INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TYROSINE PROTEIN KINASES (TK) are involved in signal transduction mediated by many hormone and growth factor receptors that regulate mitogenesis or cell growth. In addition, these enzymes play an important role in the regulation of ion channels such as L-type Ca²⁺ channels (I_Ca), which are regulated by TK in various cell types, including pituitary GH₃ cells (3), retinal pigment epithelial cells (21), smooth muscle cells (8), and cardiac myocytes (6, 24, 25). TK can regulate I_Ca by direct phosphorylation of the -subunit of Ca²⁺ channels, as is the case of the nonreceptor tyrosine kinases c-Src and focal adhesion kinase in smooth muscle cells (8). The enzymes can also regulate I_Ca indirectly by modulating the activity of various signaling pathways such as those resulting in the stimulation of protein kinase C (PKC) or cAMP-dependent protein kinase. For instance, in guinea pig ventricular myocytes, the effect of TK on I_Ca results predominantly from regulation, by the enzymes, of the sensitivity of Ca²⁺ channels to -adrenergic receptor stimulation (6).

In human atrial myocytes, as in those of other species, the L-type Ca²⁺ current is the target of various neurotransmitters, hormones, and therapeutic agents whose effects often involved serine/threonine phosphorylation via activation of cAMP- or cGMP-dependent protein kinases or PKC. However, I_Ca regulation by second messengers differs in several aspects between human atrial myocytes and myocytes from other tissues or species, as illustrated by the effects of serotonin (17). We have also reported that phosphodiesterase (PDE) types 2 and 3 participate in the basal PDE activity involved in the regulation of I_Ca of human atrial myocytes but not in those of other animal species studied (19, 11).

This species and tissue specificity of I_Ca regulation, together with the multiple effects of TK on this current, prompted us to examine whether I_Ca in human atrial myocytes is also regulated by the enzymes and, if so, by what mechanism. We examined whole cell patch-clamped myocytes treated with various pharmacological tools modulating not only TK but also serine/threonine kinases. We found that TK regulates I_Ca in human atrial myocytes, an effect that appears to involve a cooperation between PKC and TK.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cardiac myocyte preparation. Specimens of human right atrial appendages were obtained from 42 patients (15-81 yr of age) undergoing heart surgery for coronary artery insufficiency (n = 22), mitral valve disease (n = 11), aortic valve disease (n = 7), or congenital heart defect (n = 2). Most patients received a pharmacological treatment that was stopped at least 10 h before surgery (Ca²⁺-channel blocker, -adrenergic antagonist, diuretics, angiotensin-converting enzyme inhibitors, or nitric oxide donor). All patients except one were in sinus rhythm. Myocytes were enzymatically isolated as previously described (5). Briefly, small pieces of atrial appendage were cut up and washed in Ca²⁺-free Krebs-Ringer solution containing (in mM) 35 NaCl, 4.75 KCl, 1.19 KH₂PO₄, 16 Na₂HPO₄, 10 HEPES, 10 glucose, 25 NaHCO₃, 134 saccharose, and 30 2,3-butanedione monoxime (BDM) (pH 7.4 adjusted with NaOH), gassed with 95% O₂-5% CO₂ and maintained at 37°C. BDM, a compound known to have reversible effects on cardiac cellular electrophysiology (4), was used to prevent tissue injury during cutting (16). Pieces were reincubated in the same solution but without BDM and containing 200 IU/ml collagenase (type IV, Sigma Chemical, St. Louis, MO) and 6 IU/ml protease (type XXIV, Sigma Chemical). After 30 min of digestion, the enzyme solution was replaced by the same solution containing only collagenase (400 IU/ml). Isolated myocytes were incubated at 37°C with continuous gassing with 21% O₂-5% CO₂ for at least 1 h before use.

