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(Circulation Research. 1996;78:717-723.)
© 1996 American Heart Association, Inc.

Articles

Effect of Membrane Potential on the Initiation of Acetylcholine-Induced Ca²⁺ Transients in Isolated Guinea Pig Coronary Myocytes

Vladimir Y. Ganitkevich, Gerrit Isenberg

From the Department of Physiology (V.Y.G.), University of Cologne (Germany) and the Department of Physiology (G.I.), University of Halle (Germany).

Correspondence to Dr V.Y. Ganitkevich, Department of Physiology, University of Cologne, Robert-Koch-Str. 39, 50931 Köln, Germany.

	Abstract

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Abstract The muscarinic stimulation of single voltage-clamped coronary arterial smooth muscle cells of the guinea pig was used to evaluate the effect of membrane potential on the inositol 1,4,5-tris-phosphate (IP₃)–mediated changes of ionized [Ca²⁺] in the cytoplasm (Ca²⁺ transient) measured with indo 1. When applied at the membrane potential of -50 mV, 10 µmol/L acetylcholine (ACh) induced a [Ca²⁺]_i increase after the mean latency of 2.6±0.9 s. The latency was reduced to 1.1±0.3 s when the same dose was applied at a holding potential of +50 mV. In paired experiments in the same cells, the latency of response at +50 mV was reduced by a factor of 2.2±0.3 compared with the response at -50 mV. Supramaximal [ACh] (100 µmol/L) induced Ca²⁺ transients with a 0.4±0.1-s latency, which was independent of membrane potential. When applied repetitively at -50 mV, ACh induced Ca²⁺ transients with a progressively reduced amplitude and slower rate of rise. Depolarization to +50 mV accelerated the rate of rise of the Ca²⁺ transient by a factor of 3.4±0.4 without affecting the amplitude. The modulation of the initiation of Ca²⁺ transient by a 100-mV depolarization can be explained by an approximately threefold increase in the rate of IP₃ accumulation.

Key Words: acetylcholine • membrane potential • Ca²⁺ transients

	Introduction

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In many types of smooth muscle cells, agonists (like ACh) induce the increase in [Ca²⁺]_i via the release of IP₃ from the lipids of the plasma membrane and following IP₃-induced Ca²⁺ release (IICR).^{1 2} Mostly, the [Ca²⁺]_i signaling is studied without control of membrane potential, ie, in cells that are not voltage-clamped. Recently, it became clear that the membrane potential is, at least in some cells, of importance for IP₃-mediated Ca²⁺ transients. It was shown that depolarization accelerates the PIP₂ breakdown,³ that hyperpolarization inhibits the IP₃ production,^{4 5} and that the membrane potential modulates the [Ca²⁺]_i in the presence of agonist.⁶ However, the consequences of the putative voltage dependence of IP₃ accumulation for the initiation of the Ca²⁺ transient in intact cells are unknown. In the present study, we examined the effect of membrane potential on the initiation of the ACh-induced Ca²⁺ transient in single isolated voltage-clamped coronary smooth muscle cells.

Assuming that IP₃-induced Ca²⁺ release starts when some "critical" IP₃ concentration is reached,^{7 8} we used the latency of IICR as an indirect measure of the IP₃ accumulation rate. Once IICR has started, it is subjected to a feedback by the intracellular mechanisms involved in Ca²⁺ signaling.^{9 10 11} We attempt to reduce the influence of positive feedback through released Ca²⁺ by depleting the IP₃-sensitive Ca²⁺ store with repetitive ACh applications. Under these conditions, the rate of [Ca²⁺]_i rise was used as the approximate measure of the IP₃ accumulation. Both approaches give similar results: the initiation of the Ca²⁺ transient is modified by a 100-mV depolarization in a way that can be explained by an approximately threefold increase of the IP₃ accumulation rate.

