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MS 0349 Received 22 November 1999; accepted after revision 16 February 2000.
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ABSTRACT |
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-phorbol 12-myristate 13-acetate (PMA, 100 pM to 200 nM), also stimulated Ca2+ spiking and this effect was additive with a submaximal concentration of AVP (50 pM). The PKC inhibitors Ro-31-8220 (1 µM) and calphostin C (250 nM) completely blocked the stimulation of Ca2+ spiking by either PMA or AVP.
, , 
, 
, 
, 
and 
isoforms of PKC were detected in A7r5 cells by Western blot analysis. Time-dependent redistribution of PKC-, - and - isoforms between the membrane and cytosolic fractions occurred in response to 100 pM AVP. Pretreatment for 24 h with 1 µM PMA downregulated expression of PKC- and -, but not PKC-, and prevented the Ca2+-spiking responses to either 1 nM PMA or 100 pM AVP. Neither the release of intracellular Ca2+ by 1 µM AVP nor the increase in [Ca2+]i in response to elevated extracellular [K+] was prevented by the PMA pretreatment.
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INTRODUCTION |
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Arg8-vasopressin (AVP) is a peptide hormone that is released from the posterior pituitary gland into the systemic circulation in response to a decrease in blood pressure or an increase in plasma osmolality. A baroreceptor response to a large decrease in blood volume may cause plasma concentrations of AVP to increase from a few picomolar to a few hundred picomolar, resulting in arterial constriction and increased peripheral vascular resistance. The potent vasoconstrictor actions of AVP have been attributed to its activation of phospholipase C and the resulting release of Ca2+ from intracellular stores within vascular smooth muscle cells.
A7r5 cells are a smooth muscle cell line derived from embryonic rat aorta (Kimes & Brandt, 1976). As in other vascular smooth muscle preparations, stimulation of A7r5 cells with AVP results in an increase in [Ca2+]i by release of Ca2+ from intracellular stores and increased Ca2+ influx (Byron & Taylor, 1995). However, the concentration of AVP required for half-maximal release from intracellular Ca2+ stores is approximately 5 nM, much too high to account for the vasoconstrictor effects of the picomolar concentrations of AVP found in the systemic circulation.
A7r5 cells exhibit spontaneous Ca2+ spikes in the absence of AVP. The mechanism by which this Ca2+-spiking activity is generated involves activation of L-type voltage-sensitive Ca2+ channels and is independent of the release of Ca2+ from intracellular stores (Byron & Taylor, 1993). Ca2+ spiking in vascular smooth muscle may lead to rhythmic vasomotion of small resistance arteries and arterioles. Vasomotion has been observed in vivo as well as in a number of arterial preparations in vitro and is believed to be important in the regulation of regional tissue blood supply (Nicoll & Webb, 1955) as well as contributing to peripheral vascular resistance (Gratton et al. 1998).
Most of our knowledge of spontaneous vasomotor activity is derived from studies of the contraction of vessel segments and strips of vascular tissue. Information about the mechanisms involved in generating and regulating this activity is limited because of the presence of multiple cell types in tissue preparations and an inability to dissociate changes in [Ca2+]i from changes in sensitivity of the contractile mechanisms. However, in recent years, measurements of [Ca2+]i in populations of cultured vascular smooth muscle have revealed that these cells, in isolation from other cell types, retain the ability to spontaneously generate transient increases in [Ca2+]i (Ca2+ spikes) that correspond to the spontaneous electrical activity of the plasma membrane (Weissberg et al. 1989; Byron & Taylor, 1993).
We have previously shown that the frequency of Ca2+ spiking in A7r5 cells is exquisitely sensitive to concentrations of AVP found in the systemic circulation (Byron, 1996). The present study examines in more detail the signal transduction pathways involved in the stimulation of Ca2+ spiking by AVP, focusing specifically on the role of PKC.
The PKC family of lipid-regulated serine/threonine kinases comprises at least eleven identified isoforms of PKC, which have been grouped into three classes. Conventional PKCs (, I, II and ) are activated by Ca2+ and/or by diacylglycerol (DAG) and phorbol esters. Novel PKCs (, , 
and 
) are Ca2+-independent isoforms that are also activated by DAG and phorbol esters, whereas atypical PKCs (, ) are independent of both Ca2+ and DAG/phorbol esters (Ron & Kazanietz, 1999). Activation of conventional and novel PKCs by DAG or phorbol esters is thought to involve a conformational change that leads to a subcellular redistribution or translocation of the PKC from cytosolic to membrane compartments.