Current measurements. Currents were recorded with the patch-clamp technique in the whole cell configuration using borosilicate glass pipettes with a tip resistance of 1-2 M connected to the input stage of a patch-clamp amplifier (Axoclamp 200A, Axon Instruments). Currents filtered at 5 kHz were digitized by a Labmaster (Lab Rac, Scientific Solution) and stored on the hard disk of a personal computer. Data were acquired and analyzed using a program written for our laboratory (Acquis, G. Sadock). Resistance in series was compensated for to obtain the fastest capacity transient current. Membrane capacitance was calculated using the fit of the capacity transient decay. Current recording was performed 1 min after the patch was broken to obtain a steady-state intracellular dialysis, and the average duration of the experiments was around 20 min. Rundown of I_Ca usually occurred 10 min after the patch was broken. Experiments were carried out at room temperature (22-24°C).

Solutions and reagents. The compositions of the standard solutions used were as follows (in mM): normal Tyrode solution, 136 NaCl, 5.4 KCl, 2 CaCl₂, 10 glucose, 1.06 MgCl₂, 0.33 NaH₂PO₄, and 10 HEPES, pH adjusted to 7.4 with NaOH; I_Ca recording solution, normal Tyrode solution with NaCl replaced by tetraethylammonium; and pipette solution, 130 CsCl, 2 MgCl₂, 10 HEPES, 15 EGTA, 10 glucose, and 3 MgATP, pH adjusted with 7.2 with CsOH. A multibarrel system allowed exchange of the fluid solution bathing the myocyte within 2 s. Genistein, lavendustin A, tyrphostin 23, and acetylcholine were dissolved in distilled water and kept as stock solutions at 20°C; staurosporine, 4-phorbol 12,13-didecanoate (4-PDD), and phorbol 12-myristate 13-acetate (PMA) were dissolved in ethanol and stored as stock solutions at 20°C. Isoproterenol was diluted in the Tyrode solution. The protein kinase inhibitor was directly dissolved in pipette solution. In some experiments, myocytes were preincubated with the PKC inhibitor calphostin C, which, to be activated, required that myocytes were maintained for 30 min under ultraviolet light. All drugs were purchased from Sigma Chemical, except for isoproterenol (Sanofi Winthrop, France).

Data analysis. Depolarizing voltage pulses were delivered at 0.1 Hz. The amplitude of peak I_Ca was measured as the difference between the amplitude of the peak inward current and that recorded at the end of the 350-ms test pulses. Concentration-response curves were fitted as follows: E = E_max[D]/([D] + EC₅₀), where E is the percentage change in I_Ca, E_max is the maximal response induced by the drug, and [D] is the concentration of genistein, to estimate the EC₅₀.

Statistical analysis. Values are expressed as means ± SE; n indicates the number of experiments. Paired Student's t-test was used to determine the statistical significance of differences between means obtained before and after the effects of a given drug. One-way ANOVA was used to determine the statistical significance of differences between means obtained under different experimental conditions. P values <0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TK inhibitors stimulate I_Ca in human atrial myocytes. Figure 1A shows an example of the effects of external application of 50 µM genistein on the I_Ca elicited by 350-ms depolarizing test pulses from 60 mV to 0 mV. In the majority of cells studied (84%, n = 53), genistein stimulated I_Ca (132 ± 18%, n = 28, P < 0.001), an effect that was sometimes preceded by a slight inhibition of the current. In the remaining 16% of myocytes (n = 9), genistein inhibited I_Ca or had no significant effect. There was no clear relationship between the source patient's clinical data and the effects of genistein. The stimulatory effect of genistein developed slowly, reaching steady-state after ~1 min (Fig. 1B), and was not reversible on drug washout during the time of the experiments. The increase in I_Ca on genistein application was associated with a shift in the current-voltage relationship toward a negative potential of ~10 mV (Fig. 1C). The EC₅₀ of genistein on the I_Ca was 53 µM, a value close to the IC₅₀ of genistein on TK (Fig. 1D) (2). At low concentration (<20 µM), genistein only inhibited I_Ca. Lavendustin A and tyrphostin 23, other TK inhibitors structurally distinct from genistein (14), also stimulated I_Ca and shifted the current voltage-relationship leftward (116 ± 18%, n = 5, P < 0.05; 60 ± 6%, n = 23, P < 0.001, respectively) (Fig. 2). The smaller increase of I_Ca on tyrphostin exposure may reflect the fact that this compound is more specific for distinct TK compared with the broad-spectrum TK inhibitors genistein and lavendustin A (14, 18). It should be noted that in our experimental conditions, low concentrations (<0.01%) of DMSO altered the human atrial myocyte I_Ca and thus ruled out experiments with the inactive genistein analog daidzen, a compound solely soluble in DMSO.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   A: superimposition of L-type Ca2+ current (ICa) traces elicited by a test potential from 60 to 0 mV recorded with Tyrode external solution (, control) and with same solution containing 50 µM genistein (). B: time course of changes in ICa on genistein exposure, where x-axis indicates time from beginning of current recording. Cell capacitance, 70 pF. C: current density-voltage relationships of ICa recorded in control conditions and on genistein exposure. Each point is average current density in 10 cells. * P < 0.05; ** P < 0.01 vs. control. D: concentration-dependent effect of genistein on ICa. Each point is mean from 5-15 cells.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Superimposition of ICa traces recorded in control conditions and in presence of 50 µM lavendustin A (A) or 50 µM tyrphostin 23 (B). Cell capacitances, 83 and 62 pF in A and B, respectively. C: percent change in ICa in response to genistein, lavendustin A, and tyrphostin 23 exposure. * P < 0.05; *** P < 0.001 vs. control.

