©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein-tyrosine Kinases Activate while Protein-tyrosine Phosphatases Inhibit L-type Calcium Channel Activity in Pituitary GH Cells (*)

(Received for publication, June 13, 1995; and in revised form, February 1, 1996)

Mauro Cataldi Maurizio Taglialatela Salvatore Guerriero Salvatore Amoroso Gaetano Lombardi Gianfranco di Renzo (1) Lucio Annunziato (§)

From the Section of Pharmacology, Department of Neurosciences, University of Naples Federico II, 80131 Naples, Italy and the School of Pharmacy, University of Catanzaro, 88021 Catanzaro, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The aim of this study was to evaluate the effect of protein-tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) inhibitors on Ca channels in GH(3) cells. The activity of Ca channels was monitored either by single-cell microfluorometry or by the whole-cell configuration of the patch-clamp technique. Genistein (20-200 µM) and herbimycin A (1-15 µM) inhibited [Ca] rise induced either by 55 mM K or 10 µM Bay K 8644. In addition, genistein and lavendustin A inhibited whole-cell Ba currents. By contrast, daidzein, a genistein analogue devoid of PTK inhibitory properties, did not modify Ca channel activity. The inhibitory action of genistein on the [Ca] increase was completely counteracted by the PTP inhibitor vanadate (100 µM). Furthermore, vanadate alone potentiated [Ca] response to both 55 mM K and 10 µM Bay K 8644. The possibility that genistein could decrease the [Ca] elevation by enhancing Ca removal from the cytosol seems unlikely since genistein also reduced the increase in fura-2 fluorescence ratio induced by Ba, a cation that enters into the cells through Ca channels but cannot be pumped out by Ca extrusion mechanisms. Finally, in unstimulated GH(3) cells, genistein caused a decline of [Ca] and the disappearance of [Ca] oscillations, whereas vanadate induced an increase of [Ca] and the appearance of [Ca] oscillations in otherwise non-oscillating cells. The present results suggest that in GH(3) cells PTK activation causes an increase of L-type Ca channel function, whereas PTPs exert an inhibitory role.


INTRODUCTION

It has been largely demonstrated that the activity of L-type Ca channels can be regulated by different types of kinases, such as protein kinase A (PKA) (^1)(1, 2) and protein kinase C (PKC)(3, 4) . These two kinases phosphorylate serine (Ser) and threonine (Thr) residues on the alpha- and beta-subunits of these channel proteins(5, 6) . Recently, a great deal of interest in the literature has been devoted to another class of kinases, the protein-tyrosine kinases (PTKs)(7, 8, 9) . These enzymes, which exist both in transmembrane receptor-linked (7) or non-transmembrane forms(8, 9) , phosphorylate tyrosine (Tyr) residues on several cellular proteins. Since it has been recently reported that in non-excitable cells such as T-lymphocytes the overexpression of PTK activity, obtained transfecting these cells with the PTK-encoding oncogene v-src, induces a remarkable increase of basal and stimulated [Ca] levels(10) , it appeared of interest to explore the possibility that PTKs could modulate the activity of L-type Ca channels. For this purpose, the effect of the specific PTK inhibitors genistein(11, 12) , herbimycin A (13) , and lavendustin A (14) on the function of L-type Ca channels was evaluated in pituitary GH(3) cells (15) by single-cell microfluorometry and patch-clamp electrophysiology.

On the other hand, since PTK activity is functionally counteracted by protein-tyrosine phosphatases (PTPs)(16, 17) , the possible effect of the PTP inhibitor vanadate (18) on L-type Ca channels was also investigated.


EXPERIMENTAL PROCEDURES

Cell Culture

GH(3) cells were obtained from Flow Laboratories (Irvine, Scotland) and grown on plastic dishes in Ham's F-10 medium (Life Technologies, Inc., San Giuliano Milanese, Italy) with 15% horse serum (Flow, Irvine, Scotland), 2.5% fetal calf serum (HyClone, Logan, UT), 100 IU of penicillin/ml, and 100 µg of streptomycin/ml. Cells were cultured in a humidified 5% CO(2) atmosphere. Culture medium was changed every 2 days. For microfluorometric studies, cells were seeded on glass coverslips (Fisher) coated with poly-L-lysine (30 µg/ml) (Sigma). All the experiments were performed 2-4 days after seeding. The cells were at a culture passage between 34 and 60.

