Bay K 8644 enhances Ca2+ channel activities in embryonic chick heart cells without prolongation of open times

European Journal of Pharmacology, 203 (1991) 307-310 0 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50 ALXXWS ~14299991~727N

307

EJP 20934

Noritsugu

Tohse, Laura Conforti

and Nicholas

Sperelakis

Department of Physiologyand Biophysics,UniLrrsity of Cincinnati, College of Medicine, Cincinnati, OH, U.S.A. Received 13 August 1991, accepted 20 August 1991

The effects of the Ca’+ channel agonist, Bay K 8644, on the slow (L-type) Ca 2+ channels was examined in young (Iday-old} embryonic chick heart cells, which naturally exhibit long-lasting openings. Bay K 8644 (5 &MI increased (a) the peak amplitude of the ensemble-averaged current by 3.9 f 0.9-fold (mean f SE.) and (b) the maximal number of simultaneous opening from 2.6 f 0.4 to 4.4 & 0.9. Bay K 8644 had no effect on the unitary conductance (27 pS in control), and relatively little effect in the open-close kinetic analysis. The mean open times were 4.2 ms and 5.2 ms, in control and Bay K 8644, respectively. These results suggest that the agonistic effect of Bay K 8644 involves a mechanism other than open-time prolongation, such as activation of silent channels. Heart cells (cultured); Heart cells (embryonic); Patch clamp analysis; Ca’+ channels (L-type); Single channel currents; Ca2+ channel agonists

Molecular mechanisms of modulation af the slow (L-type) Cazf channels by dihydropyridine (DHP) Ca”’ agonists are of current interest, because DHPs are useful in clinical medicine and investigation of molecular structure of the channel. In analysis of single-channel activities, previous reports agree that DHP Ca2+ agonists prolong the open-times of the Ca”’ channels, which are very short in the control condition (Hess et al., 1984; Kokubun and Reuter, 1984; Caffrey et al., 1986; Hoshi and Smith, 1987; Lacerda and Brown, 1989). This prolongation of mean open-time resulted in an increase in open probability, producing an increase in macroscopic Ca2+ current (agonistic effect). Recently, we reported that long-lasting openings of the Ga*+ channels occur naturally in young (3-day-old~ embryonic chick heart cells (Tohse and Sperelakis, 19901. These channels were completely blocked by nifedipine, indicating that they were L-type (slow) Ca2+ channels, and they had a slope inductance of 26 pS. Kinetics of these channels are nearly identical with those observed in presence of the Ca2+ agonist, Bay K

Correspondence to: N, Sperelakis, Department of Physiology and 231 Biophysics, College of Medicine. University of Ciwiznali, Bethesda Avenue, Cincinnati, OH ~52~7-057~, U S.A. Tel. 1 515 558 5636.

8644, in previous reports on mammalian heart cells (Hess et al., 1984; Kokubun and Reuter, 1984; Lacerda and Brown, 1989). In the present report, we studied the effect of Bay K 8644 on the Ca2+ channels in young embryonic chick heart cells using celi-attached patch clamp. Our experiments demonstrate that in these embryonic heart cells Bay K 8644 produces a Ca’+-agonistic effect without prolongation of the open-times. One possibili~ is that Bay K 8644 activates silent (non-function~ng~ channels.

2.1. Cell culture preparation Cultures of single cells were prepared from 3-day-old embryonic chick hearts by a method similar to that described previously (Sada et al., 1988). In brief, 12 dozen fertilized White Leghorn chick embryos were incubated for 3 days at 37S”C, and the hearts were sterilely removed and collected in a balanced salt solution (ice cold). Tissue digestion was accomplished by gentle stirring in a Ca2+- and Mg2*-free Ringer solution containing 0.01% trypsin (Sigma Chemical, U.S.A.). The cell suspensions were harvested at 5min intervals, and coilected cells were washed three times in culture medium (Mf99, GIBCO, U.S.A.) containirg 10% fetal bovine serum. The cells were plated on glass

dishes (35 mm) at a concen’ cells/ml. The cultures were maintained at 37°C and pH 7.4 in a moist-air CO, incubator.

cower slips in plastic

Petri

?T _.^. Cell-attached patch-clamp recordings were made using a patch-clamp amplifier (EPC-7, List, F.R.G.) and standard techniques (Hamill et al., 1981). Tine cells were bathed in an external solution containing 140 mM

KCI, 10 mM glucose, 10 mM EGTA, and 5 mM Hepes (titrated to pH 7.4 with KOH). Patch pipettes had resistances of 3-10 MR when filled with a solution containing 50 mM BaCl,, 70 mM choline-Cl, and 10 mM HEPES (titrated to pH 7.4 with tristhydroxymethyl)aminomethane base). Experiments were carried out at room temperature (25-27°C). The current signal were filtered with a cutoff frequency of 1 kHz (&pole Bessel) and sampled at 3 kHz. The storing and analysis of the digitized signals were carried out using PCLAMP

