AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 293: H204-H214, 2007. First published March 2, 2007; doi:10.1152/ajpheart.00669.2006
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Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries

T. Kamishima,1 T. Burdyga,2 J. A. Gallagher,1 and J. M. Quayle1

Departments of 1Human Anatomy and Cell Biology and 2Physiology, School of Biomedical Sciences, University of Liverpool, Liverpool, United Kingdom

Submitted 23 June 2006 ; accepted in final form 27 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The role of caveolins, signature proteins of caveolae, in arterial Ca2+ regulation is unknown. We investigated modulation of Ca2+ homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca2+ transients were simultaneously measured with voltage-clamped single cells. Membrane depolarization triggered Ca2+ current and increased intracellular Ca2+ concentration ([Ca2+]i). After repolarization, elevated [Ca2+]i returned to the resting level. Ca2+ removal rate was determined from the declining phase of the Ca2+ transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to amino acid residues 82–101 of caveolin-1, significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55–74 of caveolin-3, also significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, and cyclosporine A, inhibitors for calmodulin, Ca2+/calmodulin-dependent protein kinase II, and calcineurin, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca2+ ATPase inhibitor, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca2+ removal of resistance artery smooth muscle cells.

caveolin-1 scaffolding domain peptide; caveolin-3 scaffolding domain peptide; calmodulin; calcium/calmodulin-dependent protein kinase II; calcineurin

CAVEOLAE ARE OMEGA-SHAPED plasmalemmal invaginations that occupy up to 20% of the total cell surface area in smooth muscle cells (6) (for review, see Ref. 34). Lately, the importance of caveolae in cell signaling has become apparent. First, it has been shown that a variety of key proteins, including receptors and ion channels, are located within caveolae (34). Apart from bringing receptors and effectors together into a confined microdomain, this topological arrangement may be useful because caveola invaginations also provide the shortest distance between the cell membrane and intracellular organelles (25). Second, it has been shown that the signature proteins of caveolae, the caveolins (22, 35), interact with a wide range of proteins involved in cell signaling (30, 34). To date, three caveolin genes [caveolin-1 (Cav1), caveolin-2 (Cav2), and caveolin-3 (Cav3)] have been identified (30, 34). Caveolins are the central component of caveolae, not only because they are required for caveola formation but also because they contain a 20-amino acid sequence, termed a scaffolding domain (24), which plays a key role in regulating signal transduction cascades. It is now known that many, but not all, caveolin-interacting proteins contain motifs of {Phi}X{Phi}XXXX{Phi} or {Phi}XXXX{Phi}XX{Phi} ({Phi} is an aromatic amino acid) recognizing caveolin scaffolding domains (6). This relatively loose requirement on the part of partner proteins may partially explain the relative promiscuity of caveolins with a wide range of targets. Presently, it is understood that caveolin scaffolding domains act as docking sites to anchor various proteins within caveolae and regulate a variety of signaling molecules negatively or positively (5).

The recent creation of Cav1 knockout mice has uncovered a multitude of roles played by Cav1 in vascular contractility (8, 32). First, endothelium-dependent relaxation of aorta was potentiated in knockout mice. Second, contractile responses of small arteries to vasoconstrictors were significantly reduced. Third, the frequency of spontaneous transient outward currents (STOCs), an important determinant of myogenic tone of resistance arteries (29) (see also DISCUSSION), was significantly lowered in cells from Cav1-null mice. In a separate study, it has been reported that aortae of Cav1-knockout mice showed an enhanced Ca2+ response to endothelin-1 (15). Moreover, chemical loading of Cav1 scaffolding domain peptide (Cav1 SDP, corresponding to Cav1 amino acids 82–101) into aorta significantly inhibited protein kinase C-mediated contraction (16). These observations strongly indicate that Cav1 is important in regulating vascular tone. Furthermore, the hypothesis that Cav1 may be important in Ca2+ homeostasis has been suggested based on the immunocytochemical finding that various Ca2+ regulatory proteins were localized with Cav1 in esophageal sphincter (7). Also, in nonsmooth muscle cells, Cav1 has been implicated in Ca2+ homeostasis (12, 17). However, to date, Ca2+ handling processes that may be coordinated by Cav1 have not been examined in smooth muscle cells, including arterial smooth muscle cells. Furthermore, it has been shown that Cav3, a prominent caveolin isoform in the heart (39), is also expressed in arterial vasculature (13). However, the function of Cav3 in arterial smooth muscle Ca2+ handling also remains unknown.