Effects of genistein on I_Ca are independent of cAMP. The slowly developing stimulatory effects of TK inhibitors on I_Ca, which were accompanied by a leftward shift in the current-voltage relationship, suggested that these compounds affected the phosphorylation status of Ca²⁺ channels. However, the observation that 1 µM isoproterenol still caused an enhancement of I_Ca prestimulated by TK inhibitors (83 ± 23%, n = 8, P < 0.01) argues against the involvement of a cAMP-dependent regulatory pathway (Fig. 3, A and B). In addition, acetylcholine (10⁶ M), a muscarinic agonist that reversed the stimulatory effect of isoproterenol on I_Ca, did not prevent genistein-induced enhancement of the current, indicating that the effect of the TK inhibitor was independent of adenylyl cyclase activity (n = 13) (Fig. 3C). Finally, in cells dialyzed with an internal solution containing the cAMP-dependent protein kinase inhibitor at a concentration of 20 µM, genistein, contrary to isoproterenol, stimulated I_Ca (130 ± 19%, n = 5, P < 0.05) (Fig. 3D).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   A: time course of changes in ICa elicited by test pulses to 0 mV on genistein (50 µM) exposure and isoproterenol (1 µM) treatment in presence of genistein, where x-axis indicates time from beginning of current recording. Cell capacitance, 96 pF. B: percent increase in ICa in response to genistein and genistein plus isoproterenol. ** P < 0.01. C: isoproterenol increases peak ICa (measured at 0 mV and plotted against time), an effect reversed by acetylcholine, whereas genistein still stimulated ICa. Cell capacitance, 80 pF. D: in a myocyte loaded with cAMP-dependent protein kinase inhibitor peptide, genistein, but not isoproterenol, stimulated ICa. Cell capacitance, 81 pF.