It is well known that activation of G-protein-coupled receptors by vasoconstrictor hormones, such as AVP, angiotensin II, endothelin and noradrenaline, leads to activation of PKC. Early studies implicated PKC in mitogenic responses (Rozengurt, 1986) as well as in the regulation of vascular smooth muscle contractility (Rasmussen et al. 1984). With the more recent identification of multiple PKC isoforms with distinct tissue distributions and regulatory properties (Hofmann, 1997; Ron & Kazanietz, 1999), the specific PKC isoforms that are activated by vasoconstrictors and their roles in specific signalling pathways are just beginning to be characterized. Our findings suggest a central role for PKC in the novel signalling pathway linking physiological concentrations of AVP with an increase in the frequency of Ca2+ spiking.
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METHODS |
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Materials
Cell culture media were from Gibco-BRL or MediaTech (Herndon, VA, USA). Fura-2 AM, fura-2 pentapotassium salt and Pluronic F127 were from Molecular Probes, Inc. Anti-PKC antibodies were from Transduction Laboratories (San Diego, CA, USA). AVP was from Sigma. 4-Phorbol 12-myristate 13-acetate, 4-phorbol-12,13-didecanoate and Ro-31-8220 were from Calbiochem. Calyculin A was from RBI/Sigma.
Cell culture
A7r5 cells are well suited to studies of vascular smooth muscle physiology because, unlike primary vascular smooth muscle cultures, they retain a differentiated phenotype in culture, including stable expression of smooth muscle-specific proteins (Kimes & Brandt, 1976; Solway et al. 1995), electrophysiological characteristics of vascular smooth muscle such as the ability to generate spontaneous action potentials and Ca2+ spikes (Kimes & Brandt, 1976; Knot et al. 1991; Byron & Taylor, 1993), and coupling of adjacent cells by gap junctions (Moore et al. 1991). As a pure vascular smooth muscle cell preparation, A7r5 cells have been used extensively to study signal transduction and electrophysiological characteristics of vascular smooth muscle.
A7r5 cells were cultured as described previously (Byron & Taylor, 1993). Cells were subcultured onto rectangular (9 mm × 22 mm, no. 1½) glass coverslips or plastic tissue culture dishes (Corning, Acton, MA, USA). Confluent cells up to the 30th passage were used 2-5 days after plating.
[Ca2+]i measurements
Coverslips were washed twice with control medium (135 mM NaCl, 5·9 mM KCl, 1·5 mM CaCl2, 1·2 mM MgCl2, 11·5 mM glucose, 11·6 mM Hepes, pH 7·3) and then incubated in the same medium with 2 µM fura-2 AM, 0·1 % bovine serum albumin and 0·02 % Pluronic F127 detergent (Poenie et al. 1986) for 90-120 min at room temperature (20-23°C) in the dark. After loading, the cells were washed twice and incubated in the dark in control medium (or pretreated with drugs) for 1-5 h prior to the start of the experiment (calphostin C pretreatment required exposure to fluorescent room lights). Fura-2 fluorescence was then measured in cell populations with a Perkin-Elmer LS50B fluorescence spectrophotometer. This instrument is equipped with a rotating filter wheel, which was used to alternate 340 and 380 nm excitation; emitted fluorescence (at 510 nm) was collected at 0·5 s intervals. A coverslip was mounted vertically on a 30 deg angle to the light path in a cuvette, which was continuously perfused with media. A four-way valve mounted just above the cuvette allowed rapid switching of solutions; replacement of the medium bathing the cells had a half-time of approximately 20 s. The excitation light illuminated an area of approximately 30 mm2 on the coverslip for recording of fluorescence from several thousand cells. Background fluorescence was determined at the end of the experiment by quenching the fura-2 fluorescence for 10-15 min in the presence of 1 µM ionomycin and 6 mM MnCl2 in Ca2+-free medium. After background fluorescence was subtracted, the ratio of fluorescence at 340 nm to that at 380 nm was calculated and calibrated in terms of [Ca2+]i.