5rM Bay K 5644

A

n = 29

(x 103 5

‘I

.I/ 4

Control

I++,

01 Control

Amplitude (pAI

Bay K

8644

5lJM

Fig. I. Effects of 5 PM Bay K 8644 on the L-type Ca?+ channel in 3-day-old embryonic chick heart cells. (A) Representative traces out of 29 records in the absence and presence of Bay K 8644. The single-channel currents were elicited by 300 ms depolarizing test pulses to !J mV from a holding potential of - 80 mV (shown in upper traces), at a rate of 0.5 Hz. The superfusion with Bay K 8644 produced the additional openings of the channels. (B) Ensemble-averaged currents from the 29 consecutive traces in the absence and presence of Bay K 8644. Bay K 8644 markedly increased the ensemble-averaged current. (C) Changes in the amplitude histogram by Bay K 8644. Data were obtained from the same cell shown in Panels A and B. The control histogram is given as the thin line, and that of Bay K 8644 is given as bold liue. The interval between adjacent peaks indicates the unit amplitude. Bay K 8644 markedly increased the number of simultaneous openings of the CaZ” channels. (D) Increase in the maximal number of simultaneous openings by Bay K 8644. Each type of symbol indicates different cells.

309

(program from Axon Instruments, U.S.A.). All values are presented as means + S.E. In the experiments with external perfusion of Bay K 8644, in which the same cell was used as the controi, the paired Student’s t-test was used to evaluate the statistical significance of differences between means. Vaiues of P < 0.05 were considered to indicate significance.

Bay K 8644 stimulated the Ca”’ channel current in young embryonic (3-day-old) chick heart cells. Figure 1A shows inward Ba2+ currents carried through the single Ca2+ channels. In the control condition (left panel), either one, two, or three channels were simultaneousiy open. As in our previous report (Tohse and Sperelakis, 19901, the channels exhibited long-lasting openings. Addition of 5 FM Bay K 8644 to the external perfusing solution (right panel) enhanced the openings of the Ca’+ channeh, the maximal number of simultaneous openings being increased to nine. Bay K 8644 acted on the Ca2+ channels in the patch membrane presumably by migrating in the plane of the membrane from outside the patch to the patch or by entering intracellularly and diffusing to the inner surface of the patch. The ensemble-averaged current from these channel activities was markedly increased by Bay K 8644 (fig. 1B). The peak amplitude of the current was increased 7.6-fold. The average increase in peak current in seven cells was 3.9 + 0.9-fold (P < 0.05). Figure 1C shows the amplitude histogram of all recordings of the same cell in fig. 1A. Bay K 8644 increased the number of peaks in the amplitude histogram. The unit amplitude, which is expressed as the current interval between peaks, is almost identical in the absence and presence of Bay K 8644. We examined the current/voltage relation of the unitary Ca2+ currents in 21 cells. The sfope conductance was 27.2 f 0.8 (n = 15) and 26.7 f 1.5 pS In = lo), in the absence and presence of Bay K 8644, respectively. Figure 1D summarizes the changes in the maximal number of simultaneous channel openings observed in seven cells. In all cells, the number of active channels was increased by Bay K 8644: the mean values are 2.6 + 0.4 and 4.4 + 0.9 (P < 0.051, in the absence and presence of Bay K 8644, respectively. We examined the effects of Bay K 8644 on the kinetics of the single C:a’+ channel. Single-channel kinetics could not be measured during superfusion with Bay K 8644, because of the increase in the tl;aximaI number of simultaneous openings. Therefore, we compared the data obtained using pipettes with and without 5 PM Bay K 8644 (different celis were used in the two conditions). Data in the two conditions were obtained only from patches in which single-channel activi-

A

Control

y K 8644

0 mV

B

30 -Ima 2 pA

0 25

2s

Open Tlmcs Pmrt

T, = 1.8 ms

Tr

w, * 3754

wq - 229

7,

7~124.9

= 11.8 mr

w, - 938

ri,

f 2.3 ms mr

6 57

T, -

a.8 m5

i, =10.0ms

R =

0.25

R = 0.25

0

0

0

SO Clomd tlmsa

insl

50

Fig. 2. Open-close kinetics of the CaZf channel in the absence and presence of 5 *M Bay K 8644. iA) The repre~ntative behavior of the Ca** channels in the absence (left column) and presence of 5 PM Bay K 8644 (right column). The single-channel currents were elicited by 150 ms (or 300 ms) test pulses to 0 mV from a holding potential of -80 mV. Broken lines indicate zero current levels. (B) Open-time histograms obtained from nine difterent patches (control) and five patches (Bay K 8644). Both histograms were fitted by two ex~nential com~nents (number of events = W, /T, +expf - t/r,)+ W2/r2 *exp(-t/rl)I. i, is the mean open-time. R is Wz/W,. CC) Closed.time histograms obtained from nine different patches (control) and three patches (Bay K 8644). Both histograms were fitted by two exponential components. i, is the mean closed-time.