	Materials and Methods

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Guinea pigs (200 to 400 g) were killed by cervical dislocation. Single myocytes were enzymatically isolated from the left coronary artery as described before.¹² Briefly, the artery was cut out from the heart and placed for 40 to 60 minutes in a nominally Ca²⁺-free PSS (see below). Thereafter, the artery was placed for 20 to 30 minutes in 1 mL of this solution complemented with 1 mg/mL collagenase (0.5 to 0.7 U/mg, Serva) and 1 mg/mL elastase (130 to 200 U/mg, Serva). The suspension of isolated cells was obtained by pipetting the tissue with a wide-bore Pasteur pipette. Cells were placed in Ca²⁺-free PSS and stored at 4°C before use on the same day. During the experiment, the myocytes were continuously superfused with a 36°C warm PSS composed of (mmol/L) NaCl 150, CaCl₂ 2.5, MgCl₂ 1.2, KCl 5.4, glucose 20, and HEPES 5, adjusted with NaOH to pH 7.4. ACh (Sigma, dissolved in nominally Ca²⁺-free PSS or Ca²⁺-free PSS supplemented with 0.1 mmol/L EGTA) was applied to a cell through a four-barrel pipette as described previously,⁶ and the solution exchange rate was adjusted to be completed within <0.2 s (as measured by application of high-K⁺ solution and the change of the K⁺ current). The voltage-clamp method for measuring membrane currents and the microfluorospectroscopy for measuring [Ca²⁺]_i have been described previously.⁶ Briefly, the isolated cells were loaded with 100 µmol/L K₅–indo 1 through patch electrodes of 3- to 5-M resistance. Whole-cell membrane currents were recorded with an RK-300 amplifier (Biologic), filtered at 1 kHz, and stored on an IBM-compatible host computer. For microfluorospectroscopy, the cells loaded with indo 1 through the patch pipette were excited at 340 nm. Emitted light in bands from 395 to 425 nm and from 450 to 490 nm was collected by a pair of photomultipliers (Hamamatsu Photonics). After filtering at 20 Hz, the fluorescence ratio F₄₁₀/F₄₇₀ was obtained on-line by an analog divider (Burr Brown DIV100). The background fluorescence was electronically subtracted in the cell-attached mode. [Ca²⁺]_i was evaluated off-line, using an intracellular calibration procedure.

The pipettes were filled with a K⁺ electrode solution containing (mmol/L) KCl 140, Na₂ATP 2, MgCl₂ 3, HEPES 10, and K₅–indo 1 0.1, adjusted with NaOH to pH 7.2. Where indicated, 60 µmol/L of PKC inhibitor H-7 (Sigma) was added to the intrapipette solution. Another PKC blocker, GF 109 203X (Boehringer), was dissolved as 2 mmol/L stock solution in dimethyl sulfoxide and was added to the extracellular solution (final concentration, 0.1 µmol/L). All experiments were performed at 36°C. When adequate, the results were expressed as mean±SD.

	Results

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Effects of Varying the Holding Membrane Potential Between -50 mV and +50 mV on ACh-Induced Ca²⁺ Transients
Fig 1A shows the typical response of a coronary myocyte held at -50 mV to a 50-s application of 10 µmol/L ACh in a Ca²⁺-free solution supplemented with 0.1 mmol/L EGTA. After application of 10 µmol/L ACh, [Ca²⁺]_i increased not immediately but after a delay of several seconds, and it peaked within 5 s to 1.8 µmol/L. The ACh-induced [Ca²⁺]_i response often started with a slow "foot," which is difficult to quantify; therefore, the latency was measured as the time between the start of the ACh application and the time when the [Ca²⁺]_i response reached 20% of its peak. This latency varied between 0.6 and 4 s in individual cells held at -50 mV, with a mean value of 2.6±0.9 s (n=35). In 60% of cells (n=21), a foot (Fig 1A and 1B) at the beginning of the Ca²⁺ transient was observed.¹¹ The [Ca²⁺]_i increase was slow (<0.1 µmol/L per second) at the beginning of the Ca²⁺ transient (Fig 1A and 1B). Subsequently, the rate of [Ca²⁺]_i increase was accelerated (maximal rate of rise, up to 10 µmol/L per second; reached 3.6 s after the start of ACh application). This acceleration indicates the involvement of the positive feedback. In the other 40% of the cells, the rise of [Ca²⁺]_i was fast from the very beginning (Fig 1C), and no clear foot could be distinguished in these cells. Correspondingly, these cells showed shorter latencies. Within 2 to 4 s after ACh application, [Ca²⁺]_i peaked to values between 1 and 2 µmol/L. From its peak, [Ca²⁺]_i fell down fast initially and then more slowly (Fig 1A). During this slow decay in the presence of ACh, [Ca²⁺]_i is thought to be extruded from the cell, since subsequent ACh applications induced greatly attenuated Ca²⁺ transients (not shown). Therefore, short (up to 10-s) ACh applications were routinely used in the following experiments, which induced much more reproducible Ca²⁺ transients.