Intracellular Calcium Measurements

Intracellular calcium levels were measured using a microfluorometric technique, as reported previously(19) . Briefly, the cells, grown on glass coverslips, were loaded with 5 µM fura-2/AM for 1 h at room temperature in Krebs-Ringer saline solution (5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl(2), 1.5 mM CaCl(2), 10 mM glucose, 0.2% bovine serum albumin, and 10 mM Hepes/NaOH, pH 7.4). At the end of fura-2/AM loading, the coverslip was mounted in a perfusion chamber (Medical System Co., Greenvale, NY) on an inverted Nikon Diaphot fluorescence microscope. Throughout the experiment, the cells were superfused continuously with Krebs-Ringer saline solution using a peristaltic pump (Gilson, France) and a microtube, positioned with a macromanipulator on the cells under observation (Narishige, Japan). The perfusion medium was removed continuously from the perfusion chamber by suction using a microaspirator (Medical System Co.) connected with a vacuum pump (Hoofer, San Francisco). All drugs tested were introduced into the superfusion line using an injection loop and a two-way valve (Thomson, Springfield, VA). A 100-watt xenon lamp (Osram, Germany) with a computer-operated filter wheel bearing two different interference filters (340 and 380 nm) illuminated the microscopic field with uv light alternatively at the wavelength of 340 and 380 nm, with an interval of 500 ms between lighting at 340 and 380 nm. The interval between each couple of lighting and the next was chosen according to the experimental protocol. Emitted light was passed through a 400 nm dichroic mirror, filtered at 510 nm, and collected by a CCD camera (Photonic Science, Robertsbridge, East Sussex, UK) connected with a light amplifier (Applied Imaging Ltd., Dukesway Gateshead, UK). Images were digitized and analyzed with a Magiscan image processor (Applied Imaging Ltd.). The Tardis software (Applied Imaging Ltd.) calculated [Ca](i) corresponding to each couple of images, from the ratio between the intensity of the light emitted when the cells were lighted at 340 and 380 nm, using a calibration curve as reported previously(19) .

Electrophysiological Recordings

Ba currents were recorded using the whole-cell configuration of the patch-clamp technique. All experiments were performed at room temperature (20-22 °C) using a List EPC7 patch-clamp amplifier (Darmstadt, Germany). The patch pipette was filled with standard internal solution (110 mM CsCl, 10 mM tetramethylammonium chloride, 2 mM MgCl(2), 10 mM EGTA, 8 mM glucose, 10 mM Hepes, pH 7.3). Two mM ATP and 0.25 mM cAMP were added to the intracellular solution to prevent channel rundown. The cells were perfused continuously with a 10 mM Ba external solution (125 mM NaCl, 10 mM BaCl(2), 1 mM MgCl(2), 10 mM Hepes, 0.3 µM tetrodotoxin, pH 7.3) both with and without the drugs to be tested. The final pipette tip resistance was 3-5 megaohms when filled with the internal solution. The currents were filtered at 5 kHz. The sampling interval was 80 µs. From a holding potential of -90 mV the cells were depolarized to various potentials at a frequency of 0.2 Hz to minimize Ca channel rundown. The compensation of capacitative transients and leakage currents was performed both on-line by the clamp amplifier settings and off-line by subtracting Cd-insensitive currents (200 µM Cd).

Materials

All chemicals were of analytical grade and were purchased from Sigma. Genistein was obtained from BIOMOL Research Labs Inc. (Plymouth, PA). Lavendustin A, herbimycin A, Bay K 8644, daidzein, and fura-2/AM were purchased from Calbiochem. Nifedipine was a kind gift of Bayer AG (Germany).

Statistical Analysis

Data were analyzed by means of Student's t test for paired data or by analysis of variance followed by Scheffè test. Data are expressed as mean values ± S.E.


