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EXTRACELLULAR MATRIX, CELL INTERACTIONS
Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires, Argentina
Submitted 10 August 2006 ; accepted in final form 11 May 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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phosphatases; protein kinase A; protein kinase C; epidermal growth factor
Changes in [Ca2+]i may be due to release from the intracellular Ca2+ stores or influx from the extracellular medium through the opening of plasma membrane Ca2+ channels.
Pituitary cells are equipped with voltage-dependent Ca2+ channels (VDCC), which mediate Ca2+ influx in response to membrane depolarization (20, 41, 47). VDCC are multimeric proteins composed of 
1-, 
-, 2
-, and 
-subunits (6). The pore-forming 1-subunit accounts for the voltage dependence of the channel. These channels are classified on the basis of the gene encoding the pore subunit and their electrophysiological and pharmacological properties (44). The L-type Ca2+ channel is the primary voltage-dependent Ca2+-influx pathway in many excitable and other secretory cells. Its pore-forming 1-subunit contains NH2- and COOH-terminal cytosolic domains, which are potential targets for protein phosphorylation (44). Therefore, the activity of L-type Ca2+ channels can be regulated by different types of kinases, such as PKA and PKC. Both are Ser/Thr kinases that have been reported to mediate activation and/or inhibition of L-type Ca2+ channel function, depending on the tissue, by phosphorylation of the 1-subunit and its auxiliary subunits (27, 29).
An emerging body of evidence suggests that VDCC are also regulated by phosphorylation on tyrosine residues (43, 50). Several growth factors activate receptor tyrosine kinases (RTKs) and trigger complex intracellular signal transduction pathways, finally leading to cell proliferation in different cell types (35). Ca2+ entry from extracellular medium is known to be a widespread signal that plays a key role in these events. However, the nature of the Ca2+ channels involved and a possible regulation through direct channel phosphorylation by RTKs remain controversial and differ among tissues. On the other hand, a direct Ca2+ channel regulation by the c-Src family of cytosolic tyrosine kinases (TKs) has been suggested, but contradictory results have been reported (38, 52).
Less is known about the role of VDCC regulation by TKs in pituitary GH3 cells, a clonal cell line established in 1965 by A. H. Tashjian, Jr., from a pituitary tumor carried in a 7-mo-old female Wistar-Furth rat (46) that secretes growth hormone and prolactin.
It has been reported that TK activation increases L-type Ca2+ channel function, whereas tyrosine phosphatases play an inhibitory role in this cell line (5). However, the nature of the TKs remains unknown.
Because 
10 different types of Ca2+ channel 1-subunits (6) have been cloned and characterized and their expression differs among tissues, L-type Ca2+ channel regulation is tissue specific. With regard to GH3 cells, different pore-forming 1-subunits of L-type Ca2+ channels were expressed: 1.1, 1.2, and 1.3 (20).
The aim of our study was to evaluate the participation of several kinases in the depolarization of Ca2+ channels in GH3 cells. [Ca2+]i was measured second-by-second via a spectrofluorometric method with fura 2-AM as a fluorescent indicator. KCl (12.5 mM) was used as the depolarization stimulus to activate and open VDCC and, thereby, enhance [Ca2+]i in pituitary cells (22).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Cells were incubated in a humid atmosphere of 5% CO2-95% O2 at 37°C. The incubation medium was changed every 2–3 days. Cells were harvested once per week by treatment with phosphate-buffered saline containing trypsin (2.5 mg/ml; GIBCO), and reseeding was carried out at 20% of the original density.
Drugs. Chelerythrine, genistein, and okadaic acid (sodium salt) were obtained from Alomone Labs (Jerusalem, Israel) and tyrphostin AG-1478 and a specific Src kinase inhibitor (PP1) from Biomol Research Laboratories (EI-275, Plymouth Meeting, PA). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified.
