold higher in the caveolae membrane compared to non-caveolae plasma membrane. Immunofluorescence shows that both the Ca2+-ATPase and the IP3 receptor-like proteins are frequently arranged in linear arrays on the surface or along the edges of the cell, which is the characteristic surface distribution of caveolae in these cells (7). Cell fractionation has also found that members of the Transient Receptor Potential (TRP) family of capacitative Ca2+ entry (CCE) channels are enriched in caveolae (8–10) and may be linked to Ca2+ oscillations (9). The caveolae marker protein caveolin has been found in a multimolecular complex with TRP3, Gαq/11, PLCβ, IP3R and a Ca2+-ATPase (8). Therefore, caveolae contain molecular machinery that can both sequester and discharge Ca2+ into the cytoplasm of the cell.

Caveolae are sites of Ca2+ entry

When intracellular Ca2+ stores in the ER become depleted, a complex cellular process is initiated to replace the lost Ca2+ using extracellular sources. This store-dependent Ca2+ influx, referred to as capacitative Ca2+ entry (CCE) or store-operated Ca2+ entry (SOC), operates in most cells, although Ca2+ selectivity and the current size seem to depend on the cell type. In endothelial cells, for example, IP3-sensitive channels with larger conductance and somewhat less cation selectivity have been detected (11).

Popescu's work predicts that caveolae are preferred sites of extracellular Ca2+ entry in response to ER depletion. To test this prediction directly, the GFP-based Ca2+ sensor yellow cameleon was targeted to caveolae of endothelial cells to monitor local [Ca2+] in response to depletion of ER Ca2+. The results of these studies clearly showed that under these conditions caveolae are preferred sites of Ca2+ entry (12). Ca2+ entry detected by the yellow cameleon in caveolae was markedly inhibited by: modifiers of caveolae structure (cholesterol oxidase, methyl-β-cyclodextrin); IP3 receptor inhibitors (xestospongin, 2-APB); modifiers of the cytoskeleton (colcemide, jasplakinolide); and phosphatases inhibitors (calyculin A, okadaic acid).

The mechanism of CCE activation at the plasma membrane (PM) in response to depletion of ER Ca2+ stores is unknown. How cells sense the level of internal Ca2+, how this information is transmitted from the ER to the PM, and the identity of channels responsible for CCE are all unanswered questions. Several models have been proposed to explain the coupling process, including: (i) the existence of a diffusible Ca2+ influx factor of unknown molecular identity that activates CCE channels (13,14); (ii) conformational coupling between an IP3R that regulates Ca2+ release from the ER and the TRP1 (15) or TRP3 (16,17) Ca2+ entry channels at the PM; (iii) secretion-like coupling model where Ca2+ entry is regulated by the fusion of vesicles containing CCE channels with the PM when ER Ca2+ stores become depleted (18,19). The latter two models suggest a physical coupling between Ca2+ store depletion and the activation of Ca2+ entry.

Regulated Ca2+ entry through caveolae

A physical coupling between CCE channels at the cell surface and Ca2+ stores in the ER is analogous to the coupling between Ca2+ channels at T-tubule-sarcoplasmic reticulum (SR) junctions in skeletal muscle. Ca2+ entry through these permanent junctions is rapid. By contrast, activation of Ca2+ entry in cells lacking T-tubules is initiated rapidly but develops slowly over the next 10–30 s in response to store depletion. It may be a slower process because coupling requires the establishment of connections between the ER lumen and the extracellular space. There is no evidence that CCE depends on a direct connection between the ER and the plasma membrane, but caveolae are well suited to function as an intermediate.

The secretion-like coupling model (18) proposes there is a membrane trafficking step, similar to exocytosis, involved in capacitive Ca2+ entry. The membrane compartment that mediates this traffic is thought to couple the entry of Ca2+ through the plasma membrane to the delivery of Ca2+ to the ER as well as cytosol. In Xenopus oocytes, this process is inhibited by constitutively active Rho, botulinum neurotoxin A and dominant negative SNAP-25 (19). The known endocytic and exocytic properties of caveolae (20) match the characteristics of the ‘secretion-like’ compartment proposed by Patterson et al. (18). First, caveolae contain all of the molecular machinery necessary to engage in membrane traffic, including α-SNAP (21), elements of the SNARE complex (21) and dynamin (22,23). They are normally enriched in RhoA (24), which may be involved in controlling the organization of the cytoskeleton from this location. Quantitative measurements of receptor-mediated uptake by caveolae show that caveolae vesicles either recycle back to the cell surface directly or move into the cell (20). The internalization step is regulated by PKCα (25) and tyrosine kinases (26), while the exocytic step is sensitive to the phosphatase inhibitor okadaic acid (27). Okadaic acid, like the calyculin A, inhibits the Ca2+ entry detected by yellow cameleon in caveolae (12). Phosphatase inhibitors may prevent the exocytic step required to bring caveolae vesicles containing the channels to the cell surface. Finally, the caveola is the only known endocytic vehicle capable of delivering molecules to the ER. This includes molecules as diverse as caveolin-1 (28), SV40 virus (26), autocrine motility factor (29) and cholera toxin (30).