Calibration of fura-2 fluorescence in terms of [Ca2+]i was carried out as described previously (Byron & Villereal, 1989) using solutions of known Ca2+ concentration to construct a standard curve. The Ca2+ concentration of the standard solutions was calculated using MaxChelator software (version 6.60) which accounts for binding of Ca2+ to each constituent of the solution. For analysis of fluorescence ratios recorded from cells, the equation [Ca2+]i = KD ((r - Rmin)/(Rmax - r)) (Grynkiewicz et al. 1985) was fitted to the standard curve using SigmaPlot software (SPSS Inc., Chicago, IL, USA) and used to convert ratios (r) into [Ca2+]i. In situ calibration of fura-2 fluorescence by direct determination of minimum and maximum ratios (Rmin and Rmax, respectively; Grynkiewicz et al. 1985) from within cells yielded similar calibrated values (curve-fit values for Rmin, Rmax, KD and for in vitro and in situ calibrations were: Rmin, 0·59, 0·72; Rmax, 36·02, 38·5; KD, 141·9 nM, 135 nM; , 19·44, 18·5, respectively). Traces shown are representative of at least three similar experiments.
Immunoblotting to determine expression and translocation of PKC isoforms
A7r5 cells were grown to confluence on 100 mm plastic tissue culture dishes, washed and allowed to equilibrate for 2 h in control medium at room temperature. The medium was then aspirated and 0·5 ml lysis buffer added (1 % Triton X-100, 50 mM NaF, 2 mM EGTA, 2 mM EDTA, 500 µM sodium orthovanadate, 10 µg ml-1 aprotinin, 10 µg ml-1 leupeptin, 0·5 mM phenylmethylsulfonyl fluoride, 20 mM Tris, pH 7·4). The dish was immediately placed onto liquid nitrogen. The cells were then allowed to thaw on ice (
15 min), scraped off the dish, transferred into a microcentrifuge tube and briefly sonicated. The protein concentration in cell lysates was measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). To determine the expression of different PKC isoforms, 40 µg of protein was subjected to SDS-PAGE, electrophoretically transferred to a nitrocellulose membrane, and immunoblotted with monoclonal antibodies for different isoforms of PKC (, , , , , and ). After blotting, the membrane was washed and treated with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG). The different isoforms of PKC were visualized using enhanced chemiluminescence reagents (Amersham), exposed to Hyperfilm (Amersham), and quantified by laser densitometry.
To measure translocation of specific PKC isoforms from cytosolic to membrane compartments after treatment for varying times with 100 pM AVP, cells were lysed with lysis buffer without Triton X-100. Cell lysates were then centrifuged at 100 000 g for 30 min at 4°C and the supernatant saved as the cytosolic fraction. The pellet was then resuspended in lysis buffer with 1 % Triton X-100, and sonicated. The resuspended pellets were incubated in a shaking ice bath for 30 min, centrifuged at 14 000 g for 10 min, and the supernatant saved as the membrane fraction. Aliquots of the cytosolic and membrane fractions containing 40 µg of protein were subjected to electrophoresis and immunoblotting for isoforms of PKC as described above. Translocation of a given isoform was defined as an increase in immunoreactivity associated with the membrane fraction with a concomitant decrease in the cytosolic fraction.
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RESULTS |
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Evidence for activation of a serine/threonine kinase by low [AVP]
In the absence of stimulation, A7r5 cells exhibit sporadic low-frequency (on average, < 0·1 min-1; Byron, 1996) Ca2+ spikes. Concentrations of AVP 
10 pM do not normally affect this spontaneous activity (Fig. 1A and D). However, the serine/threonine phosphatase inhibitor calyculin A was found to dramatically sensitize A7r5 cells to AVP, resulting in a robust Ca2+-spiking effect at concentrations of AVP as low as 5 pM (Fig. 1B). Calyculin A alone did not stimulate Ca2+ spiking (Fig. 1C), suggesting that the substrate for the calyculin A-sensitive phosphatase is only phosphorylated in the presence of AVP.