PKC-dependent effects of genistein. We next examined whether PKC and TK regulated I_Ca cooperatively. As shown in fig. 4A, in a myocyte pretreated for at least 30 min with the PKC inhibitor staurosporine (100 nM), genistein had no effect on I_Ca, whereas the current was still stimulated by isoproterenol, ruling out a rundown of the channels. These experiments were repeated with 18 myocytes, and the results are indicated in Table 1. Similar suppression of the effect of genistein was also obtained using another PKC inhibitor, calphostin C [2 ± 13 vs. 128 ± 10% in calphostin C (n = 7) and control conditions (n = 7), respectively, P < 0.01]. The effect of genistein was then studied in myocytes pretreated with PMA to stimulate PKC. In 77% of the cells studied, PMA had a biphasic effect on I_Ca, characterized by an initial increase in amplitude (75 ± 17%, n = 15, P < 0.05) (22) followed by a decrease at ~5 min (36 ± 5%, n = 15, P < 0.001) (Fig. 4B and Table 1). The fall in I_Ca observed after prolonged PMA application was distinct from that observed during classic rundown of L-type Ca²⁺ channels, with a steeper slope of current decline in PMA than in control conditions (2.8 ± 0.3 vs. 1.3 ± 0.4 ms¹, P < 0.05) (26). In addition, PMA had no significant inhibitory effect on I_Ca in myocytes pretreated with staurosporine (n = 5; Fig. 4C). Moreover, the inactive analog of PMA, 4-PDD, had no effect on I_Ca (2.7 ± 0.4 pA/pF vs. 2.8 ± 0.4 pA/pF in control conditions and after 4-PDD exposure, respectively, n = 8) and did not prevent that of PMA (47 ± 5% of I_Ca inhibition caused by PMA in 4-PDD-treated cells, n = 8). At the steady state of I_Ca inhibition by PMA, application of genistein still caused a marked increase in I_Ca (Fig. 4B), and comparison of the effect of genistein on I_Ca in control and PMA-treated myocytes showed a higher percentage of current enhancement in the latter (323 ± 25% vs. 120 ± 13%, n = 8, P < 0.05) (Table 1). Finally, pretreating myocytes with genistein reduced the inhibitory effect of PMA on I_Ca [percentage of I_Ca inhibition caused by PMA: 40 ± 5% in control (n = 15) vs. 20 ± 7% in genistein (n = 15), P < 0.05].

View larger version (14K): [in this window] [in a new window]   Fig. 4.   A: time course of changes in ICa (test pulses to 0 mV) on exposure to genistein and genistein plus isoproterenol in myocytes pretreated with 100 nM staurosporine, where x-axis indicates time from beginning of current recording. Cell capacitance, 55 pF. B and C: time course of changes in ICa in response to phorbol 12-myristate 13-acetate (PMA) application and genistein application in presence of PMA in control (B; cell capacitance, 67 pF) and staurosporine-treated myocytes (C; cell capacitance, 124 pF), where x-axis indicates time from beginning of current recording. Gen, genistein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We found that various agents known to inhibit TK, such as genistein, lavendustin A, and tyrphostin 23, stimulated I_Ca in human atrial myocytes. The effects of these compounds on the I_Ca appear to result from their ability to inhibit TK, because 1) they increased the I_Ca at concentrations known to inhibit TK activity, and 2) these compounds, which are structurally unrelated, all had the same effect on I_Ca, suggesting a common mechanism of action (2, 14, 7).

In human atrial myocytes, TK inhibition is accompanied by an apparent change in the degree of phosphorylation of L-type Ca²⁺ channels, as indicated by the slow increase in current amplitude and the shift of the voltage-relationship toward negative potentials (10). Clearly, this effect of TK inhibitors on I_Ca cannot be attributed to the modulation of cAMP-dependent processes, especially because inhibition of cAMP-dependent protein kinase did not suppress the effect of genistein. However, our results do not eliminate the possibility that in human atrial myocytes, as in guinea pig ventricular myocytes, TK regulate the -adrenergic responsiveness of Ca²⁺ channels (6).