RESULTS

Inhibition of Ca Channel Activity by Genistein, Herbimycin A, and Lavendustin A

In GH(3) cells genistein, which inhibits PTKs by competing with ATP for the binding on these enzymes(11) , caused a dose-dependent (20-200 µM) inhibition of [Ca](i) elevation elicited by a superfusion medium containing 55 mM K (Fig. 1, A-C). The apparent IC of genistein effect (30 µM) on [Ca](i) is similar to that on PTK activity(11) . Herbimycin A, another PTK inhibitor that acts on these enzymes by a completely different mechanism, namely by direct binding on its reactive SH groups(13) , exerted a similar concentration-dependent inhibition on the [Ca](i) increase induced by 55 mM K (Fig. 1D).


Figure 1: Effect of genistein and herbimycin A on K-induced [Ca]increase in GH(3) cells. Panel A shows the effect on [Ca] of two consecutive 55 mM K pulses delivered with a 10-min interval. During the interval between the pulses, cells were perfused with Krebs-Ringer saline solution. Panel B shows the effect of genistein (200 µM), added to the perfusion 7 min before and throughout the whole second K pulse. The mean peak after the second 55 mM K pulse was significantly lower than the first one (p < 0.01). In addition, genistein significantly reduced basal [Ca] (124 ± 4 versus 104 ± 3 nM Ca; p < 0.01). In panel C the concentration dependence of the inhibitory effect of genistein on the [Ca] increase induced by 55 mM K is represented. Each point is the mean of 10-30 single-cell recordings. The solid line is the fit of the experimental points to the equation y = max/(1+(x/K), where K is the K for the block and n is the Hill coefficient. Panel D shows the effect of different concentrations of herbimycin A on the 55 mM K-induced [Ca]increase. * = p < 0.01 versus control group.



On the other hand, when GH(3) cells were superfused with two 10 µM consecutive pulses of the dihydropyridine activator of L-type Ca channels Bay K 8644(20, 21) , two equivalent elevations of [Ca](i) occurred (Fig. 2A). However, if genistein (200 µM) was superfused 5 min before the second pulse with the L-type Ca channel activator, a 40% reduction of the [Ca](i) increase was observed (Fig. 2B).


Figure 2: Effect of genistein on [Ca] increase induced by 10 µM Bay K 8644 in GH(3) cells. Panel A shows the effect on [Ca] of two 10 µM Bay K 8644 pulses delivered with an approximately 25-min interval. During the resting period between the two stimulations, the cells were perfused with Krebs-Ringer saline solution. Panel B shows the effect of 200 µM genistein added to the superfusion medium 5 min before and throughout the second Bay K 8644 pulse. Each trace is the mean of at least 30 single-cell recordings obtained during a single experiment representative of at least three other experimental sessions.



To identify more directly the target of PTK inhibition, Ba currents through Ca channels were recorded in GH(3) cells by means of the whole-cell configuration of the patch-clamp technique. From the holding potential of -90 mV, test potentials above -60 mV elicited large inward Ba currents, which peaked around -35 mV (Fig. 3E). At all the test potentials the currents displayed less than 10% inactivation during the 100-ms pulse duration (Fig. 3A). These properties suggest the presence of a large population of L-type Ca channels. This was further confirmed by the ability of the selective L-type Ca channel blocker nifedipine to inhibit approximately 80% of the whole-cell Ba currents (Fig. 4C). Perfusing GH(3) cells with the PTK inhibitor genistein (100 µM) caused a 50% reduction of the currents at all potentials tested (Fig. 3B). Complete suppression of the currents was achieved with 200 µM Cd (Fig. 3C). Upon extensive washout (5 min) Ba currents recovered (Fig. 3D). The extent of genistein-induced inhibition of Ba currents was comparable to that observed in microfluorometric studies (Fig. 4D). Lavendustin A (25 µM), another PTK inhibitor which could not be studied microfluorometrically because of its intrinsic fluorescence, also inhibited Ba currents (Fig. 4, A and D). By contrast, daidzein, the inactive analogue of genistein(12) , did not exert any influence on Ba currents (Fig. 4, C and D). It should be underlined that although nifedipine inhibition of Ba currents occurred with a very short latency (10 s), the effect of genistein required a longer period of time (30 s) (Fig. 4E).