[Ca2+]i measurements. Fura 2-AM (fura 2-tetra-acetoxymethyl ester; Molecular Probes, Eugene, OR) was used as a fluorescence indicator. Cells were harvested and washed in medium and then redispersed and incubated in a buffered saline solution (BSS; in mM: 140 NaCl, 3.9 KCl, 0.7 KH2PO4, 0.5 Na2HPO4·12H2O, 1 CaCl2, 0.5 MgCl2, and 20 HEPES, pH 7.5) in the presence of 2 mM fura 2-AM, 10 mM glucose, and 0.1% BSA. Cells were incubated for 30 min at 37°C in an atmosphere of 5% CO2, during which fura 2 is trapped intracellularly by esterase cleavage. Cells were then washed twice in BSS without fura 2-AM and brought to a density of 2 x 106 cells/ml BSS. Fluorescence was measured in a spectrofluorometer (Jasco, Tokyo, Japan) equipped with an accessory to measure Ca2+ with continuous stirring (model CA-261), a thermostat adjusted to 37°C, and an injection chamber. [Ca2+]i was registered every second by exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 505 nm was measured. In this way, light intensities and the ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/F380) were followed. Drugs were injected (5 µl) into the chamber as a 100-fold concentrated solution without interruption of recording. The preparation was calibrated by determination of maximal fluorescence induced by 0.1% Triton X-100 and minimal fluorescence in the presence of 6 mM EGTA (pH adjusted to >8.3). [Ca2+]i was calculated according to Grynkiewicz et al. (23). Values were corrected for dye leakage as described elsewhere (21, 23) and for autofluorescence using unlabeled cells. Dye leakage and autofluorescence were minimal.
The response of GH3 cells to 12.5 mM KCl in buffer with or without Ca2+ is shown in Fig. 1. In the latter case, cells were incubated with fura 2-AM, washed, and resuspended in BSS without CaCl2, and 1 min later, 60 µM EGTA was added, as previously described (22, 45).
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The peak [Ca2+]i response, plateau phase, and basal [Ca2+]i were compared by paired t-test (see Figs. 1, 3, 4, 5, 
7, and 9). The effects of AG-1478, vanadate, and H89 on peak values or plateau levels were analyzed by one-way ANOVA followed by Tukey's honestly significant difference test. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Changes in [Ca2+]i may be due to Ca2+ release from the intracellular Ca2+ stores or influx from the extracellular medium through the opening of plasma membrane Ca2+-permeable channels. To show the importance of extracellular Ca2+ influx in the K+ response, we demonstrated that the GH3 cells' response to depolarization was lost in Ca2+-free medium (P = 0.00013; Fig. 1A, EGTA). The absence of the peak and plateau phases in the absence of extracellular Ca2+ indicates that the [Ca2+]i response to K+ represents Ca2+ entry. Furthermore, baseline [Ca2+]i decreased in Ca2+-free medium (P = 0.0015; Fig. 1A, inset).
To evaluate the contribution of L-type channels to the Ca2+ influx in GH3 cells, we tested the effect of nifedipine, a dihydropyridine that inhibits L-type Ca2+ channels, on Ca2+ influx evoked by 12.5 mM KCl. Nifedipine significantly decreased baseline [Ca2+]i by 24.3 ± 2.4% (P = 0.00041; Fig. 1B, inset, at 2 min), confirming an active participation of L-type Ca2+ channels in the maintenance of basal [Ca2+]i. On the other hand, nifedipine markedly (but not totally) decreased K+-induced Ca2+ mobilization: maximal peak values were 308.3 ± 22.9% with buffer and 140.8 ± 9.9% with nifedipine (P = 2.4 x 10–5), and plateau (average at 6.5–8 min) values were 235 ± 20.3% with buffer and 123 ± 4.2% with nifedipine (P = 0.00046; Fig. 1B). In a different set of experiments in which nifedipine was applied 1 min after K+-induced depolarization, the return of [Ca2+]i to basal values was striking and the plateau response was totally abolished (Fig. 1C), showing that the contribution of the L-type Ca2+ channel to Ca2+ influx is essential and more important during the plateau phase than in the peak phase. The average plateau values at 3.05–5 min were 227.2 ± 16.1 with buffer and 97.2 ± 5.9% with nifedipine (n = 7, P = 0.00018).
It has been demonstrated that L-type Ca2+ channels can be regulated by several types of kinases in different tissues (7, 12, 28, 36). Previous studies confirmed that PKA and PKC phosphorylate serine and threonine residues on the - and -subunits of the L-type Ca2+ channel (34, 36, 53). Therefore, to evaluate the contribution of PKA to depolarization-induced Ca2+ entry in GH3 cells, we tested the effect of N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide hydrochloride (H89), a selective PKA inhibitor, on Ca2+ mobilization evoked by 12.5 mM KCl.