We sought to investigate the role of caveolins in Ca2+ regulation of smooth muscle cells from cerebral resistance arteries. In particular, we were interested in the possible role of caveolins in Ca2+ clearance. We determined membrane current and Ca2+ transient simultaneously from voltage-clamped single smooth muscle cells. We investigated the effect of Cav1 and Cav3 Abs and Cav1 and Cav3 SDPs on Ca2+ removal rate. We present evidence that Cav1 and Cav3 regulate [Ca2+]i over the physiologically important range in resistance artery smooth muscle cells. Our results also show that the effect of functional inhibition of Cav1 and Cav3 were mimicked by inhibitors of Ca2+-dependent enzymes. Our findings are consistent with the hypothesis that caveolins regulate Ca2+ handling of arterial smooth muscle cells by providing docking sites for Ca2+-dependent enzymes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. Male Sprague-Dawley rats (200–300 g) were made unconscious by exposure to a rising concentration of CO2 and euthanized by exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedure) Act (1986) under license from the Home Office, United Kingdom. All procedures involving animals were reviewed and approved locally by the University of Liverpool. We dissociated single smooth muscle cells from superior cerebral arteries (<200 µm diameter) (19) by using a two-step enzymatic treatment (21). The arteries were first digested for 25 min at 35°C with 1.7 mg/ml papain and 0.7 mg/ml dithioerythritol in a low-Ca2+ solution containing (in mM) 134 NaCl, 5 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 10 glucose, and 0.2 EDTA (pH adjusted to 7.3 at room temperature using NaOH). Next, the arteries were further digested for 20 min at 35°C with 1.7 mg/ml collagenase (type F) and 1 mg/ml hyaluronidase (type I-S) in the low-Ca2+ solution. Single smooth muscle cells were obtained by triturating the arteries with a fire-polished Pasteur pipette.

Immunocytochemistry. Single smooth muscle cells were allowed to settle onto size 1 cover glasses and fixed with 2% paraformaldehyde in PBS containing (in mM) 2.7 KCl, 1.5 KH2PO4, 137 NaCl, and 8 Na2HPO4 (pH 7.4). Unreacted aldehyde was quenched with 100 mM glycine buffer (pH 7.4), and cells were permeabilized with 0.1% Triton X-100 in PBS. Mouse monoclonal primary antibodies against Cav1 (Cav1 Ab), Cav2 (Cav2 Ab), and Cav3 (Cav3 Ab) were diluted by 200-, 200-, and 500-fold, respectively, with antibody diluting solution. Antibody diluting solution was made with 2% goat serum, 1% BSA, and 0.05% Triton X-100 in sodium citrate buffer containing (in mM) 150 NaCl and 15 Na3 citrate (pH 7.2). Primary antibodies were visualized with fluorescent anti-mouse secondary antibody (Alexa fluor 488) diluted 500-fold. To examine nonspecific binding of the secondary antibody, the control cells were treated with secondary antibody alone. We used a Leica (SP2 AOBS) confocal microscope to detect fluorescent signals.

Electrophysiology and microfluorometry. Simultaneous measurement of membrane current and Ca2+ transient was performed as previously described (21). A conventional whole cell clamp technique was used to record membrane currents. Whole cell membrane currents were amplified by an Axopatch 200B amplifier (Axon Instruments), filtered at 1 kHz, and sampled at 5 kHz using pCLAMP 7 (Axon Instruments). The cells were voltage clamped at –70 mV, and depolarization to 0 mV was applied for duration of 1.8 s to activate voltage-dependent Ca2+ currents. The Ca2+-sensitive dye fura 2 was included in the pipette solution to report [Ca2+]i. A single cell was alternately illuminated for 10 ms with UV light at 340 and 380 nm (bandpass 8 nm). Emission signals were obtained at 510 nm (bandpass 40 nm). Background fluorescence was measured for each cell after the formation of a gigaohm seal and subtracted from the measurements obtained during the experiments. The composition of extracellular solution was (in mM) 120 NaCl, 20 tetraethylammonium chloride, 1.1 MgCl2, 10 HEPES, 3 CaCl2, and 30 glucose (pH 7.4). The composition of intracellular solution was (in mM) 145 CsCl, 3 MgCl2, 3 Na2ATP, 10 HEPES, and 0.05 fura 2 pentapotassium salt (pH 7.2). In all experiments, including control experiments, cells were dialyzed with the pipette solutions for at least 10 min before commencement of measurements. Cav1 Ab (200-fold dilution), Cav1 SDP (10 µM; residues corresponding to amino acids 82–101, DGIWKASFTTFTVTKYWFYR), Cav3 Ab (500-fold dilution), Cav3 SDP (10 µM; residues corresponding to amino acids 55–74, DGVWRVSYTTFTVSKYWCYR), calmodulin inhibitory peptide (50 nM), and autocamtide-2-related inhibitory peptide (10 µM) were applied by inclusion in the intracellular solution. Cyclosporine A (10 µM) and cyclopiazonic acid (CPA; 10 µM) were included in the extracellular solution. All agents except Cav1 Ab and Cav3 Ab were made as stock solutions using vehicles recommended by the manufactures and diluted at least 1,000-fold in the intracellular or extracellular solutions. All experiments were performed at room temperature. Detailed methods of Ca2+ removal analysis are described elsewhere (20).

Statistics. Data are expressed as means ± SE of n cells. The number of rats used is also mentioned in the text. When appropriate, one-way ANOVA, followed by Tukey's test as a post hoc analysis, was used to examine significant differences for multiple comparisons.