ties were observed. Figure 2A shows representative examples in the absence (control, left panel) and presence of Bay K 8644 (right panel). In both cells, the Ca” channels showed long-lasting openings. Figure 28 shows the open-time histograms of the Ca” channels in the absence (left graph) and presence of Bay K

t graph). Data were collected from nine control, and from five patches for Bay K 86=&kThese histograms were well fitted by two exponential components (Tohsc and Sperelakis. 1990). The time constants of the fast (7,) and slow (TV) components were not changed by Bay K 8644. The ratio (RI itude of the slow component (W2) to the fast ent (W,) reflects the incidence of long-openings. because the time constants were not changed (see Iegend of fig. 2). R is also identical (0.25) in the absence and presence of Bay K 8644. indicating that Bay K 8644 did not facilitate the occurrence of the long-lasting openings. The mean open-time (I,,) of the Ca” channels was 4.2 ms (control) and 5.2 ms (Bay K 8644). Figure 2C shows the closed-time histograms. Data were collected from nine patches for control and f=om three patches for Bay K 8644. B;;, K 8644 did not produce a marked change in any parameters, although the time constant of the slow component was prolonged in the presence of Bay K 8644.

findmg, showing that the maximal number of simultaneous channel openings was increased by Bay K 8644, can be explained by either possibility. It seems likely that Bay K 8644 activates non-functional channels, although we cannot exclude the other possibility. Because, if Bay K 8644 increased the opening probability of each channel, it would be expected that intervals between two consecutive burstings would be shortened by Bay K 8644. Such a shortening should decrease the slow time constant of the closed-time histogram; in contrast, Bay K 8644 slightly increased this slow time constant. In conclusion, our results indicate that the Ca2+= agonistic action of Bay K 8644 includes multiple mechanisms, and that this embryonic preparation is a good model for exploring the second mechanism without masking by the open-time prolongation.

Acknowledgements We thank Drs. Y. lnoue and I.R. Josephson for helpful discussions, Susan Osborn and Lisa Wuksick for preparation of the cell cultures. This work was supported by National Heart, Lung and Blood Institute Grant HL-31942 (U.S.A.).

Since the first report of Hess et al. (1984) that Ca’+ channel agonists prolonged open-times of the Ca’+ channels in adult guinea-pig and frog heart cells, many subsequent reports reproduced this finding in various adult and neonatal heart cells (Kokubun and Reuter, 1984; Lacerda and Brown, 1989). However, Brown et al. (1984) reported that the Ca’+ agonist CGP 28392 enhanced the Ca’+ channel activities of neonatal rat heart cells without affecting the mean open-time. Recently. we (Tohse and Sperelakis, 1990) reported that the L-type Ca’+ channels of young (3-day-old) embryonic chick heart cells exhibited long-lasting openings having similar properties to those observed in adult mammalian heart cells in the presence of DHP Ca’+ agonist, and suggested that these heart cells seem to naturally possess a mechanism (or substance) for producing the long openings. In the present study, although Bay K 8644 failed to prolong the opening of these Ca’+ channels, the Ca” agonist did produce marked agonistic effects on the ensemble-averaged currents. These results suggest that the Ca’+=agonistic action includes a mechanism other than prolongation of the open-times. There are two possibilities: (1) enhancement of availability of each Ca2+ channel (i.e., increase probability of openings), and (2) activation of additional non-functional Ca2+ channels. The present

eferences Brown, A.M., D.L. Kunze and A. Yatani, 1984, The agonist effect of dihydropyridines on Ca channels, Nature 31 I, 570. Caffrey, J.M.. I.R. Josephson and A.M. Brown, lY86, Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells, Biophys. J. 49, 1237. Hamill, O.P., A. Marty. E. Neher B. Sakmann and F.J. Sigworth, 1981, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfhigers Arch. 391, X5. Hess, P., J.B. Lansman and R.W. Tsien. 1984, Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists, Nature 311, 538. Hoshi, T. and S.J. Smith. 1987, Large depolarization induces long openings of volta,e-dependent calcium channels in adrenal chromaffin cells, J. Neurosci. 7, 571. Kokubun, S. and H. Reuter, 1984, Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells, Proc. Natl. Acad. Sci. U.S.A. 81. 4824. Lacerda, A.E. and A.M. Brown, 1989, Nonmodal gating of cardiac calcium channels as revealed by dihydropyridines, J. Gen. Physiol. 93, 1243. Sada, H.. M. Kojima and N. Sperelakis, 1988. Use of single heart cells from chick embryos for the Na+ current measuremants. Mol. Cell. Biochem. 80, 9. Tohse. N., N. Sperelakis, 1990, Long-lasting openings of single slow (L-type) Ca’+ channels in chick embryonic heart cells, Am. J. Physiol. 259, H63Y.