Application of 0.1, 1, 10, 50, and 100 µM H89 1 min before addition of K+ significantly reduced depolarization-induced Ca2+ mobilization. The effect was a concentration-related event, and the higher doses (50–100 µM) abolished Ca2+ entry totally, suggesting that PKA exerted a strong and positive control on VDCC opening (Fig. 2, A and C).
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These results show not only a positive effect of PKA on depolarization-induced Ca2+ mobilization but, also, suggest a marked effect on the regulation of channel inactivation because of the important PKA-induced modulation of L-type Ca2+ channel function in GH3 cells.
PKC activation by phorbol esters has been well reported (4). To ascertain whether phorbol ester-sensitive PKC is essential not only in opening of VDCC by depolarization but, also, in regulation of the open state during the plateau phase, we studied the effect of PMA applied before and after K+ on K+-induced Ca2+ mobilization in GH3 cells. Application of 100 nM PMA 2 min before addition of K+ partially, but significantly, reduced the peak and plateau depolarization-evoked increase in [Ca2+]i: relative peak values were 294.9 ± 21.4% with PMA and 358.3 ± 17.9% with buffer (P = 0.0020), and average plateau values at 4.5–6 min were 275.5 ± 13.4% with buffer and 192.4 ± 12.6% with PMA (P = 3.97 x 10–5; Fig. 3A). Furthermore, when PMA was applied 1 min after the K+ stimulus, the total Ca2+ influx during the subsequent minutes of the plateau phase was significantly reduced: average influx at 4–6 min was 214 ± 11.8% with K+ and buffer and 152 ± 2.8% with K+ and PMA (P = 0.0021; Fig. 3B). These observations were confirmed using the selective PKC inhibitor chelerythrine (Fig. 3): K+-induced Ca2+ influx was markedly increased when PKC action was inhibited with the application of 1 µM chelerythrine 2 min before the K+ stimulus. In chelerythrine-treated cells, [Ca2+]i reached a peak of 488.9 ± 17.5% vs. 342.9 ± 22.2% in buffer-treated cells (P = 0.0019). The average of the plateau values was increased by chelerythrine to 375.6 ± 10.2% vs. 271.4 ± 16.9% in buffer-treated cells (P = 0.00050). These results suggest an inhibitory influence of PKC on VDCC function not only on the activation induced by depolarization but, also, on the maintenance of the open state. Even though chelerythrine did not affect baseline [Ca2+]i, PMA evoked a transient increase in basal [Ca2+]i during the first 2 min (P = 0.0060; Fig. 3A, inset).
To investigate the involvement of TKs on VDCC activity in GH3 cells, we first studied the effect of genistein (a TK inhibitor that acts by binding to the ATP site of the TK) on depolarization-induced [Ca2+]i mobilization. Figure 4 shows that application of 25 µM genistein 2 min before the K+ stimulus significantly reduced the depolarization-induced Ca2+ influx. The average plateau [Ca2+]i values were 233 ± 11.4% with genistein and 307 ± 17.9% with buffer (P = 0.0076). Moreover, baseline [Ca2+]i was reduced by 18% immediately after genistein (P = 0.00073; Fig. 4, inset). This result implies that TKs are involved in the maintenance of baseline [Ca2+]i as well as in the depolarization-induced Ca2+ entry in GH3 cells.
Inasmuch as several growth factors have been reported to induce Ca2+ influx in many cell types, activating their specific receptors with intrinsic TK activity (RTKs), we studied the involvement of growth factor receptors in modulating the activation of membrane Ca2+ channels in GH3 cells. Addition of 10 ng/ml epidermal growth factor (EGF; Fig. 5A) or 30 ng/ml type A vascular endothelial growth factor (VEGF; Fig. 6B) to the cell suspension enhanced K+-induced Ca2+ mobilization. The average plateau values were 350 ± 12.4% with EGF vs. 284 ± 6.5% with buffer (P = 0.0021) and 325 ± 9.6% with VEGF vs. 275 ± 9.6% with buffer (P = 0.0037). On the other hand, 10 ng/ml insulin-like growth factor-I (IGF-I; Fig. 5C) did not modify the depolarization-induced increase in [Ca2+]i. None of these growth factors had an effect on basal Ca2+ levels (not shown).