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Fura-2-loaded A7r5 cell monolayers were exposed to low concentrations of AVP alone (A), AVP plus 5 nM calyculin A (B, cells were pretreated with calyculin A for 1 h prior to the start of recording) or calyculin A alone (C, cells were pretreated with calyculin A for 1 h prior to the start of recording). The boxes at the top of each panel indicate the duration of exposure to the agents. D, summarized results (means ± S.E.M.) from three experiments. The frequency of Ca2+ spiking was measured during the last 5 min in each concentration of AVP (asterisks denote significant difference from control, P < 0·05, Student's t test). | ||
Activation/inhibition of PKC
PKC is a serine/threonine kinase that might account for the stimulation of Ca2+ spiking by AVP. We tested the PKC activator PMA and found that at concentrations as low as 100 pM, PMA dramatically enhanced AVP-stimulated Ca2+ spiking (Fig. 2A). The frequency of Ca2+ spiking in the presence of 50 pM AVP was significantly increased (325 %) by 100 pM PMA (2·1 ± 0·4 spikes min-1 in AVP alone vs. 6·8 ± 0·5 spikes min-1 in AVP + PMA, means ± S.E.M., n = 4, P < 0·01, Student's paired t test). PMA alone was also sufficient to stimulate Ca2+ spiking. Low concentrations of PMA (0·1-1 nM) produced sustained Ca2+ spiking after a variable delay. The delay decreased and the frequency of spiking increased with increasing [PMA], but the Ca2+-spiking response became transient at high [PMA] (10- 200 nM) and was followed by a decline in [Ca2+]i to a sustained level that remained elevated above baseline (Fig. 2B). An inactive phorbol ester, 4-phorbol-12,13-didecanoate (1 nM), did not stimulate Ca2+ spiking (not shown).
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A, a low concentration of PMA (100 pM, filled boxes) reversibly enhances the Ca2+-spiking response to submaximal [AVP] (50 pM, open box). B, [Ca2+]i recordings from different coverslips exposed to increasing concentrations of PMA (filled boxes) illustrating a concentration-dependent decrease in latency and increase in frequency of Ca2+ spiking. The Ca2+-spiking response to 10-200 nM PMA was transient and was followed by a decline in [Ca2+]i to a sustained plateau. | ||
The specific PKC inhibitors Ro-31-8220 (1 µM) and calphostin C (250 nM) prevented PMA-stimulated Ca2+ spiking, as well as the stimulation of Ca2+ spiking by AVP (Fig. 3). Complete inhibition of Ca2+ spiking was observed whether AVP and PMA were administered together (Fig. 3B) or individually (Fig. 3D and F). The mean steady-state frequency of Ca2+ spiking in response to 1 nM PMA alone was 3·5 ± 0·5 spikes min-1 (n = 5) and in response to 100 pM AVP alone was 2·9 ± 0·4 spikes min-1 (n = 7). No spiking was observed in response to either agent in the presence of 1 µM Ro-31-8220 (n = 2 for PMA, n = 4 for AVP) or 250 nM calphostin C (n = 3 for both PMA and AVP; both Ro-31-8220 and calphostin C significantly inhibited both PMA- and AVP-stimulated Ca2+ spiking, P < 0·01, Student's t test). In control experiments, Ro-31-8220 (1 µM) did not inhibit Ca2+ spiking induced by 1·5 mM BaCl2 (not shown), and calphostin C (250 nM) did not inhibit the elevation of [Ca2+]i in response to increased extracellular [K+] (not shown), arguing against non-specific effects of these drugs on Ca2+ transport processes.
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A, control recording from a coverslip treated with 1 nM PMA (open box) and 50 pM AVP (filled box). B, cells were pretreated for 1 h with 1 µM R0-31-8220, then stimulated with 1 nM PMA (open box) and 50 pM AVP (filled box) in the presence of Ro-31-8220 (1 µM). C, control response to 1 nM PMA (filled box). D, response to 1 nM PMA in cells pretreated for 1 h with 250 nM calphostin C. E, control response to 100 pM AVP (filled box). F, cells were pretreated for 1 h with 250 nM calphostin C, then stimulated with 100 pM AVP (filled box) in the presence of 250 nM calphostin C (open box). A 1 h pretreatment with calphostin C (e.g. D) was sufficient to block the Ca2+-spiking response to either PMA or AVP (the drug did not have to be present during the recording). | ||
Expression and translocation of PKC isoforms
Western blot analysis using isoform-specific anti-PKC monoclonal antibodies detected immunoreactivity of , , , , , and isoforms of PKC (Fig. 4). The expression of PKC- in A7r5 cells (50 µg protein loaded) was extremely low compared with a positive control (rat brain homogenate, 2 µg protein loaded).