Figure 3: Genistein inhibits voltage-dependent Ba currents in GH(3) cells. The same cell was recorded in control solution (panel A), 3 min after the exposure to 100 µM genistein (panel B), 1 min after the exposure to 200 µM Cd (panel C), and after 5 min of washout in 10 mM Ba control extracellular solution (panel D). The holding potential was -90 mV, and 100-ms depolarization steps from -80 to +25 in 15-mV increments were delivered. The data are shown without any leak subtraction procedure. Panel E shows the current to voltage (I/V) relationship for genistein-induced inhibition of voltage-dependent Ba currents. Current values were taken at the end of the depolarizing steps. Each point is the mean of three different cells recorded in the same experimental conditions. The data have been normalized to the peak value of the control I/V (-35 mV) for each cell, to facilitate comparison.




Figure 4: Comparison among the effects of genistein, lavendustin A, daidzein, and nifedipine on Ba currents in GH(3) cells. Panel A shows single-current traces obtained from a cell depolarized to -40 mV from a holding potential of -90 mV. As indicated, the same cell was subsequently recorded in control solution, after a 2-min exposure to 25 µM lavendustin A, and after a 1-min exposure to 200 µM Cd. Panel B shows single-current traces obtained from a cell depolarized to -30 mV from a holding potential of -90 mV. As indicated, the same cell was subsequently recorded in control solution, after a 3-min exposure to 100 µM daidzein, after a 3-min exposure to 100 µM genistein, and after a 1-min exposure to 200 µM Cd. Panel C shows single-current traces obtained from a cell depolarized to -40 mV from a holding potential of -90 mV. As indicated, the same cell was subsequently recorded in control solution, after a 2-min exposure to 5 µM nifedipine, and after a 1-min exposure to 200 µM Cd. Each trace is shown without any leak subtraction procedure. In panel D is reported the percent of inhibition of the Ba currents at -30 mV by 25 µM and 100 µM genistein, 25 µM lavendustin A, 100 µM daidzein, and 5 µM nifedipine. Each point is the mean ± S.E. of at least four separate experiments. * denotes p < 0.01. Panel E, time course of nifedipine and genistein inhibition of Ba currents. Inward Ba currents were elicited by depolarizing pulses to -30 mV from a holding potential of -90 mV every 5 s in two separate cells. After the first three pulses in control solution, the perfusion solution was changed with the respective drug-containing one, and the time course of inhibition was followed for both 5 µM nifedipine and 100 µM genistein. The two cells shown are representative of at least five experiments, each giving comparable results.



Effect of Genistein on the Fura-2 Fluorescence Ratio Increase Induced by Extracellular Ba

It is well known that Ba ions enter into the cells through Ca channels, bind fura-2, increase its fluorescence ratio, and cannot be extruded through Ca efflux pathways(22, 23) . For this reason, an increase in the fura-2 fluorescence ratio after extracellular Ba exposure is a specific index of cation influx through Ca channels. When GH(3) cells were superfused in a Ca-free medium, two consecutive exposures to 1 mM extracellular Ba caused comparable increases in the fura-2 fluorescence ratio. When genistein (200 µM) was superfused 5 min before and during the second Ba exposure, a significant decrease in the fura-2 fluorescence ratio occurred. In fact, the S(2)/S(1) ratio was 0.99 ± 0.001 in control cells and 0.73 ± 0.001 in genistein-treated cells (p < 0.01).

The PTP Inhibitor Vanadate Enhances the [Ca](i) Increase Elicited by L-type Ca Channel-activating Stimuli and Reverses Genistein Inhibition of 55 mM K-induced [Ca](i) Increase

When the PTP inhibitor vanadate (100 µM) was superfused for 15 min before the 55 mM K pulse, a 30% increase of the [Ca](i) response was observed (Fig. 5A). A similar potentiation of the [Ca](i) response was also observed when the cells were exposed to 10 µM Bay K 8644 (Fig. 5B).