AG-1478, a selective EGF receptor (EGFR) TK inhibitor, decreased the depolarization-induced Ca2+ entry (Fig. 6, A and C). The peak [Ca2+]i mobilization decreased to 254 ± 9.4% in cells treated with 1 µM AG-1478 vs. 345 ± 4.5% in buffer-treated cells (P = 0.015), and the average relative plateau value was reduced to 165.3 ± 6.8% in cells treated with 1 µM AG-1478 and 181.0 ± 6.9% in cells treated with 500 nM AG-1478 vs. 212.8 ± 6.8% in buffer-treated cells. Moreover, when AG-1478 was applied 1 min after K+ stimulus, total Ca2+ influx during the subsequent 4 min of the plateau phase was significantly reduced at 200, 500, and 1,000 nM AG-1478 (Fig. 6, B and D; P < 0.002).
Non-RTKs, such as pp60 c-Src (c-Src), have also been involved in the control of Ca2+ signal transduction pathways, including interaction with Ca2+ channels in different tissues (38, 52). To study whether the depolarization-induced activation of VDCC involves c-Src kinase activity, we examined the effect of a selective c-Src kinase inhibitor, type 1 protein phosphatase (PP1), on depolarization-induced Ca2+ mobilization. Unexpectedly, PP1-induced inhibition of c-Src activity enhanced depolarization-induced Ca2+ mobilization. The maximal peak value was 408.9 ± 17.3% in PP1-pretreated cells vs. 334.5 ± 22.3% in buffer-treated cells (P = 0.0023), and the total plateau values during the following 1.5 min were 339.5 ± 11.6% in PP1-pretreated cells and 278.8 ± 16.9% in cells treated with buffer (P = 0.0103, Fig. 7).
To ascertain whether phosphatases affect VDCC activity in GH3 cells, we first studied the effect of orthovanadate, a tyrosine phosphatase inhibitor, on depolarization-induced [Ca2+]i mobilization. Figure 8, A and C, shows that different concentrations of orthovanadate decreased basal Ca2+ levels significantly (P = 2.14 x 10–6, for the effect of treatment). However, when K+ was applied, the absolute increase (difference between peak and basal values) in depolarization was not altered by vanadate, even though absolute peaks were lower. On the other hand, when vanadate was applied 1 min after the K+ stimulus, the plateau phase was immediately reduced in a concentration-related manner (Fig. 8, B and D). The average of plateau values between 3.05 and 5.05 min were 245 ± 11.8% with buffer and 158.2 ± 6.7% with 1 mM vanadate (P = 0.00022).
Finally, we studied the effect of okadaic acid, an inhibitor of Ser/Thr phosphatases, on Ca2+ channel function. Application of 1 or 50 nM okadaic acid 2 min after the K+ stimulus did not modify basal Ca2+ levels or K+-induced Ca2+ mobilization at 4 min (data not shown). Nevertheless, 20 min of pretreatment with 50 nM OK enhanced depolarization-induced Ca2+ influx. Averages of the plateau values at 25–26 min were 302.69 ± 6.5% with 50 nM okadaic acid and 235.5 ± 6.7% with buffer (P = 0.0077; Fig. 9).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Spontaneous oscillations in [Ca2+]i have been shown in the majority (
70%) of GH3 cells. The pattern of these oscillations varies considerably from cell to cell (42, 47), and these Ca2+ oscillations are completely abolished during perfusion with medium containing no added Ca2+, confirming that Ca2+ influx is necessary for the maintenance of basal [Ca2+]i and Ca2+ oscillations.