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A, Western blot analysis of A7r5 cell homogenates reveals the presence of PKC- | ||
Activation of PKC is generally associated with translocation of the kinase from the cytosol to membranes. Translocation of specific isoforms of PKC was measured by treating A7r5 cells with 100 pM AVP for varying times, fractionating the cells to produce fractions enriched in cytosol or membranes, and determining relative amounts of PKC-, -, - or - in these fractions by Western blotting. To examine which isoforms might be involved in the stimulation of Ca2+ spiking by AVP, we focused on the conventional and novel PKC isoforms, because these isoforms should be sensitive to phorbol esters and might therefore account for the stimulation of Ca2+ spiking by PMA. The results, presented in Fig. 5, show that 100 pM AVP induced an increase in PKC-, - and - in the membrane fraction with a concomitant decrease in these isoforms in the cytosolic fraction. PKC- was rapidly translocated to the membrane within 1 min of the start of AVP treatment, reached a maximum at 5 min, and declined to its basal distribution by 20 min (Fig. 5A). In contrast, translocation of PKC- and - was slower, not reaching a statistically significant redistribution until after 10 and 5 min of AVP treatment, respectively (Fig. 5B and C). PKC- translocation was transient, declining towards its basal distribution by 30 min, whereas PKC- translocation to the membrane fraction was sustained for at least 30 min. In two experiments, PKC- was detected only in the cytosolic fraction and was not affected by treatment for up to 30 min with 100 pM AVP (not shown). A representative time course for the stimulation of Ca2+ spiking by 100 pM AVP in Fig. 5D shows that this response began after a lag of several minutes (4·2 ± 0·6 min, n = 34) and was generally sustained for as long as AVP was present (30-60 min in several experiments).
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, - and - from cytosol to membranes in response to 100 pM AVP
A7r5 cell monolayers were treated for varying times with 100 pM AVP, lysed, and separated into cytosolic and membrane fractions. The amount of PKC- | ||
Downregulation of PKC prevents AVP- or PMA-stimulated Ca2+ spiking
Chronic treatment with a high concentration of PMA is often used as a tool for downregulating PKC. We examined the effects of a 24 h pretreatment with 1 µM PMA or vehicle (control) on the expression of PKC-, - and - protein in cytosolic and membrane fractions. This PMA pretreatment dramatically reduced the detectable immunoreactivity of PKC- and - in both cytosol and membrane fractions (Fig. 6). In contrast, PKC- immunoreactivity was only moderately reduced, but was completely restricted to the membrane. Examination of [Ca2+]i responses to 100 pM AVP or 1 nM PMA in PMA-pretreated A7r5 cells revealed a complete inhibition of Ca2+-spiking responses to either agent (Fig. 7C and D). In three paired experiments measuring the Ca2+-spiking responses to 1 nM PMA or 100 pM AVP with vehicle (control) or 1 µM PMA pretreatment, the frequency of Ca2+ spiking in response to 1 nM PMA in control cells was 2·56 ± 0·29 spikes min-1 and in control cells in response to 100 pM AVP was 2·90 ± 0·41 spikes min-1; no spiking was observed in response to either agent in the PMA-pretreated cells (significant inhibition, P < 0·001, Student's t test). PMA pretreatment did not, however, prevent the release of Ca2+ from intracellular stores by 1 µM AVP (Fig. 8A), or the increase in [Ca2+]i induced by high extracellular [K+] (Fig. 8B), suggesting that the PMA pretreatment did not interfere with other AVP-stimulated signalling processes or alter the Ca2+ transport mechanisms present in the cells.