Figure 5: Enhancement by 100 µM vanadate of [Ca] response to two different L-type Ca channel-activating stimuli. Panels A and B represent the mean traces of [Ca] response to two consecutive stimuli with 55 mM K and 10 µM Bay K 8644, respectively. Vanadate (100 µM) was superfused 20 min before and throughout the second stimulus. Each trace is the mean of at least 30 single-cell recordings obtained during a single experiment representative of at least three other experimental sessions. In addition, vanadate significantly (p < 0.01) increased mean basal [Ca] after its addition to the medium (107.9 ± 2.7 versus 83.8 ± 1.9 nM in panel A and 124.3 ± 5.7 versus 93.1 ± 3.3 nM in panel B).



In addition, the superfusion of GH(3) cells with 100 µM vanadate for 15 min completely abolished the inhibition of the [Ca](i) response to 55 mM K which follows the exposure of these cells to 200 µM genistein for 2 min (Fig. 6, A and B).


Figure 6: Reversal by 100 µM vanadate of genistein-induced inhibition of 55 mM K-elicited [Ca]increase. In panel A, genistein (200 µM) was added 2 min before the second 55 mM K stimulus. In panel B, 100 µM vanadate was superfused for 20 min before throughout the second 55 mM K pulse. Genistein was added to the superfusion medium 2 min before the second 55 mM K pulse. Each trace is the mean of at least 30 single-cell recordings obtained during a single experiment representative of at least three other experimental sessions.



Effect of the PTK Inhibitor Genistein and of the PTP Inhibitor Vanadate on Basal [Ca](i) in Resting GH(3) Cells

In unstimulated conditions, 20% (12/53) of GH(3) cells displayed oscillations of [Ca](i), defined as an increase of [Ca](i) above the mean of the basal values + 2 S.D. occurring with a frequency higher than one peak every 3 min. The remaining cells (41/53, i.e. 80%) that did not display these characteristics were defined as non-oscillating. In non-oscillating cells, the superfusion with 200 µM genistein caused a 30% decline of basal [Ca](i) (Fig. 7A). In oscillating cells, this PTK inhibitor produced the interruption of [Ca](i) oscillations and a decline of baseline [Ca](i) values (Fig. 7B). By contrast, the PTP inhibitor vanadate (100 µM) induced an increase in the frequency (1.11 ± 0.2 versus 0.6 ± 0.06 peaks/min; p < 0.05) and amplitude (45.9 ± 3.8 versus 33.1 ± 3.4% increase over basal values; p < 0.05) of [Ca](i) oscillations in spontaneously oscillating GH(3) cells. In addition, vanadate induced the appearance of [Ca](i) oscillations in 66.6% of GH(3) cells that were non-oscillating (frequency: 1.32 ± 0.1 versus 0.2 ± 0.02 peaks/min, p < 0.05; amplitude: 44.7 ± 4.1 versus 37.2 ± 3.4% increase over basal values, p < 0.05) (Fig. 7, C and D).


Figure 7: Effect of the PTK inhibitor genistein and of the PTP inhibitor vanadate on [Ca]in non-oscillating and oscillating GH(3) cells. Typical response of a non-oscillating (panel A) and an oscillating (panel B) GH(3) cell superfused with genistein (200 µM for 250 s) and vanadate (100 µM for 20 min) (panels C and D). All traces shown in the figure are single-cell recordings and are representative of the pattern of 53 cells exposed to vanadate and 57 cells superfused with genistein, recorded in at least three experimental sessions.




DISCUSSION

The results of the present study, obtained by means of single-cell microfluorometry and whole-cell patch-clamp techniques, demonstrate that the activity of Ca channels in GH(3) cells can be influenced by the interplay between PTK and PTP activity: PTK activation seems to cause an increase, whereas PTP activation appears to exert an inhibitory role on this ion channel.