In GH3 cells exposed to depolarization induced by 12.5 nM K+, there was an immediate spike increase in [Ca2+]i followed by a sustained plateau. Changes in [Ca2+]i may be due to Ca2+ release from the intracellular Ca2+ stores or Ca2+ influx from the extracellular medium through the opening of plasma membrane Ca2+-permeable channels. We evaluated the effect of Ca2+-free medium on the response of GH3 cells to depolarization: the lack of the peak and plateau phases in the absence of extracellular Ca2+ indicates that the [Ca2+]i response to K+ represents Ca2+ entry. The significant reduction of the maximum peak value in nifedipine-pretreated cells shows the importance of the contribution of L-type Ca2+ channels to depolarization-induced Ca2+ and, furthermore, suggests that nifedipine-insensitive channels may also participate in K+-induced Ca2+ entry. These results are consistent with the finding that the GH3 cell line expresses different types of VDCC and, primarily, the L-type VDCC, as shown by Glassmeier et al. (20). Using the whole cell configuration of the patch-clamp technique and Ba2+ as the charge carrier, these authors described in detail the voltage-activated Ca2+ channel current components in GH3 cells. In addition to the L-type Ca2+ channel, three other high-voltage-activated Ca2+ channel current components were distinguished pharmacologically: 
-agatoxin IVA-sensitive current (22%, P-type), -conotoxin MVIIC-sensitive current (18%, Q-type), and toxin-resistant current (24%). Glassmeier et al. demonstrated that 49% of the total Ba2+ current amplitude in basal conditions is mediated by L-type Ca2+ channels, but they showed that nifedipine totally blocked action potential firing in GH3 cells. In our present study, application of nifedipine after K+-induced depolarization completely inhibited the plateau response. This may imply that the contribution of the L-type Ca2+ channel is the most important factor in the maintenance of depolarization-induced Ca2+ influx but that other VDCC, as well as the L-type Ca2+ channels, participate in the immediate peak [Ca2+]i response.
Numerous studies have attempted to unravel the mechanisms of VDCC modulation. Phosphorylation is one of the most common mechanisms that modifies ion channel activity. Moreover, L-type Ca2+ channel function is highly regulated by hormones and neurotransmitters, mainly through the activation of kinases and phosphatases.
Phosphorylations by PKC and/or PKA are important regulatory pathways; they show widely variable effects on L-type Ca2+ channel activity, depending, among others factors, on the tissue. Moreover, cross talk between PKA and PKC pathways in the modulation of channel function has been demonstrated (28, 53). These mechanisms have been studied in detail in different tissues, especially cardiomyocytes, but there is little information about kinase regulation of depolarization-induced Ca2+ influx in GH3 cells. Haymes et al. (24) demonstrated that PKC activation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate decreased depolarization-induced Ca2+ influx in GH3 cells. Using PMA and a PKC antagonist, we have demonstrated that PKC has an inhibitory influence on VDCC function and, furthermore, that PKC participates in the depolarization-induced activation, as well as maintenance, of the open state of the channels. These results were confirmed using the selective PKC inhibitor chelerythrine, which produced a clear increase in depolarization-induced Ca2+ influx.
MacEwan and Mitchell (33) demonstrated that Ca2+ influx through L-type Ca2+ channels in rat anterior pituitary cells can be modulated by PKC in opposite ways, depending on the experimental model. Activators of PKC, similar to phorbol esters, enhanced K+-induced 45Ca2+ influx in dispersed anterior pituitary cells. On the contrary, in the GH3 tumor cell line, the same authors showed, in accordance with our results, that phorbol esters inhibited K+-induced 45Ca2+ influx. Nevertheless, in this last situation, the cells were subjected to longer (10-min) incubations with phorbol esters, and we have shown that the effect is immediate. We previously demonstrated an overall alteration in Ca2+ metabolism in tumoral pituitary cells (13). Furthermore, different PKC isoforms are present in each tissue, and each isoform displays differential sensitivity to phorbol esters (32). The -, -, -, 
-, 
-, and 
-isoforms of PKC are present in rat anterior and posterior pituitary tissue, as well as in the GH3 cell line, but in different proportions (32). In particular, it has been demonstrated that the enhancement of prolactin secretion in tumoral pituitary cell lines is related to overexpression of PKC- (1). It may be possible that different PKC isoforms and different regulatory mechanisms (activation/inhibition) on VDCC are involved in the opposite effects observed in normal and tumoral pituitary cells.
On the contrary, a PKA antagonist, H89, had an inhibitory effect on K+-evoked Ca2+ influx, suggesting a positive contribution of PKA to depolarization-induced Ca2+ entry through VDCC in GH3 cells. Moreover, the high doses of H89 totally abolished Ca2+ entry. When applied after K+ stimulation, H89 reduced K+-evoked Ca2+ influx and significantly reduced the total Ca2+ entry during the plateau phase, and we previously showed that this influx primarily depended on the L-type Ca2+ channel.