Drugs. Fura 2 pentapotassium salt and Alexa fluor 488 secondary antibody were obtained from Molecular Probes. Papain was purchased from Worthington Biochemical. Calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, CPA, and cyclosporine A were obtained from Calbiochem. Cav1 Ab, Cav2 Ab, and Cav3 Ab were purchased from BD Transduction Laboratories. Cav1 SDP and Cav3 SDP were synthesized by Sigma Genosys. All other agents were purchased from Sigma or BDH.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Immunostaining of Cav1, Cav2, and Cav3 in cerebral artery smooth muscle cells. It has been previously reported that ferret aortic smooth muscle cells were positively stained with Cav1 Ab, as visualized by fluorescent probes (16). Therefore, we first examined whether cerebral arterial smooth muscle cells also express Cav1. As shown in Fig. 1A, rat cerebral artery smooth muscle cells were stained positively with Cav1 Ab. When the cells were treated with the secondary antibody alone, virtually no signal was detected (Fig. 1D). The staining pattern in our cells was very similar to that of ferret aortic smooth muscle cells (16). Contrary to Cav1 staining, the visualization of Cav2 Ab produced a substantially dimmer image (Fig. 1B), whereas that of Cav3 Ab was indistinguishable from that with Cav1 Ab (Fig. 1C). Because Cav1 and Cav3 seemed prominently expressed in our cells, we sought to examine the possible involvement of these proteins in Ca2+ homeostasis.


Figure 1
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  		Fig. 1. Confocal images of cells dissociated from rat superior artery. Cells were immunostained in the presence of mouse monoclonal primary antibodies against caveolin-1 (Cav1 Ab; A), Cav2 Ab (B), and Cav3 Ab (C) or in the absence of primary antibody (D), using Alexa fluor 488 as fluorescent probe. Scale bar = 10 µm.

 
Effect of Cav1 Ab and Cav1 SDP on Ca2+ removal. The Ca2+ handling patterns of rat cerebral resistance artery smooth muscle cells were previously reported (20). However, it was necessary to perform control experiments with a condition matching our later experiments. Because the experiments designed to test the effect of antibodies and peptide blockers (see below) would require prolonged dialysis, we conducted the control experiments in the same condition. Thus the control cells were dialyzed with the control pipette solution for 10 min before the commencement of the measurements. Figure 2 shows an example of a control Ca2+ transient (Fig. 2A) triggered by a depolarization to 0 mV from a holding potential of –70 mV (Fig. 2B). Figure 2 also shows Ca2+ current recordings with two time scales [Fig. 2C, left, shares same time scale with Fig. 2, A and B, whereas Fig. 2C, right, is shown with an expanded time scale of 1 s (depicted by dotted line)]. With the use of the same preparation, the inward current was previously identified as an L-type, voltage-dependent Ca2+ current because it was blocked by the voltage-dependent Ca2+ channel antagonist nimodipine (19) and enhanced by the agonist BAY K 8644 (20). Ca2+ removal rate was calculated as the negative derivative of the polynomial fit to the declining phase of the Ca2+ transient during repolarization (see Ref. 20). It has been shown previously that virtually no Ca2+ influx or release occurs during repolarization, and thus Ca2+ removal rate can be accurately examined from the declining phase of the Ca2+ transient (20). Also, by the end of a 1.8-s depolarization to 0 mV, Ca2+ channels were largely inactivated due to Ca2+- and voltage-dependent inactivation, as seen by the lack of tail current (Fig. 2C, right). Thus the declining phase of the Ca2+ transient was fitted with a ninth-order polynomial equation, and Ca2+ removal rate was calculated as the negative derivative of the fit. A high-order polynomial fit has been previously shown to fit different shapes of Ca2+ transients effectively (18) and does not require any assumptions regarding the Ca2+ removal pathways. Instead, it simply reflects the underlying shape of the Ca2+ transients while removing superimposed experimental noise. Ca2+ removal rate is shown as a function of time, where time 0 is the beginning of the repolarization (Fig. 2D), or as a function of measured [Ca2+]i (Fig. 2E). As reported previously, the latter expression of Ca2+ removal rate has been particularly useful in the investigation of Ca2+ removal pathways, including the sarcoplasmic reticulum Ca2+ pump and Na+/Ca2+ exchange (20). Contrary to previous work (20), we did not detect a clear delayed acceleration in Ca2+ removal rate manifesting as an upward hump in the Ca2+ removal profile. Because the same arteries from the same strain of rats were used in both studies, this may be because of the prolonged dialysis in our experiments.


Figure 2
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  		Fig. 2. Ca2+ transient and Ca2+ current induced by depolarization in rat cerebral artery smooth muscle cell. Control Ca2+ transient (A) and Ca2+ currents (C) were triggered by a depolarization to 0 mV from a holding potential of –70 mV (B). Solid scale bar corresponds to 10 s and applies to traces in A, B, and C, left. Current recording in C, right, is the same current recording with an expanded time scale of 1 s shown with the dotted line. Vm, membrane potential; I, current. Ca2+ removal rate is shown as a function of time, where time = 0 is the beginning of the repolarization (D), or as a function of measured intracellular Ca2+ concentration ([Ca2+]i; E).