View larger version (18K): [in this window] [in a new window]   Figure 1. A, Ca2+ transient in a coronary myocyte induced by a 50-s application of 10 µmol/L ACh. ACh was applied together with Ca2+-free PSS containing 0.1 mmol/L EGTA. Membrane potential was held at -50 mV. Lower trace is the membrane current. Upper trace is the pen recording of the fluorescence ratio F410/F470; note the nonlinear [Ca2+]i scale. B, Example of Ca2+ transient with a slow "foot" preceding a rapid [Ca2+]i rise. C, Example with no apparent foot. Panels A, B, and C are from different cells. The duration of ACh application is indicated above the traces.

After receptor occupation by the agonist, the latency of the Ca²⁺ transient is mainly due to the time required by PLC to cleave PIP₂ and to liberate IP₃.¹³ That is, other steps are thought to be considerably faster.^{14 15 16 17 18 19 20} The Ca²⁺ release starts when [IP₃] reaches some critical level.^{7 8} The hypothesis that the membrane potential influences the rate of IP₃ accumulation predicts that under conditions of constant SR Ca²⁺ load, the latency of the response depends on membrane potential. Depolarization reduced the latency by accelerating IP₃ accumulation so that critical [IP₃] could be reached earlier; hyperpolarization of the cell membrane increased the latency. Thus, the effect of the membrane potential on the latency of the IICR was analyzed in the first series of experiments.

When the cell was held at +50 mV, the resting [Ca²⁺]_i was not modified. ACh application induced Ca²⁺ transients with an amplitude very similar to the one at -50 mV; however, the latency of the response was markedly reduced. In 11 cells, a latency of 1.1±0.3 s was measured at +50 mV, on average. The variability of latencies between cells (see above) complicated the analysis; therefore, the effect of membrane potential on latency was tested in the same cell.

Fig 2A shows two superimposed ACh-induced Ca²⁺ transients recorded at holding membrane potentials of -50 mV and +50 mV in the same cell. The first application of ACh at -50 mV triggered the Ca²⁺ transient after a latency of 3.4 s. There was a clear foot during the rising phase of the [Ca²⁺]_i response (Fig 2A). Subsequently, membrane potential was stepped to +50 mV, and 1 minute later, the ACh application was repeated. The Ca²⁺ transient then had a slightly lower amplitude, suggesting some deprivation of SR with releasable Ca²⁺ after the first transient. However, the latency of the ACh response was then reduced to 1.6 s, and the foot became less distinguishable. In this cell, the latency was reduced by a factor of 2.1 by stepping the membrane potential 100 mV in a more positive direction. In seven cells studied with the same protocol, the latency of the Ca²⁺ transient was reduced by a factor of 2.2±0.3.