Our finding that the effects of TK inhibitors on I_Ca were modulated by compounds known to alter PKC activity suggests a link between these two enzymes. It is unlikely that staurosporine or calphostin C inhibited the effect of genistein via an effect of genistein on PKC, because 1) the genistein concentration used was much lower than that required for PKC blockade (2), and 2) lavendustin A, which is devoid of any significant PKC-inhibiting action (7), had the same effect as genistein on the I_Ca. Similar cases of cooperative regulation of ion channels by PKC and TK have been observed in other cell types. For instance, in rat and human retinal epithelial cells, genistein stimulates L-type Ca²⁺ channels when PKC is prestimulated with PMA (21). Furthermore, the G protein-coupled m₁-muscarinic acetylcholine receptor inhibits Kv1.2 channels expressed in Xenopus oocytes and in cell lines through a phospholipase C (PLC)/PKC-dependent mechanism that controls direct tyrosine phosphorylation of K⁺ channels (9). These effects involved a cytosolic proline-rich tyrosine kinase (PYK2), which can be activated by phosphorylation in response to various stimuli such as intracellular Ca²⁺ and PKC activation (13, 20). Interestingly, in cat atrial myocytes it has been reported that a cytosolic (nonreceptor) TK may be responsible for the inhibition of Ca²⁺ channels and, in turn, for the increase in I_Ca caused by genistein (24). Our observation that TK inhibitors stimulate I_Ca in whole cell patch-clamped human atrial myocytes may appear to conflict with the presence of a cytosolic TK, which should be dialyzed by the patch pipette; however, it is possible that cytosolic TK is located in the proximity of ion channels in fuzzy subsarcolemmal spaces poorly accessible to dialysis (1). In cat atrial myocytes, inhibition of PKC does not suppress the effect of genistein on the I_Ca, suggesting that distinct soluble TK isoforms regulate I_Ca in cat and human atrial myocytes or that the nature of the stimuli (increase in intracellular Ca²⁺ concentration) that activate soluble TK varies with the species or pathophysiological conditions. In addition to soluble TK stimulated by PKC, other mechanisms might link the two enzyme activities. For instance, tyrosine phosphorylation of G_q/11 protein facilitates coupling between G_q/11 protein and glutamate receptor 1 expressed in Chinese hamster ovary, leading to activation of a PLC-dependent signaling pathway (23). However, genistein still increased I_Ca in myocytes pretreated with PMA, which argues against the possibility that TK modulates PKC through a permissive effect on G protein coupling to a phospholipase, for instance. Finally, the observation that PMA still inhibited the current in myocytes pretreated with genistein, although less efficiently compared with the control, indicates that only part of the effect of PKC on L-type Ca²⁺ channels depends on TK activation. The relatively low density of I_Ca in staurosporine-treated myocytes appears to oppose the possibility of a tonic regulation of I_Ca by PKC via their coupling with TK. However, it is possible that staurosporine has some cellular toxicity, including disruption of the cytoskeleton (15) and resulting in a decreased density of I_Ca that was not caused by a rundown of the channel, as indicated by the persistence of the stimulatory effect of isoproterenol on I_Ca in staurosporine-treated myocytes.

The inhibitory effect of genistein on I_Ca observed in a smaller percentage of cells that, in some cases, preceded the increase in current may be caused by activation of receptor-bound TK distinct from the cytosolic form, as reported in cat atrial myocytes (24). It is also possible that the inhibitory effect of genistein is in part TK-independent and caused by a direct effect of the compound on Ca²⁺ channels. This is suggested by the observation that the inhibition of I_Ca by genistein was a rapid process that did not occur with the other TK inhibitors tested and that also was observed at a concentration at which genistein is a weak TK inhibitor (Fig. 1D). In guinea pig ventricular myocytes, both genistein and its inactive analog daidzen inhibit I_Ca, indicating that this effect is related not to suppression of TK activities but to direct effects of these drugs on L-type Ca²⁺ channels (6, 25). Our results, which are similar to those obtained in cat atrial myocytes but different from those obtained in guinea pig ventricular myocytes, point to the tissue and species specificity of L-type Ca²⁺ channel regulation by TK. Distinct regulatory mechanisms of I_Ca in human atrial myocytes are already known with regard to Ca²⁺ channel coupling to 5-HT₄ receptors (17) and the effects of PDE inhibitors (19, 11); furthermore, the specificities of I_Ca regulation in human atrial myocytes may also be influenced by pathophysiological conditions (12).

Our observation of tonic I_Ca regulation by TK in human atrial myocytes raises important questions as to the significance of this regulatory process. Because L-type Ca²⁺ channels play a central role in excitation-contraction coupling of atrial myocytes (5), regulation of their activity by TK may have profound implications for the electrical and mechanical activity of these cells. In addition, it is conceivable that cellular Ca²⁺ influx regulation by TK contributes to modulation of tonic phenomena such as gene expression and cardiac phenotypic plasticity.