Based on these considerations, an updated secretion-like coupling model is presented in Figure 1. Under resting conditions (A), a population of caveolae vesicles containing the regulatory machinery (IP3R, Ca2+-ATPase, TRP channels, etc.) is located beneath the plasma membrane in juxtaposition to the ER. The depletion of ER Ca2+ causes these caveolae to fuse with the plasma membrane (B), thereby depositing at the cell surface the channels and regulatory machinery necessary for the entry of extracellular Ca2+. Some Ca2+ may be delivered to both the cytosol, where it initiates various signaling cascades (see below), and to nearby ER cisterna. There may even be special contact sites between the cytoplasmic surface of caveolae and the ER that facilitate Ca2+ delivery. In some cases, internalizing caveolae may capture bulk phase extracellular Ca2+ and deliver it directly to the ER (C) (see below).

image

Figure 1. A role for caveolae in capacitative Ca2+ entry. Populations of caveolae contain-ing the channels and necessary regulatory machinery for CCE are sequestered in the cytoplasm subtending the plasma membrane. (A) Cells are stimulated to release ER Ca2+, leading to Ca2+-depleted ER. (B) The caveolae vesicles move to the cell surface, where they fuse and deposit the CCE machinery. These caveolae then deliver Ca2+ directly to nearby ER, possibly by making direct contacts with the ER. (C) Alternatively, depletion of ER Ca2+ stimulates caveolae inter-nalization. The vesicles that form trap bulk extracellular Ca2+ and deliver it to the ER, either by fusion as shown or by forming junctional contacts with the ER as depicted in (B).

Ca2+-Regulated Signal Transduction from Caveolae

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ca2+-Regulated Signal Transduction from Caveolae
  5. Conclusion
  6. Acknowledgments
  7. References

There is considerable evidence that caveolae compartmentalize signal transduction at the cell surface (31,32). Not only are caveolae enriched in a variety of well-characterized signaling modules, but also specific signaling activities have been mapped to these domains. One of the better studied classes of signaling activities is that involving Ca2+.

Control of ER Ca2+ release by caveolae

If caveolae are involved in replenishing ER Ca2+ stores, they might also regulate the reciprocal process, namely Ca2+ release from the ER. Recent confocal microscopy studies have shown that spatially and temporally organized Ca2+ waves originate in caveolin-rich regions at the edges of endothelial cells in response to IP3-mobilizing agonists such as ATP and bradykinin. From these sites, the wave propagates throughout the cell (33). These ATP-induced Ca2+ waves were inhibited by microinjecting heparin, a potent IP3R inhibitor. The initiation sites were restricted to a subpopulation of caveolin-1 rich regions on the surface, suggesting there is heterogeneity in the signaling capabilities of each caveola. Remarkably, when caveolae redistribute to the rear of migrating cells (34), sites of wave initiation co-redistribute. Caveolae may function, therefore, as containers that carry signaling machinery to different locations in the cell. Discrete sites of Ca2+ wave initiation have also been localized in ascidian eggs (35), pancreatic acinar cells (36,37) and hepatocytes (38), although there was no evidence in these studies that caveolae were involved.

The ability of caveolae to compartmentalize signaling machinery may explain how they mediate the initiation of Ca2+ waves. The most critical factor governing the initiation of Ca2+ release is the potency of the ER IP3R (39). Cytosolic Ca2+ biphasically regulates the sensitivity of IP3R to IP3. Elevation of local Ca2+ concentrations to 100–300 nm causes IP3 receptors to become more sensitive to IP3, while micromolar Ca2+ decreases their sensitivity (40). By contrast, the concentration of Ca2+ in the lumen of the ER affects the sensitivity of IP3R to IP3 (41, 42). As the concentration of Ca2+ in the ER lumen exceeds its buffering capacity, the IP3R becomes more sensitive to IP3, so that small increases in cytosolic Ca2+ greatly potentiate Ca2+ release (43). Therefore, the concentrations of Ca2+ in both the cytoplasm and the ER are key factors that regulate the sensitivity of ER IP3R, which, in turn, determines where IP3R will initiate Ca2+ release. Based on these considerations, there are at least four, not necessarily mutually exclusive, ways that caveolae might regulate sites of Ca2+ wave initiation: (a) as sites where regulatory IP3 is released; (b) locally released IP3 binds to IP3R in caveolae and causes entry of extracellular Ca2+ into the cytoplasm at that site; (c) [Ca2+] on the cytoplasmic face of ER IP3 receptors that are near caveolae may sensitize these receptors to IP3; (d) caveolae have a direct role in loading the ER with Ca2+. Evidence in support of each model is discussed below.