 
Next, we examined how, if at all, the Ca2+ removal profile may be modified by disruption of Cav1. In cardiac myocytes, it has been shown that the inclusion of Cav3 Ab abolished the isoproterenol-induced enhancement of Na+ current (40). Thus we hypothesized that application of the Cav1 Ab, which was effective in immunostaining in our cells, may functionally disrupt Cav1. Figure 3A shows one such experiment, in which Cav1 Ab was included in the pipette solution and dialyzed for 10 min. Ca2+ transient recovery was slower in the presence of Cav1 Ab. As previously reported in cardiac myocytes, the mechanism by which caveolin antibody exerts its effect is unknown (10). We therefore sought to determine whether the apparent change in Ca2+ removal profile can be repeated by the application of Cav1 SDP. Figure 3B shows one such experiment. The Ca2+ transient recovered slowly when the cell was dialyzed with 10 µM Cav1 SDP. Thus two Cav1 inhibitory agents based on different principles, an immunoglobulin and a short peptide, produced similar results. Figure 3C summarizes Ca2+ removal rate, expressed as a function of measured [Ca2+]i at 25 nM intervals, for the control cells, Cav1 Ab-treated cells, and Cav1 SDP-treated cells. Multiple statistical comparisons for these results, along with the results that will be described later, were carried out, and the outcome is also shown in the Fig. 3C. The application of Cav1 Ab or Cav1 SDP reduced Ca2+ removal rate up to a [Ca2+]i of 300 nM with little effect at higher [Ca2+]i. It is unlikely that the observed reduction in Ca2+ removal rate in Cav1 Ab- and Cav1 SDP-treated cells is due to unmasking of previously silent Ca2+-permeable cation channels (3). In the cells treated with Cav1 Ab and Cav1 SDP, depolarizing the membrane potential to 100 mV, a potential close to the equilibrium potential for Ca2+, during the declining phase of the Ca2+ transient had no effect on Ca2+ removal profile (data not shown). If Ca2+ influx through cation channels produced the apparent inhibition of Ca2+ removal rate, depolarization to equilibrium potential for Ca2+ where no net Ca2+ influx occurs should have facilitated a decline of the Ca2+ transient. Thus these results indicate that Cav1 regulates Ca2+ clearance in cerebral resistance artery smooth muscle cells.


Figure 3
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  		Fig. 3. Modulation of Ca2+ transients and Ca2+ removal by Cav1 Ab and Cav1 scaffolding domain peptide (SDP). A: Ca2+ transient from a cell dialyzed with Cav1 Ab. Scale bar = 10 s. B: Ca2+ transient from a cell dialyzed with 10 µM Cav1 SDP. Scale bar = 10 s. C: summary of Ca2+ removal rates obtained from control cells ({circ}; n = 8 from 5 rats), Cav1 Ab-treated cells (bullet; n = 7 from 4 rats), and Cav1 SDP-treated cells ({blacksquare}; n = 11 from 7 rats), expressed as a function of measured [Ca2+]i at 25 nM intervals. Statistical significance was detected with ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 at the appropriate [Ca2+]i. Application of Cav1 Ab or Cav1 SDP significantly altered Ca2+ removal rate up to [Ca2+]i of 300 nM, with little effect at higher [Ca2+]i.

 
Effect of Cav3 Ab and Cav3 SDP on Ca2+ removal. Next, we sought to examine whether functional disruption of Cav3 affects Ca2+ homeostasis in arterial smooth muscle cells. First, Cav3 Ab was introduced into the cell interior from the patch pipette, and Ca2+ transients were examined after 10-min dialysis. Figure 4A shows one such experiment. In the presence of Cav3 Ab, the Ca2+ transient recovery occurred more slowly. Thus we also investigated whether the apparent change in Ca2+ removal profile can be replicated by the application of Cav3 SDP. Figure 4B shows one such experiment. Once again, the Ca2+ transient recovered slowly when the cell was dialyzed with 10 µM Cav3 SDP. Figure 4C summarizes Ca2+ removal rate, expressed as a function of measured [Ca2+]i at 25 nM intervals, for Cav3 Ab-treated cells and Cav3 SDP-treated cells. Results from control experiments are also shown for comparison. Multiple statistical comparisons for these results were carried out, and the results are also shown in Fig. 4C. In the presence of Cav3 Ab, a significant difference was detected up to a [Ca2+]i of 350 nM. In the presence of Cav3 SDP, a significant difference was observed up to a [Ca2+]i of 300 nM. Neither Cav3 Ab nor Cav3 SDP had a significant effect on Ca2+ removal rate at higher [Ca2+]i. These results suggest that Cav3 also regulates Ca2+ clearance in cerebral resistance artery smooth muscle cells.


Figure 4
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  		Fig. 4. Modulation of Ca2+ transients and Ca2+ removal by Cav3 Ab and Cav3 SDP. A: Ca2+ transient from a cell dialyzed with Cav3 Ab. Scale bar = 10 s. B: Ca2+ transient from a cell dialyzed with Cav3 SDP. Scale bar = 10 s. C: summary of Ca2+ removal rates obtained from control cells ({circ}; n = 8 from 5 rats), Cav3 Ab-treated cells (bullet; n = 14 from 10 rats), Cav3 SDP-treated cells ({blacksquare}; n = 17 from 12 rats), expressed as a function of measured [Ca2+]i at 25 nM intervals. Statistical significance was detected with ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 at the appropriate [Ca2+]i. Application of Cav3 Ab significantly altered Ca2+ removal rate up to [Ca2+]i of 350 nM, whereas that of Cav3 SDP did so up to [Ca2+]i of 300 nM.