View larger version (20K): [in this window] [in a new window]   Figure 2. The latency of the Ca2+ transient is voltage and concentration dependent. A, ACh (10 µmol/L) was applied twice to the same cell. At first, ACh was applied at -50 mV; it induced a Ca2+ transient after a latency of 3.4 s. Then, the membrane potential was stepped to +50 mV, and 1 minute later, ACh was applied again. The Ca2+ transient started after a latency of 1.6 s. The traces were superimposed for better comparison. B, Holding potential was set at +50 mV. Initially, 1 µmol/L ACh induced a Ca2+ transient with a prominent "foot." Thirty seconds later, application of 10 µmol/L ACh induced a Ca2+ transient with a markedly shorter latency and without a foot. The traces were superimposed for better comparison. The duration of ACh application is indicated.

IP₃ is not the only second messenger produced during the ACh action. Cleavage of PIP₂ by PLC results in activation of PKC through the membrane-bound second messenger DAG. PKC activation was shown to inhibit the agonist-induced IP₃ accumulation in various types of cells, including smooth muscle cells.²¹ Therefore, the effect of PKC inhibitors (GF 109 203X and H-7) on the initiation of the ACh-induced Ca²⁺ transient was studied. Cells were treated for at least 30 minutes with the membrane-permeable PKC blocker GF 109 203X (0.1 µmol/L). Then the whole-cell measurements were performed with an intrapipette solution containing 60 µmol/L of H-7. ACh-induced Ca²⁺ transients were measured at least 3 minutes after establishment of the whole-cell mode. In five cells, the latency of 2.2±0.4 s was measured at -50 mV. At +50 mV, it was reduced to 1.0±0.3 s. Both values were not significantly different from the control experiments without PKC inhibitors. Thus, the inhibition of PKC does not have an immediate effect on the latency of the ACh-induced Ca²⁺ transients and on its modulation by the membrane potential.

ACh-Induced Ca²⁺ Transients and Their Voltage Dependence at a Varied [ACh]
Since the latency is known to be a function of receptor occupancy,^{8 22 23} the effect of membrane potential was compared with the effect of a varied agonist concentration. When [ACh] was reduced from 10 to 1 µmol/L, the latency of the Ca²⁺ transients at +50 mV was increased from 1.1±0.3 to 4.8±1.7 s (n=5). Along with the increase of latency, a clear foot appeared with 1 µmol/L ACh (Fig 2B).

At -50 mV, 1 µmol/L ACh usually did not induce Ca²⁺ transients (Fig 3), whereas 10 µmol/L triggered full-sized responses. After stepping to +50 mV, 1 µmol/L ACh induced Ca²⁺ transients, although with a lower peak amplitude and with a slower maximal rate of rise compared with those found with 10 µmol/L ACh (800 nmol/L and 1 µmol/L per second versus 1600 nmol/L and 8 µmol/L per second). This result suggests that at low fractional receptor occupancy (ie, a low rate of IP₃ accumulation), the membrane potential can determine whether IICR will happen or not.

View larger version (11K): [in this window] [in a new window]   Figure 3. The [Ca2+]i response to 1 µmol/L ACh depends on membrane potential. Applied at -50 mV, 1 µmol/L ACh does not induce a Ca2+ transient, whereas 10 µmol/L ACh elevated [Ca2+]i to 1.8 µmol/L. Then, membrane potential was stepped to +50 mV, 1 µmol/L ACh was applied again, and [Ca2+]i was elevated to 0.8 µmol/L. Subsequent application of 10 µmol/L ACh elevated [Ca2+]i to 1.6 µmol/L. Lower trace shows the voltage protocol; middle trace, membrane current; and upper trace, [Ca2+]i in nonlinear scale. The duration of ACh application is indicated above the traces.