The hypothesis that the L-type Ca channel is the target of PTK and PTP modulation derives from the results showing that the increase of [Ca](i) elicited by the specific L-type Ca channel activator Bay K 8644 and high K concentrations was reduced by the PTK inhibitor genistein and enhanced by the PTP blocker vanadate. A further support to this idea is the ability of genistein and lavendustin A to inhibit Ba currents through Ca channels that displayed biophysical and pharmacological features of the L-type. On the other hand, the possibility that the action of PTK inhibitors is exerted on the T-type Ca channels, which have been described in GH(3) cells, seems unlikely since this Ca channel type does not play a significant role in the [Ca](i) elevation elicited by strong activating stimuli (55 mM K or Bay K 8644)(24, 25) . In addition, the biophysical features of Ba currents recorded in GH(3) cells in the present study do not show the presence of a significant population of this Ca channel type. Furthermore, the remarkable inhibition of Ba currents by the L-type blocker nifedipine suggests that the largest population of Ca channels is represented by the L-type.

The possibility that the genistein-induced reduction of the [Ca](i) increase elicited by high K concentrations could be due to an increase of Ca removal from the cytoplasm to the extracellular space or into the intracellular Ca stores seems not to be compatible with the results of the present study. In fact, genistein also reduced the entrance of Ba ions, a cation that is known to be unable to substitute for Ca in the extrusion mechanisms. In support of this interpretation, the entity of the genistein-induced inhibition of the [Ca](i) rise induced by 55 mM K and 10 µM Bay K 8644 was comparable to the inhibition observed in electrophysiological experiments.

Since it has been reported that genistein, besides inhibiting PTKs, can also block other protein kinases such as PKA and PKC(11, 12) , which are known to modulate L-type Ca channels(1, 2, 3, 4) , the possibility exists that its effects on the activity of L-type Ca channels could occur via PKA or PKC inhibition. However, this hypothesis seems unlikely since herbimycin A and lavendustin A, two other specific PTK inhibitors devoid of PKA or PKC inhibitory action (14, 26) and structurally unrelated to genistein, effectively inhibited Ca channel activity in GH(3) cells. This evidence strongly suggests that PKA or PKC inhibition is not involved in the genistein action on Ca channels. In addition, the IC for genistein inhibition of Ca channels (30 µM) was very similar to that for PTK inhibition and much lower than that for PKA and PKC blockade(11) . The specificity of genistein action on Ca channels via PTKs was confirmed further by the inability of the genistein analogue daidzein, which lacks PTK inhibitory properties, to modify Ca channel activity in electrophysiological recordings.

The existence of a PTK regulation of L-type Ca channels in GH(3) cells is also supported by the fact that PTPs, which physiologically counteract the activity of PTKs(16, 17) , exert an opposite modulation on L-type Ca channel activity. In fact, the inhibition of PTPs by orthovanadate, a well known inhibitor of these enzymes(18) , was able to enhance the [Ca](i) increase induced by high K concentrations and to counteract the inhibitory effect of genistein on this response.

The modulation exerted by PTKs and PTPs seems to occur not only when L-type Ca channels are activated by high depolarizing stimuli, but also in resting conditions. In fact, the inhibition of PTKs by genistein caused a decline of [Ca](i) and a disappearance of [Ca](i) oscillations in oscillating GH(3) cells, whereas the blockade of PTPs by vanadate induced an increase of [Ca](i) or the appearance of [Ca](i) oscillations. These findings were not unexpected since in unstimulated conditions, L-type Ca channels of GH(3) cells are spontaneously active, as shown by the fact that spontaneous action potentials have been detected (27) and that these potentials are coupled to oscillations of [Ca](i), which can be abolished by the specific L-type Ca channel blocker nifedipine (15) .

The results of the present study showing that PTKs exert a stimulatory modulation on L-type Ca channels are in line with the recent report that genistein induces a concentration-dependent inhibition of Ca channel currents in vascular smooth muscle cells(28) . In addition, evidence has been provided that the inhibition of PTKs can also reduce Ca influx through plasma membrane ``refilling'' channels (29, 30, 31) and that different types of receptor-operated channels, like the nicotinic, N-methyl-D-aspartic acid, and -aminobutyric acid receptor channels, can be modulated by PTKs(32, 33, 34) .