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and - expression; PKC- is translocated to the membrane fraction
A7r5 cells were treated for 24 h with 1 µM PMA or vehicle (Control), then lysed and fractionated as described in Fig. 5. Western blot analysis reveals a loss of PKC- | ||
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Representative [Ca2+]i recordings from A7r5 cells treated with 100 pM AVP (A and C) or 1 nM PMA (B and D) after 24 h pretreatment with vehicle (A and B) or 1 µM PMA (C and D) revealing a complete loss of Ca2+-spiking responses in PMA-pretreated cells. | ||
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A, A7r5 cells were pretreated for 24 h with 1 µM PMA. [Ca2+]i responses reveal that despite the absence of a Ca2+-spiking response to 1 nM PMA (open box), the cells nonetheless respond to 1 µM AVP (filled box) with a robust increase in [Ca2+]i. B, increasing extracellular [K+] (from 5·9 to 59 mM, filled box) has been shown previously to produce a large increase in [Ca2+]i that can be completely inhibited by blockers of L-type voltage-sensitive Ca2+ channels (Byron & Taylor, 1993; Byron 1996). This response is preserved in A7r5 cells pretreated for 24 h with 1 µM PMA. | ||
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DISCUSSION |
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The serine/threonine phosphatase inhibitor calyculin A was found to dramatically sensitize A7r5 cells to AVP (Fig. 1). This suggests that a serine- and/or threonine-phosphorylated molecule is involved in the stimulation of Ca2+ spiking. Since calyculin A alone did not stimulate Ca2+ spiking, it may be argued that the substrate for the calyculin A-sensitive phosphatase is not phosphorylated in the absence of AVP. Furthermore, these results suggest that AVP, even at concentrations as low as 1-5 pM, activates a serine/ threonine kinase, and that the phosphorylated substrates are normally dephosphorylated by a calyculin A-sensitive phosphatase. We might speculate that, at very low [AVP] (< 10 pM), the phosphorylated product does not normally accumulate because the phosphatase activity out-balances the kinase activity. However, at higher concentrations of AVP (> 10 pM), the kinase activity is sufficiently increased to overcome the phosphatase activity, the phosphorylated substrate accumulates, and this leads to stimulation of Ca2+ spiking.
PKC may be a serine/threonine kinase that is involved in the stimulation of Ca2+ spiking by AVP because (1) Ca2+ spiking was stimulated by low concentrations ( 1 nM) of the PKC activator PMA and (2) both AVP- and PMA-stimulated Ca2+ spiking were prevented by the PKC inhibitors Ro-31-8220 and calphostin C, or by downregulation of expression of PKC isoforms by prolonged PMA pretreatment. The finding that a concentration of AVP that stimulates Ca2+ spiking induces the translocation of at least three PKC isoforms from cytosolic to membrane compartments further supports a potential role for PKC in the signalling pathway.
At least two signalling pathways activated by AVP may lead to PKC activation. It is well known that phospholipase C (PLC) is activated by AVP, though not very robustly at the concentrations of AVP that stimulate Ca2+ spiking. Nonetheless, DAG produced as a result of submaximal PLC activity may be adequate to activate one or more PKC isoforms. We also have preliminary results which suggest that AVP-stimulated Ca2+ spiking involves phospholipase D (PLD; Li et al. 1999). It is possible that DAG is produced by PLD activity and the subsequent dephosphorylation of phosphatidic acid. The roles of PLC and PLD in AVP-stimulated Ca2+ spiking are currently under investigation.
We found that submaximal concentrations of AVP and PMA were additive in stimulating Ca2+ spiking (Fig. 2A). Although this observation is consistent with the possibility that the two agents are acting through the same mechanism to stimulate Ca2+ spiking, additivity of the responses to maximal concentrations of AVP and PMA could not be assessed. High concentrations of both AVP (Knot et al. 1991; Byron, 1996) and PMA (Fig. 2B) inhibit Ca2+ spiking (> 400 pM and > 1 nM, respectively).
The stimulation of Ca2+ spiking by low concentrations of PMA implicates PKC in a signalling pathway that leads to activation of L-type voltage-sensitive Ca2+ channels. Sperti & Colucci (1987) reported that low concentrations of PMA ( 3 nM) stimulated dihydropyridine-sensitive 45Ca2+ uptake in A7r5 cells. However, low concentrations of phorbol esters have been reported to have little or no effect on L-type Ca2+ currents in A7r5 cells (Satoh & Sperelakis, 1995; Obejero-Paz et al. 1998), suggesting that the stimulation of Ca2+ spiking by PMA may not be due to a direct stimulation of L-type Ca2+ channels. PKC activation may lead to membrane depolarization by inhibition of delayed rectifier K+ currents (Clément-Chomienne et al. 1996; Shiels et al. 1998); this may then indirectly activate voltage-sensitive L-type Ca2+ channels and Ca2+ spiking.