At supramaximal stimulation (high rates of IP₃ accumulation), the latency of IP₃-induced Ca²⁺ release is known to approach its minimal value (<1 s) in different preparations.^{8 15 23} This minimal latency presumably reflects the saturation of PLC. When supramaximal concentrations of ACh (100 µmol/L) were applied at -50 mV, the Ca²⁺ transients were triggered after a latency of 0.4±0.1 s (n=8). In these experiments, ACh was dissolved in high-K⁺ solution to precisely determine the time of solution change (from the changes in K⁺ current). When 100 µmol/L ACh was applied at +50 mV, a similar latency was measured, and in paired applications to the same cell, latency of response was not influenced by stepping the holding potential from -50 mV to +50 mV (Fig 4). These results suggest that conditions that bring PLC close to saturation, ie, that reduce the latency of the Ca²⁺ transient to its minimal value, prevent the effect of membrane potential on the latency. The lack of the effect of membrane potential on the latency of [Ca²⁺]_i responses to 100 µmol/L ACh suggests that the voltage dependence of the latency measured between -50 and +50 mV (Fig 2A) is underestimated. When corrected for 0.4-s minimal latency, the above change of the holding potential reduced latency by a factor of 2.8±0.7.

View larger version (15K): [in this window] [in a new window]   Figure 4. At supramaximal 100 µmol/L ACh, the membrane potential does not modulate the initiation of the Ca2+ transient. Upper traces show Ca2+-sensitive K+ current (potentials indicated). Lower traces show [Ca2+]i in nonlinear scale. ACh was applied in solution with substitution of NaCl for KCl to indicate the moment of solution change by the change in membrane current. The duration of ACh application is indicated. The traces are superimposed for better comparison. Note that with 100 µmol/L ACh, neither the latency nor the upstroke of the Ca2+ transient was affected by changing the membrane voltage between -50 and +50 mV.

Effect of Varying of Membrane Potential on the Maximal Rate of Rise of Ca²⁺ Transients
The other method, besides assessing latency, of estimating the effect of membrane potential on IICR is to analyze the rate of rise of the Ca²⁺ transient, which is proportional to the number of simultaneously opened release channels. In these cells, ryanodine was without effect on ACh-induced Ca²⁺ transients¹¹ (authors' unpublished data, 1995); thus, Ca²⁺-induced Ca²⁺ release is not involved in the Ca²⁺ transients under study.

Once initiated, IP₃-induced Ca²⁺ release is subjected to positive feedback by cytoplasmic Ca²⁺.^{9 10} Under this condition, the rate of rise of the Ca²⁺ transient is not the measure of [IP₃] but is likely to be limited by other processes (eg, Ca²⁺-induced transition into a high-affinity state of IP₃ receptor^{24 25} ). In agreement with this idea, the maximal rate of rise was not significantly influenced by changing the membrane potential to +50 mV, providing that the interval between application was sufficiently long to allow the recovery of the peak amplitude (Fig 2A).

The influence of Ca²⁺ feedback can be reduced during desensitization of the transduction pathway, resulting, presumably, in both lower rates of IP₃ accumulation and deprivation of the SR of releasable Ca²⁺. When the SR becomes Ca²⁺-depleted, the rate of rise of the Ca²⁺ transient is expected to be less dependent on the positive feedback; hence, it may measure the IP₃ accumulation more appropriately.

When 10 µmol/L ACh was applied repetitively to the cell held at -50 mV, Ca²⁺ transients of progressively smaller amplitude were evoked (Fig 5A). Simultaneous with the reduction of the peak amplitude, the maximal rate of rise of [Ca²⁺]_i was reduced from 4.8 µmol/L per second (first transient) to 1.24 µmol/L per second (second transient) and to 0.07 µmol/L per second (fourth transient). At the same time, the latency become longer (4.5 s for fourth transient compared with 1.8 s for first transient). This result is thought to suggest that the reduction of the SR Ca²⁺ load can slow down the rise of the Ca²⁺ transient. Because the latency is prolonged in parallel, the IP₃ accumulation rate also seems to be slowed down. Then the membrane potential was stepped to +50 mV, and 10 s later, ACh was applied again (Fig 5A). The response was a Ca²⁺ transient with a very similar amplitude but with a markedly shorter latency and faster rate of rise (Fig 5A). Since the step to +50 mV did not change the resting [Ca²⁺]_i and because the peak of ACh-induced transient at +50 mV was about the same as at -50 mV, we suggest that the holding potential change does not significantly change the SR Ca²⁺ load during the time between applications. Since the SR Ca²⁺ load was not increased, then, most likely, an altered gain in feedback through cytoplasmic Ca²⁺ ions was not the reason for the acceleration of the Ca²⁺ transient at +50 mV (Fig 5). Instead, the reduction of the latency in parallel with the increase of maximal rate of rise can be explained by the assumption that the depolarization accelerates the IP₃ accumulation.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of membrane potential on the rate of rise of the Ca2+ transient. A, Four applications of 10 µmol/L ACh at -50 mV resulted in a decrease of the peak amplitude and of the rate of rise and an increase in the latency of the Ca2+ transients. Depolarization to +50 mV does not increase the peak of [Ca2+]i during the fifth transient; however, it shortens its latency and increases its rate of rise. B, Evaluation of maximal rates of rise at -50 mV and +50 mV from a similar experiment (cell different from one in panel A) is shown. Lines of maximal rate of rise are linear regression through linearized data. The slope is 0.16 µmol/L per second for -50 mV (left) and 0.52 µmol/L per second for +50 mV (right). The duration of ACh application is shown.