The results of the present study could be of interest to explain the Ca dependence of certain biological responses elicited by some growth factors(35) . In fact, the stimulation of many growth factor receptors, such as those for the epidermal growth factor, recognize as a signaling pathway the activation of a receptor-linked PTK(7) . Since the results of the present study indicated that PTK activation leads to Ca entrance through L-type Ca channels into the cells, the Ca-dependent epidermal growth factor-induced differentiation of GH(3) cells toward the lactotroph phenotype (36) could be the consequence of the activation of L-type Ca channels, especially if one considers that in a different pituitary cell line, epidermal growth factor induces an increase of [Ca](i) which is independent of phospholipase C1-dependent inositol 1,4,5-trisphosphate generation(37) .

In conclusion, all of these results suggest that L-type Ca channels are modulated by the PTK/PTP system in GH(3) cells. The molecular mechanism of this modulation remains to be clarified. However, a possible working hypothesis to explain the effect of PTK inhibitors on Ca channel function could be that phosphorylation by PTKs exerts a permissive role on the activation of Ca channels elicited by both the dihydropyridine agonist Bay K 8644 and depolarizing stimuli. Such a model has already been proposed by Armstrong et al.(2) to explain the effect of PKA on Ca channel activation.


FOOTNOTES

*
This work was supported by Consiglio Nazionale delle Ricerche Grants 93.02003-CT14, 93.04222-CT04, and 94.02525-CT04 and Ministero dell' Università e della Ricerca Scientifica e Tecnologica (by 40 and 60% grants) (to L. A. and G. d. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Section of Pharmacology, Dept. of Neurosciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Naples, Italy. Tel.: 39-81-746-3323; Fax: 39-81-746-3323; farmacol{at}ds.cised.unina.it.

(^1)
The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; PTK, protein tyrosine kinase; [Ca], cytosolic free calcium; PTP, protein tyrosine phosphatase; fura-2/AM, fura-2 acetoxymethylester.


ACKNOWLEDGEMENTS

We thank V. Grillo for technical support. We also appreciate greatly the invaluable help of Prof. Emilio Carbone, University of Turin, Italy, in the design and analysis of the electrophysiological experiments.