It has been shown that both Ser/Thr kinases, PKC and PKA, phosphorylate the -subunit of L-type Ca2+ channels, even though this is not their only target. Puri et al. (36) demonstrated that all subunits of these channels are excellent substrates for PKA and PKC. Moreover, Gerhardstein et al. (19) showed in intact cardiac myocytes that the -subunit is a substrate for PKA. The -subunit contains more than one potential consensus site for PKA-mediated phosphorylation that is not a substrate for PKC. These sites are conserved in most -subunit isoforms and, thus, represent potential sites for regulation of channel activity.
More recently, it has been reported that PKA and PKC signaling pathways converge on the cardiac L-type Ca2+ channel complex at Ser1928 to increase channel activity, and a preassociation of the channel with PKC isoforms has been suggested (53). In the same way, direct phosphorylation of dihydropyridine-sensitive Ca2+ channels from skeletal muscle by PKA or PKC resulted in an activation of the channels that was observed as an increase in the rate and extent of Ca2+ influx (7, 12, 27). Nevertheless, our results clearly demonstrate opposite effects of PKC and PKA on depolarization-induced VDCC activation in GH3 cells.
The Ser/Thr phosphatases PP1 and PP2A, but not PP2B or PP2C, have been demonstrated to regulate PKA-stimulated L-type Ca2+ channels (11). It is well known that okadaic acid inhibits the Ser/Thr phosphatases PP2A (IC50 0.1 nM), PP1 (IC50 10 nM), and, also, although much less potently, PP2B (calcineurin, IC50 5 µM) (10, 40). Under our experimental conditions, 50 nM okadaic acid increased K+-evoked Ca2+ influx in GH3 cells. At the concentration used, PP2A and PP1 were inhibited, and those conditions favored Ca2+ entry, probably related to enhancement of PKA action. Nevertheless, the effect of okadaic acid was only observed in 20 min of preincubation assays.
Even though an active and direct participation of TKs in L-type Ca2+ channel function in different tissues was demonstrated (29, 38, 49, 52), less is known about VDCC regulation through tyrosine phosphorylation in GH3 cells. Cataldi et al. (5) showed that TK inhibition reduced L-type Ca2+ channel activity evoked by 55 mM K+ in GH3 cells. This led us to investigate possible TKs involved in VDCC activation. Genistein, a TK inhibitor, decreased total Ca2+ influx evoked by 12.5 mM K+ by 33%, indicating an involvement of TK in depolarization-induced VDCC activation. However, we observed that sodium orthovanadate, a protein tyrosine phosphatase inhibitor, not only decreased basal Ca2+ levels in GH3 cells but, also, significantly decreased Ca2+ influx in the plateau phase when it was applied after K+ depolarization, suggesting that different kinases might have differential effects on VDCC.
Several growth factor receptors have intrinsic TK activity, and it has been reported that they can regulate ion channel function (9, 18, 39). Tyrosine phosphorylation has been set forth as the mechanism involved in this regulation, because it was demonstrated that the effect of growth factors on channel activity can be blocked by TK inhibitors (9, 39). In GH3 cells, EGF and VEGF increased depolarization-evoked Ca2+ influx. Furthermore, the tyrphostin AG-1478, which specifically inhibits the EGFR kinase activity by competing for ATP binding, inhibited depolarization-evoked Ca2+ influx. Moreover, when AG-1478 was applied after the K+ stimulus, it significantly reduced the total Ca2+ influx during the plateau phase, suggesting a stimulatory effect of EGFR on L-type Ca2+ channel activity in GH3 cells, even in the absence of EGF. It is well known that EGFR can be activated by six ligands (EGF, transforming growth factor-, amphiregulin, heparin-binding EGF-like growth factor, -cellulin, and epiregulin) (14) and that, at least, transforming growth factor- is expressed in GH3 cells (15). Moreover, EGFR can be transactivated by other signaling pathways (14), as we demonstrated in pituitary cells (45). This might explain the mechanism of the inhibitory effect of AG-1478 on VDCC activity in GH3 cells.