Evaluation of computer playbacks (Fig 5B) suggests that stepping from -50 mV to +50 mV increased the maximal rate of rise of the Ca²⁺ transient from 160 to 520 nmol/L per second. Approximately the same peak of [Ca²⁺]_i (500 nmol/L) was reached at -50 mV within 7 s and at +50 mV within 2 s after the addition of ACh. However, the maximal rate of [Ca²⁺]_i rise did not occur at some definite level of [Ca²⁺]_i. For example, in the cell shown in Fig 5B, the maximal rate of rise of the Ca²⁺ transient was reached 4.4 s after the addition of ACh at -50 mV and 0.7 s at +50 mV (at 240 and 170 nmol/L [Ca²⁺]_i, respectively). Thus, we did not observe in this experiment some critical [Ca²⁺]¹¹ associated with the maximal rate of [Ca²⁺]_i rise. However, the global indo 1 signal does not report [Ca²⁺]_i in the vicinity of IP₃-activated release channels.

This protocol was applied totally to five cells. In summary, changing the holding potential from -50 mV to +50 mV accelerated the maximal rate of rise of the Ca²⁺ transient by a factor of 3.4±0.4 after it was slowed down by repetitive ACh applications.

	Discussion

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The results of the present study confirm and put forward the idea that the membrane depolarization facilitates the IP₃-mediated Ca²⁺ release. In the present study, we evaluate this idea in terms of the latency of the IICR, and we show that this parameter becomes shorter with increasing [ACh] and with more positive membrane potentials.

As a second messenger, IP₃ diffuses much faster than Ca²⁺ does.^{26 27} Despite its fast diffusion at 283 (µmol/L)² per second, it is not possible to exclude that spatial inhomogeneities in [IP₃] occur initially (in the subsecond range) at a high degree of PLC activation. [IP₃] inhomogeneities would be reported by the IICR, however, not by the chemical determination of IP₃. [IP₃] was not measured in the present study; hence, the interpretation of the membrane potential effect on IICR depends on the assumption used.

Presumably, the IP₃-mediated Ca²⁺ release starts when [IP₃] reaches some threshold level.^{7 8 14} With this assumption, the IP₃ accumulation to the threshold level is related to the time between receptor occupation and the beginning of the IICR, ie, latency of the [Ca²⁺]_i response. Whether IICR will be initiated or not depends on the rate of IP₃ production and the rate of IP₃ degradation (ie, the lifetime of IP₃). If the rate of IP₃ production overrides the rate of degradation to such an extent that accumulation to the critical concentration occurs, then the IICR begins. Once initiated, IICR is modified by the feedback of the released Ca²⁺,^{9 10 11} which is supposed to transfer the IP₃ receptor into a state of higher affinity to IP₃.^{24 25} Another possible feedback presumably involved in IICR is the PKC-mediated inhibition of IP₃ accumulation.²¹ However, to be activated by DAG, PKC needs to be translocated to the membrane. This process seems to be considerably slower than the time scale of the latency measurements.²⁶ Feedbacks involving Ca²⁺-activated PKC isoforms are unlikely to be of importance for the latency measurements, since the latency was defined as the time until moderate (20%) increase of [Ca²⁺]_i. In agreement with this, our experiments showed no significant effect of PKC inhibitors on the latency of the [Ca²⁺]_i response. This supports the idea that the latency can be related to the accumulation of IP₃ to threshold concentration.