The inhibition of Ca2+ spiking by high concentrations of PMA or AVP suggests that PKC may also play a role in a negative feedback pathway that limits AVP-induced Ca2+ signals. This possibility is supported by other studies which have shown that dihydropyridine-sensitive 45Ca2+ uptake and L-type Ca2+ currents in A7r5 cells were inhibited by chronic (1 h) exposure to high concentrations of PMA (IC50 = 25 nM) or acute exposure (10 min) to 100 nM AVP (Galizzi et al. 1987; Van Renterghem et al. 1988).
Several PKC isoforms are expressed in A7r5 cells. Expression of PKC-, -, -, - and - has been reported by other groups (Boscoboinik et al. 1994; Wang et al. 1997; Ricciarelli et al. 1998; Kaplan-Albuquerque & DiSalvo, 1998). We also detected PKC- immunoreactivity, as did Kaplan-Albuquerque & DiSalvo (1998). However, the latter study attributed it to cross-reactivity of the anti-PKC- antibodies with PKC-. We found that PKC- and PKC- immunoreactivity had different basal distributions (PKC- was only cytosolic) and PKC-, but not PKC-, translocated from cytosol to membrane in response to 100 pM AVP.
The stimulation of Ca2+ spiking by PMA implicates a phorbol ester-sensitive PKC isoform in the signalling pathway. We found that PKC-, - and - were all translocated from cytosolic to membrane compartments in response to AVP. Our results do not conclusively determine which of these isoforms is/are important. Pretreatment with 1 µM PMA resulted in downregulation of expression of PKC- and -, but not PKC-, and loss of Ca2+-spiking responses, perhaps implicating PKC- or - in the pathway. However, the distribution of PKC- was also altered following PMA pretreatment and this might have resulted in the loss of the Ca2+-spiking response. The time course for translocation of PKC- to the membrane fraction was similar to the time course for stimulation of Ca2+ spiking by 100 pM AVP - both responses were sustained for at least 30 min. PKC- has been implicated in signalling by vasoconstrictor agonists, including the Ca2+-independent modulation of contractility (Walsh et al. 1996), stimulation of mitogen-activated protein kinases by angiotensin II (Malarkey et al. 1996) and inhibition of delayed rectifier K+ channels by angiotensin II (Clément-Chomienne et al. 1996). Molecular approaches that alter the expression or activity of each of the individual PKC isoforms will be required to determine unambiguously which are essential for AVP-stimulated Ca2+ spiking.
In summary, our results indicate that physiological concentrations of AVP activate several PKC isoforms and that one or more of these isoforms is essential for the stimulation of Ca2+ spiking in A7r5 cells. Previous work from our own and other laboratories has demonstrated that L-type voltage-sensitive Ca2+ channels are essential for the Ca2+-dependent action potentials that underlie the Ca2+ spiking in A7r5 cells (Kimes & Brandt, 1976; Van Renterghem et al. 1988; Marks et al. 1990; Knot et al. 1991; Byron & Taylor, 1993; Byron, 1996). Action potentials recorded from vascular smooth muscle cells in vivo (Nicoll, 1975; Gokina et al. 1996) correlate with rhythmic vasomotion. Numerous studies have established that vasomotion depends on activation of L-type Ca2+ channels (Colantuoni et al. 1984; Taga et al. 1990; Raddino et al. 1996) and can be stimulated by vasoconstrictor agonists, including AVP (Gerstberger et al. 1987; Fujii et al. 1990; Raddino et al. 1996). The modulation of this activity by AVP may represent a novel signalling pathway in which PKC plays a central role in the regulation of vascular smooth muscle excitability by a vasoconstrictor agonist.
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REFERENCES |
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The authors gratefully acknowledge the technical support of Matt Hammoudeh and John Barakat.
This work was supported by the Eugene J. and Elsie E. Weyler Endowment for Clinical Cardiology Research, the John and Marion Falk Trust for Medical Research, and the National Heart, Lung, and Blood Institute (1R01 HL60164-01A1).
Corresponding author
K. L. Byron: Loyola University Medical Center, Cardiovascular Institute, 2160 South First Avenue, Maywood, IL 60153, USA.
Email: kbyron{at}luc.edu
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