 
Effect of inhibitors of calmodulin, Ca2+/calmodulin-dependent protein kinase II, calcineurin, and sarcoplasmic Ca2+ ATPase on Ca2+ removal rate. Although our main aim was to examine whether functional inhibition of Cav1 and Cav3 modifies the Ca2+ removal profile in our cells, we sought to investigate whether similar results may be obtained by interfering with signal transduction pathways. In this context, we were interested in testing the effect of inhibition of Ca2+-dependent enzymes on Ca2+ transients for the following reasons. First, the Ca2+ transients in the experiments above were triggered by depolarization without activation of receptors. Thus it is unlikely that G-protein-coupled, receptor-mediated enzymes such as protein kinases A and C would play major roles in the signal transduction cascade. Second, it is well documented that Ca2+-dependent enzymes facilitate Ca2+ clearance in cardiac myocytes and amphibian gastric smooth muscle cells (1, 27) (see also DISCUSSION). Indeed, as far as depolarization-induced Ca2+ transients of voltage-clamped cells are concerned, Ca2+-dependent enzymes are the sole regulatory molecules conclusively identified as facilitators of Ca2+ clearance (1, 27). We therefore examined the effect of inhibitors of Ca2+-dependent enzymes known to modulate Ca2+ homeostasis.

In our first series of experiments, we sought to investigate the role of calmodulin. Figure 5A shows the Ca2+ transient obtained from a cell treated with an inhibitor of calmodulin, calmodulin inhibitory peptide (50 nM). When the cell was dialyzed with a pipette solution containing calmodulin inhibitory peptide, recovery of the Ca2+ transient was slower than that of a control cell. Figure 5B summarizes the results from calmodulin inhibitory peptide-treated cells, along with control data for comparison. Inhibition of calmodulin significantly affected Ca2+ removal rate up to a [Ca2+]i of 425 nM, with little effect at higher [Ca2+]i.


Figure 5
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  		Fig. 5. Modulation of Ca2+ transients and Ca2+ removal by calmodulin inhibitory peptide (CIP). A: Ca2+ transient from a cell dialyzed with CIP. Scale bar = 10 s. B: summary of Ca2+ removal rates obtained from control cells ({circ}; n = 8 from 5 rats) and CIP-treated cells (bullet; n = 10 from 6 rats), expressed as a function of measured [Ca2+]i at 25 nM intervals. Statistical significance was detected with ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 at the appropriate [Ca2+]i. Application of CIP significantly altered Ca2+ removal rate up to [Ca2+]i of 425 nM, whereas little effect is observed at higher [Ca2+]i.

 
Because calmodulin is known to activate other enzymes such as Ca2+/calmodulin-dependent protein kinase II (CaMKII), we next examined the effect of CaMKII on Ca2+ removal. Cells were dialyzed with pipette solution containing autocamtide-2-related inhibitory peptide (10 µM), a CaMKII inhibitor. Figure 6A shows a Ca2+ transient obtained from one such cell. Compared with the effect of calmodulin inhibitory peptide, the effect of autocamtide-2-related inhibitory peptide was less pronounced, although the recovery of Ca2+ transient seems a little slower than that of the control cell. Figure 6B summarizes the results from autocamtide-2-related inhibitory peptide-treated cells along with control data for comparison. These results show that inhibition of CaMKII significantly affected Ca2+ removal rate up to a [Ca2+]i of 250 nM with little effect at higher [Ca2+]i.


Figure 6
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  		Fig. 6. Modulation of Ca2+ transients and Ca2+ removal by autocamtide-2 related inhibitory peptide (A-2 RIP). A: Ca2+ transient from a cell dialyzed with A-2 RIP. Scale bar = 10 s. B: summary of Ca2+ removal rates obtained from control cells ({circ}; n = 8 from 5 rats) and A-2 RIP-treated cells (bullet; n = 15 from 8 rats), expressed as a function of measured [Ca2+]i at 25 nM intervals. Statistical significance was detected with ANOVA: **P < 0.01 and ***P < 0.001 at the appropriate [Ca2+]i. Application of A-2 RIP significantly altered Ca2+ removal rate up to [Ca2+]i of 250 nM, whereas little effect is observed at higher [Ca2+]i.

 
We have examined the effect of inhibition of two major Ca2+-dependent kinases, calmodulin and CaMKII, that may regulate Ca2+ homeostasis in our cells. However, [Ca2+]i elevation may also activate Ca2+-dependent phosphatases. Thus we sought to test the hypothesis that the inhibition of calcineurin, an important Ca2+-dependent phosphatase, may modify the profile of Ca2+ removal in rat cerebral artery smooth muscle cells. Figure 7A shows the Ca2+ transients obtained from cells treated with cyclosporine A (10 µM), a blocker for calcineurin. When cells were treated with cyclosporine A, recovery of the Ca2+ transient was slower than that of control cells. Figure 7B summarizes results from cyclosporine A-treated cells with control data for comparison. These results show that inhibition of calcineurin significantly affected Ca2+ removal rate up to a [Ca2+]i of 375 nM with little effect at higher [Ca2+]i.