REFERENCES

  1. Curtis, B. M., and Catterall, W. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2528-2532 [Abstract]
  2. Armstrong, D., and Eckert, R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2518-2522 [Abstract]
  3. Marchetti, C., and Brown, A. M. (1988) Am. J. Physiol. 254, C206-C210
  4. Mac Ewan, D. J., and Mitchell, R. (1991) FEBS Lett. 291, 79-83 [CrossRef][Medline] [Order article via Infotrieve]
  5. Nastainczyk, W., Röhrkasten, A., Sieber, M., Rudolph, C., Schächtele, C., Marmé, D., and Hofmann, F. (1987) Eur. J. Biochem. 169, 137-142 [Abstract]
  6. Hell, J. W., Yokoyama, C. T., Wong, S. T., Warner, C., Snutch, T. P., and Catterall, W. A. (1993) J. Biol. Chem. 268, 19451-19457 [Abstract/Free Full Text]
  7. Schlessinger, J., and Ullrich, A. (1992) Neuron 9, 383-391 [Medline] [Order article via Infotrieve]
  8. Mustelin, T., and Burn, P. (1993) Trends Biol. Sci. 18, 215-220
  9. Ziemiecki, A., Harpur, A. G., and Wilks, A. F. (1994) Trends Cell Biol. 4, 207-212 [CrossRef]
  10. Niklinska, B. B., Yamada, H., O'Shea, J. J., June, C. H., and Ashwell, J. D. (1992) J. Biol. Chem. 267, 7154-7159 [Abstract/Free Full Text]
  11. Akiyama, T., and Ogawara, H. (1991 Methods Enzymol. 201, 362-370
  12. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262, 5592-5595 [Abstract/Free Full Text]
  13. Uehara, Y., Fukazawa, H., Murakami, Y., and Mizuno, S. (1989) Biochem. Biophys. Res. Commun. 163, 803-809 [Medline] [Order article via Infotrieve]
  14. Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshiki, K., Naganawa, H., and Takeuchi, T. (1989) J. Nat. Prod. (Lloydia) 52, 1252-1257
  15. Schlegel, W., Winiger, B. P., Mollard, P., Vacher, P., Wuarin, F., Zahnd, G. R., Wollheim, C. B., and Dufuy, B. (1987) Nature 329, 719-721 [CrossRef][Medline] [Order article via Infotrieve]
  16. Brady-Kalnay, S. M., and Tonks, N. K. (1994) Trends Cell Biol. 4, 73-76 [Medline] [Order article via Infotrieve]
  17. Walton, K. M., and Dixon, J. E. (1993) Annu. Rev. Biochem. 62, 101-120 [CrossRef][Medline] [Order article via Infotrieve]
  18. Swarup, G., Cohen, S., and Garbers, D. L. (1982) Biochem. Biophys. Res. Commun. 107, 1104-1109 [Medline] [Order article via Infotrieve]
  19. Fatatis, A., Caporaso, R., Iannotti, E., Bassi, A., di Renzo, G. F., and Annunziato, L. (1994) J. Biol. Chem. 269, 18021-18027 [Abstract/Free Full Text]
  20. Nowycky, M. C., Fox, A. P., and Tsien, R. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2178-2182 [Abstract]
  21. Enyeart, J. J., Biagi, B., and Day, R. N. (1990 Mol. Endocrinol. 4, 727-735
  22. Schilling, W. P., Rajan, L., and Strobl-Jager, E. (1989) J. Biol. Chem. 264, 12838-12848 [Abstract/Free Full Text]
  23. Kwan, C. Y., and Putney, J. W., Jr. (1990) J. Biol. Chem. 265, 678-684 [Abstract/Free Full Text]
  24. Armstrong, C. M., and Matteson, D. R. (1985) Science 227, 65-67 [Medline] [Order article via Infotrieve]
  25. Matteson, D. R., and Armstrong, C. M. (1986) J. Gen. Physiol. 87, 161-182 [Abstract]
  26. Fuzakawa, H., Li, P., Yamamoto, C., Murakami, Y., Mizuno, S., and Uehara, Y. (1991) Biochem. Pharmacol. 42, 1661-1671 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kidokoro, Y. (1975) Nature 258, 741-742 [Medline] [Order article via Infotrieve]
  28. Wijetunge, S., Aalkjaer, C., Schachter, M., and Hughes, A. D. (1992) Biochem. Biophys. Res. Commun. 189, 1620-1623 [Medline] [Order article via Infotrieve]
  29. Lee, K., Toscas, K., and Villereal, M. (1993) J. Biol. Chem. 268, 9945-9948 [Abstract/Free Full Text]
  30. Yule, D. I., Kim, E. T., and Williams, J. A. (1994) Biochem. Biophys. Res. Commun. 202, 1697-1704 [CrossRef][Medline] [Order article via Infotrieve]
  31. Sargeant, P., Farndale, R. W., and Sage, S. O. (1993) J. Biol. Chem. 268, 18151-18156 [Abstract/Free Full Text]
  32. Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235 [CrossRef][Medline] [Order article via Infotrieve]
  33. Moss, S. J., Gorrie, G. H., Amato, A., and Smart, T. G. (1995) Nature 377, 344-348 [CrossRef][Medline] [Order article via Infotrieve]
  34. Hopfield, J. F., Tank, D. W., Greengard, P., and Huganir, R. L. (1988) Nature 336, 677-680 [CrossRef][Medline] [Order article via Infotrieve]
  35. Takada, K., Amino, N., Tada, H., and Miyai, K. (1990) J. Clin. Invest. 86, 1548-1555 [Medline] [Order article via Infotrieve]
  36. White, B. A., Bauerle, L. R., and Bancroft, F. C. (1981) J. Biol. Chem. 256, 5942-5945 [Abstract/Free Full Text]
  37. Aanestad, M., Røtnes, J. S., Torjesen, P. A., Haug, E., Sand, O., and Bjøro, T. (1993) Acta Endocrinol. 128, 361-366 [Medline] [Order article via Infotrieve]

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