A number of studies indicate that the essential regulatory and substrate components necessary for PI turnover are located in caveolae. The receptors known to stimulate IP3 formation that have been localized to caveolae include the bradykinin B2 receptor (44), the endothelin ETA receptor (45), the Ca2+-sensing receptor CaSR in parathyroid cells (46), the EGF receptor (47), and the PDGF receptor (48). The critical heterotrimeric G protein Gαq/11 has also been localized to caveolae both biochemically and morphologically (49). Gαq/11 links various seven transmembrane receptors to phospholipase Cβ (PLCβ), which is present in caveolae (50). PLC, which is activated by both EGF and PDGF tyrosine kinase receptors, is also found in caveolae (48). Finally, caveolae contain ∼ 50% of the phosphatidylinositol 4,5-bisphosphate (PIP2) in the cell, and it is this pool that is specifically hydrolyzed by PLCβ to IP3 in response to either bradykinin or EGF (51). Thus, caveolae contain the machinery to produce locally IP3 that can trigger Ca2+ release from nearby segments of ER. The Ca2+ released into the cytosol propagates the wave by cooperatively accelerating Ca2+ release from adjacent segments of ER.

The presence of IP3 receptor-like protein in caveolae (4) may also be important. The locally produced IP3 could preferentially bind to these receptors and stimulate the entrance of extracellular Ca2+. The entering Ca2+ would then sensitize local IP3R in the ER membrane, thereby facilitating the release of ER Ca2+ by IP3 at these sites. Wave propagation would proceed as outlined above.

The yellow cameleon detected a higher basal Ca2+ concentration beneath caveolae membrane relative to the general plasma membrane in unstimulated endothelial cells (12). Local ER IP3 receptors near caveolae may be sensitized by the higher [Ca2+] and therefore are constitutively primed for IP3-induced Ca2+ release from the ER.

A final consideration is that the concentration of Ca2+ among ER subcompartments is heterogeneous (52). The initiation of a Ca2+ wave may require higher levels of ER Ca2+ than can be achieved by ion pumping, owing to the need for an enhanced sensitivity of the ER IP3 receptors to IP3. Caveolae vesicles are well suited for carrying extracellular levels of Ca2+ directly to nearby segments of ER by potocytosis (53) (Figure 1C). Very often, a fine network of ER is found concentrated at caveolin-rich cell edges, situated close to the plasma membrane. Therefore, caveolae vesicles would not have to travel far to deliver large amounts of Ca2+ to this population of ER.

Ca2+-dependent signal transduction from caveolae

In addition to the signals that appear to be embedded in the spike frequency of the Ca2+ wave (54), Ca2+ is a direct cofactor in many signaling processes. Caveolae appear to be important for regulating some of these Ca2+-dependent signaling activities.

The Ca2+ entering through caveolae appears to regulate the activity of several resident molecules. Ca2+ can enter during CCE (see above) or by the Na+/Ca2+ exchanger (NCX) located in caveolae (55). Entering Ca2+ is known to regulate eNOS (12) and adenylyl cyclase (56). Similar to Ca2+-dependent nitric oxide (NO) production, adenylyl cyclase type VI needs to be in cholesterol-rich domains to be regulated by CCE (56). Other examples of Ca2+-dependent signaling molecules that have been localized to caveolae include prostacyclin PGI2 synthase (57), PKCα (25) and PKCβ (48).

Caveolae were originally proposed to function in sensing mechanical stress because of their apparent linkage to the actin cytoskeleton and the ability to compartmentalize signaling molecules (31). Mechanical force is an essential modulator of cell function in many tissues, but is particularly critical in the cardiovascular system. The endothelial cells that line the vasculature rapidly respond to the variable mechanical conditions created by blood flow during the cardiac cycle. Hemodynamic shear stress on the endothelium can affect the acute control of vascular tone, regulate arterial structure, and determine the location of atherosclerotic lesions [reviewed in (58)]. Interestingly, acute changes in fluid flow in cultured endothelium will elevate intracellular Ca2+ in proportion to the amount of shear stress applied to the cells (59). A critical intermediate in shear stress-mediated vascular response is NO, which in endothelial cells is produced by eNOS. Ca2+-regulated eNOS activity is enriched 9.4-fold in caveolae membrane fractions relative to the whole plasma membrane (60).