Figure 7
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  		Fig. 7. Modulation of Ca2+ transients and Ca2+ removal by cyclosporine A. A: Ca2+ transient from a cell treated with cyclosporine A. Scale bar = 10 s. B: summary of Ca2+ removal rates obtained from control cells ({circ}; n = 8 from 5 rats) and cyclosporine A (cyclo A)-treated cells (bullet; n = 10 from 9 rats), expressed as a function of measured [Ca2+]i at 25 nM intervals. Statistical significance was detected with ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 at the appropriate [Ca2+]i. Application of cyclosporine A significantly altered Ca2+ removal rate up to [Ca2+]i of 375 nM, whereas little effect is observed at higher [Ca2+]i.

 
The results described above indicate that functional inhibition of caveolins and Ca2+-dependent enzymes significantly modified Ca2+ removal rate at a low to medium range of [Ca2+]i but had little effect on Ca2+ clearance at higher [Ca2+]i. It has been previously reported that thapsigargin or ryanodine application slowed Ca2+ removal rate at [Ca2+]i <300 nM in these cells (20). Thus sarcoplasmic reticulum Ca2+ uptake seems to be important at low [Ca2+]i (20). In the present study, application of CPA, another type of sarcoplasmic reticulum Ca2+ uptake inhibitor, produced a similar result. As shown in Fig. 8A, in the presence of 10 µM CPA, the Ca2+ transient recovered slowly. Figure 8B summarizes the results from CPA-treated cells with control data for comparison. These results show that inhibition of sarcoplasmic reticulum Ca2+ uptake significantly affected Ca2+ removal rate up to a [Ca2+]i of 250 nM with little effect at higher [Ca2+]i.


Figure 8
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  		Fig. 8. Modulation of Ca2+ transients and Ca2+ removal by cyclopiazonic acid (CPA). A: Ca2+ transient from a cell treated with CPA. Scale bar = 10 s. B: summary of Ca2+ removal rates obtained from control cells ({circ}; n = 8 from 5 rats) and CPA-treated cells (bullet n = 9 from 7 rats), expressed as a function of measured [Ca2+]i at 25 nM intervals. Statistical significance was detected with ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 at the appropriate [Ca2+]i. Application of CPA significantly altered Ca2+ removal rate up to [Ca2+]i of 250 nM, whereas little effect is observed at higher [Ca2+]i.

 
Blockade of Cav1, Cav3, calmodulin, CaMKII, calcineurin, and sarcoplasmic reticulum Ca2+ uptake significantly inhibited Ca2+ removal rate at low [Ca2+]i but not at high [Ca2+]i. Figure 9 summarizes the Ca2+ removal rate under various conditions at a measured [Ca2+]i of 250 and 600 nM. A concentration of 250 nM was chosen as an example of a low [Ca2+]i and is within the range that is physiologically important in these cells (26) (see also DISCUSSION). Ca2+ removal rate was significantly different from control when the cells were treated with Cav1 Ab (P < 0.01), Cav1 SDP (P < 0.001), Cav3 Ab (P < 0.001), Cav3 SDP (P < 0.001), calmodulin inhibitory peptide (P < 0.001), autocamtide-2-related inhibitory peptide (P < 0.01), cyclosporine A (P < 0.001), and CPA (P < 0.05). Figure 9B summarizes the Ca2+ removal rate determined at a measured [Ca2+]i of 600 nM. At this higher [Ca2+]i, no significant differences were detected among the various conditions. It is possible, however, that the higher noise level seen at higher [Ca2+]i might have made the detection of significant differences among various treatments difficult; hence, further study will be required to fully conclude that the functional inhibition of caveolins and Ca2+-dependent enzymes do not modify Ca2+ clearance when [Ca2+]i is high. Nonetheless, significant differences were detected at lower [Ca2+]i, and these results are consistent with the hypothesis that caveolin scaffolding domains act as part of a signal transduction complex for Ca2+-dependent enzymes to regulate Ca2+ clearance. Furthermore, Ca2+ removal was significantly affected by the blockade of Ca2+ uptake to the sarcoplasmic reticulum over low [Ca2+]i (Ref. 20 and this study). The range of [Ca2+]i where removal rates were affected is of importance for arterial smooth muscle cells that are unlikely to experience high [Ca2+]i (26) (see DISCUSSION). Thus the results presented here provide a useful insight to Ca2+ handling mechanisms that may be physiologically important in arterial smooth muscle cells (26).