In contrast with our results, it has been demonstrated that GH3 cells incubated for several days with 10 nM EGF responded to depolarization with a 30–65% smaller increase in 45Ca2+ uptake than untreated cells, and similar results were obtained by Fu et al. (16) in GH4C1 pituitary tumor cells. However, both studies were done in long-duration incubation conditions. Therefore, a more complex mechanism in the effect of EGF, such as protein synthesis and desensitization, may occur. In our experimental conditions, we describe an immediate and direct effect of EGF on channel activity.
It has been reported that VEGF and its receptor Flk-1 are expressed in GH3 cells (31, 48), and, furthermore, it has been shown that VEGF induces Ca2+ influx in epithelial cells. Here we show, for the first time, that VEGF receptor (VEGFR) activation stimulates depolarization-induced Ca2+ influx in GH3 cells, suggesting a positive effect of VEGFR, an RTK, on VDCC activity.
IGF-I has been involved in inhibitory and stimulatory effects on Ca2+ influx in different tissues (3, 9, 12, 39). Our failure to find an effect of IGF-I on K+-induced Ca2+ mobilization in GH3 cells suggests that the RTK effect is growth factor and tissue specific.
On the other hand, an effect of non-RTKs on channel regulation has been suggested. C-Src is a non-RTK, and it has been shown that c-Src family kinases could mediate tyrosine phosphorylation of the 1.2-subunit of L-type VDCC in the hippocampus, potentiating the channel currents (25). When we explored the c-Src contribution to VDCC activity in GH3 cells, we found that inhibition of c-Src with PP1 evoked an increase in depolarization-induced Ca2+ influx, suggesting an inhibitory effect of this kinase. Therefore, the effect of vanadate, a protein tyrosine phosphatase, which decreased VDCC activity, might be explained by the activation of endogenous c-Src kinase activity.
In vascular smooth muscle cells, genistein (a nonselective TK inhibitor), bistyrphostin (an RTK inhibitor), and PP1 inhibited L-type Ca2+ channel activity in a concentration-dependent manner, indicating a stimulatory effect of these kinases (52). On the other hand, an opposite action of endogenous c-Src was found in rabbit ear artery cells, where c-Src increased VDCC currents (51).
The inhibitory effect of this kinase on VDCC in GH3 cells described in the present study is in contrast to the positive action mediated by RTKs coupled to EGF and VEGF. In accordance with our results, two independent and competing TK signaling mechanisms that control of L-type Ca2+ current in feline atrial myocytes have been described (49): a positive action (channel activation) through membrane-bound TKs and an inhibitory effect by cytosolic TKs. Similar results were found in cardiac and smooth muscle L-type Ca2+ channel regulation (29), and, more recently, the inhibitory effect of c-Src was described in human atrial myocytes (38).
Precisely how c-Src modulates the L-type Ca2+ channel is uncertain, but functional and biochemical evidence closely links c-Src to the pore-forming 1-subunit of the L-type Ca2+ channel. c-Src has been found in immunoprecipitates of the 1.2-subunit of the L-type Ca2+ channel isolated from colonic smooth muscle (26).
Regulation of ion channel function is complex, differs among tissues, and involves many complex mechanisms, including Ser/Thr and tyrosine phosphorylations, as well as interactions between kinases. Differences among tissues in kinase activity on L-type Ca2+ channels may be due in part to the -subunit subtype expressed in each tissue and the experimental conditions that allow differences in basal kinase and phosphatase activities. At least 10 different types of Ca2+ channel 1-subunits (6), four types of -subunits (2), three types of 2-subunits (17), and five types of -subunits (30) have been cloned and characterized. However, pharmacological and electrophysiological studies have identified more subtypes of VDCC in excitable cells than the types of 1-subunits currently known (37).
In GH3 cells, expression of different pore-forming 1-subunits of L-type Ca2+ channels has been found: 1.1, 1.2, and 1.3 (20). Therefore, if we consider that more than one -subunit (and/or their splicing variants) coexist in the same tissue in combination with the variety of the other subunits, it is comprehensible that all may contribute to the pharmacological and electrophysiological diversity of Ca2+ channels in GH3 cells and to the differences between normal and tumoral cell pituitary responses.
The present study strongly demonstrates that different kinases regulate VDCC activity in the pituitary cell line GH3: PKA and RTKs, such as the VEGFR and EGFR, enhance depolarization-induced Ca2+ influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and, thereby, may participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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