The mechanism underlying Ca2+-regulated eNOS activity in caveolae appears to involve the interaction of several proteins. Caveolae are enriched in calmodulin, a critical Ca2+-binding protein that regulates eNOS activity (60). Michel et al. (61) found that up-regulation of NO production depends on eNOS binding to Ca2+-bound calmodulin, while down-regulation occurs when eNOS is bound to caveolin. This suggests that eNOS is clamped in an inactive state by a physical association with caveolin. In response to the appropriate stimulation, Ca2+ binds calmodulin and causes caveolin to dissociate from the eNOS (61, 62). Heat shock protein 90 (hsp90) may be essential for the dissociation step (63). NO production is regulated by Ca2+ influx from caveolae, but is not sensitive to Ca2+ released from intracellular stores. The precise regulation of NO production therefore appears to depend on the action of Ca2+ at the cytoplasmic face of caveolae membranes. The importance of caveolae in regulating eNOS is emphasized by the fact that caveolin-1 null mice are defective in regulating NO production (64).

Not only does Ca2+ entering through caveolae regulate eNOS, but also the locally produced NO inhibits Ca2+ entry through caveolae and stimulates uptake into the ER (65). This autoregulatory, feedback loop may involve the activation of a soluble guanylyl cyclase by NO. As [Ca2+] increases, the cyclase is translocated to caveolae (66), where locally produced cGMP may inhibit CCE channels. NO produced by eNOS in heart caveolae may also inhibit L-Type Ca2+ channels, but NO produced by nNOS colocalized with ryanodine receptors (RyR) in the SR activates RyR Ca2+ channels (67).

Another type of signaling activity where caveolae appear to be involved is in the generation of Ca2+ sparks. Ca2+ sparks are transient (30–100 ms), local (2–4 μm) intracellular sites of Ca2+ release that control excitation-contraction coupling in heart and smooth muscle. Ca2+ influx at caveolae, probably through a dihydropyridine sensitive L-type Ca2+ (DHP) channel, sensitizes RyR in adjacent SR leading to release of Ca2+ from these stores. If [Ca2+]i increases sufficiently, the cell will contract. Ca2+ sparks in cardiomyocytes occur repeatedly at defined sites enriched in caveolae (68), similar to sites of Ca2+ wave initiation in endothelial cells (34). Treatments that disrupt caveolae structure cause a decrease in spark frequency, amplitude, and spatial size (68). Loss of caveolae organization may disrupt the proper spatial relationship between Ca2+ entry sites and clusters of RyR in the SR, as occurs in cardiac hypertrophy and heart failure in hypertensive rat models (69). Ca2+ influx through caveolae may be cooperatively regulated by NCX channels (70), the Na+/K+-ATPase (71), and β-adrenergic receptor/cAMP signaling (72,73). Thus, caveolae appear to have critical functions in normal cardiac contractile function.

The proposal that caveolae modulate Ca2+ sparks in muscle agrees with the phenotype of mice lacking caveolin-1 and 3. Caveolin-3 is associated with both the plasma membrane and the developing T-tubule system in differentiating skeletal myoblasts (74). Caveolin-3 knock-out mice develop a mild to moderate cardiomyopathy with thickened left ventricle and hypertrophied cardiomyocytes, in parallel with p42/44 MAPK hyperactivation (75). In these mice, a defect in the spatial organization of Ca2+-influx/EC-coupling units due to alterations in the T-tubule system can explain the phenotype. In caveolin-1 null mice, enhanced systemic NO production resulting from regulated eNOS appears to cause dilated cardiomyopathy (76). The double KO mice have a more severe form of cardiomyopathy, suggesting that caveolin-1 and caveolin-3 act synergistically (77). Whether this cardiac phenotype is due to abnormal EC coupling, altered Ca2+ sparks or abnormal NCX activity in caveolae will need to be further examined.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ca2+-Regulated Signal Transduction from Caveolae
  5. Conclusion
  6. Acknowledgments
  7. References

The tools of modern cell biology have begun to provide information in support of Popescu's original hypothesis that caveolae were involved in regulating intracellular Ca2+. Most likely, the key functions for caveolae in Ca2+ homeostasis include: regulating the spatial organization of Ca2+ entry sites; controlling the amount of Ca2+ that is delivered at these sites; initiating Ca2+ wave formation; and modulating the multiple Ca2+-dependent signaling cascades that originate in caveolae. Defining at the molecular level exactly how caveolae carry out these multiple functions will be an important area of future investigation. Clearly, caveolae are where incoming and outgoing messages meet (31).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ca2+-Regulated Signal Transduction from Caveolae
  5. Conclusion
  6. Acknowledgments
  7. References

We would like to thank Brenda Pallares for administrative assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ca2+-Regulated Signal Transduction from Caveolae
  5. Conclusion
  6. Acknowledgments
  7. References