Figure 9
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  		Fig. 9. Comparison of Ca2+ removal rate under various conditions. A: Ca2+ removal rate determined at a [Ca2+]i of 250 nM. Starting from left, columns show the results from control cells (n = 8 from 5 rats) and cells treated with Cav1 Ab (n = 6 from 4 rats; P < 0.01), Cav1 SDP (n = 10 from 7 rats; P < 0.001), Cav3 Ab (n = 13 from 10 rats; P < 0.001), Cav3 SDP (n = 10 from 7 rats; P < 0.001), CIP (n = 9 from 6 rats; P < 0.001), A-2 RIP (n = 14 from 8 rats; P < 0.01), cyclosporine A (cyclo A, n = 9 from 8 rats; P < 0.001), and CPA (n = 6 from 5 rats; P < 0.05). B: Ca2+ removal rate determined at a [Ca2+]i of 600 nM. At this higher [Ca2+]i, no significant differences were detected among various conditions.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We provide here the first evidence that Ca2+ removal is modified by functional inhibition of Cav1 and Cav3 in arterial smooth muscle cells. Application of Cav1 Ab or Cav1 SDP significantly slowed the Ca2+ removal rate over low [Ca2+]i with little effect at higher [Ca2+]i (Figs. 3C and 9). Similarly, application of Cav3 Ab or Cav3 SDP significantly slowed the Ca2+ removal rate over low to medium [Ca2+]i with little effect at higher [Ca2+]i (Figs. 4C and 9). It should be noted that the [Ca2+]i range where Cav1 Ab, Cav1 SDP, Cav3 Ab, and Cav3 SDP were effective is important. Arterial smooth muscle cells do not normally fire action potentials but rather produce small and steady depolarizations. Under physiological conditions, cerebral resistance artery smooth muscle cells are unlikely to experience [Ca2+]i elevation going much beyond 300 nM (26). Ca2+ removal rates over this concentration range were significantly modified by the inclusion of Cav1 Ab, Cav1 SDP, Cav3 Ab, and Cav3 SDP in the pipette solution (Figs. 3C and 4C). Thus our results indicate that Cav1 and Cav3 play an important role in Ca2+ removal over physiologically relevant [Ca2+]i.

The importance of Ca2+-activated enzymes on Ca2+ removal rate has been reported in nonarterial muscle cells (1, 27), but equivalent studies in arterial smooth muscle cells are lacking. Thus, in the present study, we investigated the effect of Ca2+-dependent enzyme inhibitors on the Ca2+ clearance profile in our cells. We report that application of calmodulin inhibitory peptide significantly slowed the Ca2+ removal rate over low to medium [Ca2+]i with little effect at higher [Ca2+]i (Figs. 5B and 9). Calmodulin is a ubiquitous Ca2+-binding protein and exerts its effects directly and indirectly on various proteins, including ryanodine-sensitive Ca2+-releasing channels (2) and sarcoplasmic reticulum Ca2+ pumps (38). It has been reported that calmodulin may be regulated by Cav1 in nonsmooth muscle cells. In cultured endothelial cells and Sf-9 cells, it has been noted that Cav1 and calmodulin reciprocally regulate endothelial nitric oxide synthase (28). Crucially, the scaffolding domain of Cav1 seems important in endothelial nitric oxide synthase regulation, although it may not interact with calmodulin directly (28). This is a very complex issue to address, however, because calmodulin does not necessarily regulate the final target molecules directly but may exert its effect by regulating other Ca2+-dependent enzymes such as CaMKII and calcineurin. Nonetheless, the results presented here provide an interesting insight to the regulation of Ca2+ homeostasis by these enzymes.

In cardiac myocytes, it has been reported that CaMKII is important in facilitating Ca2+ sequestration to the sarcoplasmic reticulum (1). The role of CaMKII was ascribed to the phosphorylation of phospholamban, a small protein that tonically inhibits the sarcoplasmic reticulum Ca2+ pump (Ref. 38, but also see Ref. 23). When phosphorylated, phospholamban dissociates from the sarcoplasmic reticulum Ca2+ pump, increasing its activity (38). CaMKII has also been implicated in Ca2+-dependent acceleration of Ca2+ removal in toad gastric myocytes, although in this instance the target organelle is mitochondria (27). However, a comparable study that used peptide inhibitors to examine the role of CaMKII is yet to be carried out in arterial smooth muscle cells. We report that application of autocamtide-2-related inhibitory peptide significantly slowed the Ca2+ removal rate up to [Ca2+]i of 250 nM with little effect at higher [Ca2+]i (Figs. 6B and 9). Our results are in agreement with those of cardiac myocytes, in which CaMKII was found to be important in facilitating Ca2+ sequestration to the sarcoplasmic reticulum (1). However, it should also be noted that CaMKII does not interact with caveolin in the signal transduction pathway involved in KCl-induced contraction in ferret aorta (16). Furthermore, as discussed above, CaMKII may also be regulated by other enzymes such as calmodulin, and thus further work will be needed to fully dissect complex interaction among these enzymes.

As described earlier, the role of Ca2+-dependent protein kinases in Ca2+ removal has been examined and identified in nonarterial muscle cells (1, 27). However, the involvement of phophatases in Ca2+ clearance was not studied in these early works, although it has been suggested that calcineurin, a ubiquitous protein phosphatase, inhibits Ca2+ uptake to the sarcoplasmic reticulum in cardiac myocytes (36). We therefore examined the possibility that calcineurin may be involved in the Ca2+ removal in rat superior artery smooth muscle cells. Application of cyclosporine A significantly slowed the Ca2+ removal rate over low to medium [Ca2+]i with little effect at higher [Ca2+]i (Figs. 7B and 9). These results offer further insight to the signal transduction cascade that is important in Ca2+ clearance in arterial smooth muscle cells. The question of whether calcineurin directly affects the proteins responsible for Ca2+ removal, or requires additional second messengers, could be addressed by biochemical investigation in the future.