According to the above assumptions, the shortening of the latency by membrane depolarization is attributed to the faster rate of IP₃ accumulation. For quantification, the minimal latency, which is voltage independent and presumably corresponds to saturated PLC, should be subtracted. In reference to those corrected latency measurements, the 100-mV depolarization shortens the time to reach the threshold concentration by 2.8-fold. This estimate, however, is valid only if the IP₃ degradation rate is considerably slower. The estimate also depends critically on the precision of the estimation of minimal latency.

The results of the effect of membrane potential on the response to 1 µmol/L ACh (Fig 3) suggest that with low fractional receptor occupancy, the membrane potential can define whether IICR will start or not. With low fractional receptor occupancy, IP₃ is released at a low rate, presumably comparable to the IP₃ degradation rate. The accumulation to the threshold does not happen or requires a time so long such that a Ca²⁺ transient at -50 mV is not evoked (Fig 3). This could mean that the lifetime of IP₃ is short in coronary myocytes (in other cells, 1 to 9 s^{8 27 28} ).

The other approach used in the present analysis is the measurement of the rate of rise of "desensitized" IICR. Repeated ACh applications partially depleted the IP₃-sensitive Ca²⁺ store and, hence, should reduce the influence of the positive feedback through the released Ca²⁺. Under these conditions, the rise in [Ca²⁺]_i was markedly slowed, and it could be accelerated again by the subsequent depolarization. Because the results suggest that the SR Ca²⁺ load was not changed, this acceleration of the rate of rise of IICR by stepping to +50 mV cannot be explained by a Ca²⁺ feedback. Most likely, the rise of [Ca²⁺]_i during desensitized IICR is rate-limited by a slow accumulation of IP₃. The effect of depolarization on the maximal rate of rise of the Ca²⁺ transient (factor of 3.4) is likely to be overestimated, since the IP₃ degradation process is likely to be less important during the fast [Ca²⁺]_i (and IP₃) rise at +50 mV than during the slow process at -50 mV (Fig 5). In addition, it is possible that during the slow rise of the desensitized Ca²⁺ transient, other feedbacks (mediated through DAG-activated PKC isoforms and/or Ca²⁺-activated PKC isoforms) are of importance.

In conclusion, we have shown for coronary myocytes that the membrane voltage influences the initiation of the Ca²⁺ transient. The effect was concentration dependent: high [ACh] induced responses that were independent of the membrane potential, whereas the effect of low doses of agonist was voltage dependent. If we accept the assumptions about the rate-limiting processes involved in the initiation of the IICR, then the present results could be explained by an approximately threefold acceleration of IP₃ accumulation rate for a 100-mV change of the membrane voltage.

In vivo, even in the absence of agonists, the PLC in vascular smooth muscle has been shown to become activated by stretch.²⁹ Elevation of transmural pressure also directly activates PLC and leads to the production of IP₃.³⁰ These findings, together with our findings that the membrane potential exerts more of an effect during low rates of IP₃ accumulation, suggest a new role for PLC in coupling the changes of membrane potential to vascular tone.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine [Ca2+]i = ionized [Ca2+] in the cytoplasm DAG = diacylglycerol GF 109 203X = bisindolylmaleimide IICR = IP3-induced Ca2+ release IP3 = inositol 1,4,5-tris-phosphate PIP2 = phosphatidylinositol 4,5-diphosphate PKC = protein kinase C PLC = phospholipase C PSS = physiological salt solution SR = sarcoplasmic reticulum

Received September 19, 1995; accepted December 7, 1995.

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