The results described above provide evidence that inhibition of calmodulin, CaMKII, and calcineurin significantly reduced Ca2+ removal rates to various degrees in rat superior artery smooth muscle cells (Fig. 9A). Whether the action of these enzymes is coordinated by Cav1 and/or Cav3 through their scaffolding domains needs to be investigated in the future. It is plausible that, given the long list of proteins that are regulated by caveolins, at least some of these Ca2+-activated enzymes are coordinated by caveolins. One interesting hypothesis is that calcineurin may be colocalized with Cav1, since Cav1 and protein kinase A are colocalized (33), whereas calcineurin seems to be colocalized with protein kinase A (36) and protein kinase A-anchoring protein (31). It should be also noted that, in nonsmooth muscle cells, calmodulin may be regulated by Cav1, although calmodulin may not interact directly with the scaffolding domain (28).

As reported previously (20), the Ca2+ removal rate was suppressed when cells were treated with CPA (Fig. 8B). It has also been shown in these cells that Ca2+ clearance through Na+/Ca2+ exchange is not important, whereas that through the plasmalemmal Ca2+ pump plays a minor role at low [Ca2+]i (20). Because the functional inhibition of caveolins also affected Ca2+ removal over a low to medium [Ca2+]i range, it is possible that Ca2+ uptake to the sarcoplasmic reticulum could be regulated by caveolins. If indeed this is the case, it would have an important implication regarding the regulation of vascular contractility. In arterial smooth muscle cells from Cav1-knockout mice, it has been reported that the frequency of STOCs, caused by the opening of multiple Ca2+-dependent K channels (29), was reduced (8). STOCs are a crucial determinant of artery membrane potential (29), and understanding of STOC inhibition in Cav1-null cells will help with the elucidation of vascular tone regulation. The sequence of events that inhibit STOC frequency in Cav1-knockout mice is hypothesized as follows. The loss of caveolae results in increased distance between caveolemmal voltage-dependent Ca2+ channels and ryanodine-sensitive Ca2+-releasing channels in the sarcoplasmic reticulum (25). In this modified geometry, the occurrence of Ca2+ sparks, synchronized Ca2+ release from clusters of ryanodine-sensitive channels (29), will be reduced because elevation of Ca2+ in the subsarcolemmal domain, the activator of ryanodine-sensitive channels, will be smaller. The reduced frequency of Ca2+ sparks will lead to compromised STOCs because Ca2+ sparks trigger STOCs (25, 29). This hypothesis neatly explains the observed inhibition of STOC frequency in Cav1-null cells. However, if the absence of Cav1 also reduces Ca2+ content of the sarcoplasmic reticulum as a result of reduced Ca2+ sequestration to this organelle, the open probability of ryanodine-sensitive channels may be inhibited because these channels are regulated by the luminal Ca2+ content (14). Although it has been reported that the Ca2+ content of the sarcoplasmic reticulum was unchanged in cells that are made caveola-free with methyl-beta-cyclodextrin, a cholesterol-depleting agent, subtle changes may be difficult to detect with the methods used (25). Thus our finding may provide an additional explanation of why STOC frequency is inhibited in cells from Cav1-null mice. This hypothesis is consistent with the report that the frequency, amplitude, and width of Ca2+ sparks were reduced in cells treated with methyl-beta-cyclodextrin (Ref. 25, but also see Ref. 4). However, the identification of target molecules for caveolins would be complicated. Obviously, block of the sarcoplasmic reticulum Ca2+ pump, as seen with thapsigargin (20) or CPA (this study), would inhibit Ca2+ sequestration to this organelle. However, there are other possibilities. For example, if functional inhibition of caveolins locks ryanodine-sensitive Ca2+-releasing channels to the semiconducting state (11), Ca2+ sequestration to this organelle will be effectively blocked due to a "leaky" sarcoplasmic reticulum (20). Testing of various possible mechanisms is rather difficult, partly because regulation of sarcoplasmic reticulum Ca2+ pumps and Ca2+-releasing channels are yet to be fully elucidated (11, 38). Furthermore, the exact role of Ca2+-activated enzymes and their target molecules have not been fully understood. For example, a clear consensus regarding the role of calcineurin on various Ca2+-handling pathways has not been reached (9, 36, 37). Nonetheless, the results presented here offer new information regarding Ca2+ clearance mechanisms of arterial smooth muscle cells.

In conclusion, we have shown the evidence that Cav1 and Cav3 regulate Ca2+ homeostasis in rat cerebral resistance artery smooth muscle cells. These findings support the idea that caveolins serve an important function in the regulation of vascular contractility.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the British Heart Foundation (PG/04/130/18114).


   ACKNOWLEDGMENTS
 
We thank Dr. Helen Burrell (Human Anatomy and Cell Biology, University of Liverpool) for expert help with the confocal microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Quayle, Dept. of Human Anatomy and Cell Biology, School of Biomedical Sciences, Univ. of Liverpool, The Sherrington Bldgs., Ashton St., Liverpool L69 3GE, UK (e-mail: jquayle{at}liv.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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