Arginine Vasopressin–Induced Potentiation of Unitary L-Type Ca²⁺ Channel Current in Guinea Pig Ventricular Myocytes

Preparations

Ventricular myocytes from guinea pig hearts were obtained by an enzymatic dissociation procedure similar to that described previously.⁹ Briefly, guinea pigs weighing 250 to 350 g were anesthetized with pentobarbital sodium (40 mg/kg IP). The chest was opened, and the aorta was cannulated in situ and perfused with Tyrode’s solution before the heart was removed. Hearts were then retrogradely perfused with low-Ca²⁺ (30 μmol/L) Tyrode’s solution with collagenase (0.4 mg/mL, type I, Sigma Chemical Co) for 20 minutes by using the Langendorff apparatus. After the enzyme was washed out, the cells were dissociated in high-K⁺ low-Cl⁻ storage solution.

Electrophysiological Measurement and Data Analysis

Single L-type Ca²⁺ channel currents were recorded in cell-attached configuration¹⁰ by using an AXOPATCH-1D amplifier (Axon Instruments) at room temperature (22°C to 24°C). Pipettes were pulled from capillary tubes in a two-step process, coated with insulating varnish, and fire-polished afterward. The electrode had a resistance of 5 to 10 MΩ when the pipette was filled with the Ba²⁺ solution. The membrane potential of myocytes was depolarized to ≈0 mV by high-K⁺ solution. The electrode potential was adjusted to give a zero current between the pipette solution and the bath solution immediately before the seal formation. From a holding potential of −80 mV, patches were depolarized at 1 Hz to 0, +10, or +20 mV for 180 ms. The threshold for the activation of L-type Ca²⁺ channels was ≈−10 mV in our recording conditions. After the gigaseal formation, stability of basal Ca²⁺ channel activity was checked for at least 10 minutes. Current signals were usually filtered at 1 kHz (−3 dB, eight-pole Bessel filter) and digitized at 5 kHz by using pclamp software (Axon Instruments) on an IBM AT personal computer. After digital subtraction for capacitive and leak components, idealized recordings obtained by standard half-height criteria were used to calculate the channel open probability (Po or NPo, where Po is the probability of the channel opening and N is the number of functional channels) and to obtain averaged current tracings. Fittings for open or closed time distributions were performed with pclamp software using the maximum-likelihood method and the nonlinear least-squares fitting method. Fitted values presented in this article were those for which both methods returned consistent values. Where appropriate, numerical values are presented as mean±SD. Differences in the numerical values between two groups were evaluated by using Student’s t test. A value of P<.05 was considered significant.

[Ca²⁺]_i Measurement

In some experiments (Fig 5⇓), changes in [Ca²⁺]_i were monitored simultaneously during single-channel current recordings. This method has been described in detail in our recent report,¹¹ along with the assessment of its limitations to obtain absolute [Ca²⁺]_i values. Briefly, cells were loaded with fura 2 by exposure to 5 μmol/L acetoxymethyl ester and were placed on a stage of an inverted microscope equipped with epifluorescence optics (Diaphoto TMD, Nikon). [Ca²⁺]_i was monitored by using a dual-wavelength fluorometer (CAM-230, Japan Spectroscopic). Fluorescence was excited at wavelengths of 340 and 380 nm alternatively by using a rotating sector mirror method, with a chopper frequency set at 400 Hz. The emission of fluorescence (500 nmol/L) was sampled and processed by the CAM-230 chopper system to yield two fluorescence intensities separately. They were digitized at 5 kHz along with current signals and stored in a computer (PC9801RA, NEC). By use of locally written programs, the ratio of fluorescence intensities (R340/380) was obtained for every sweep to elicit channel activity. [Ca²⁺]_i levels were then determined by using a calibration curve obtained by in vivo calibration technique.¹²

Solutions and Drugs

The bath solution contained (mmol/L) potassium aspartate 120, KCl 20, glucose 10, EGTA 2, and HEPES 10 (pH 7.4 by KOH). The pipette solution contained BaCl₂ 100, HEPES 10 (pH 7.4 by tetraethylammonium hydroxide). AVP was obtained from Sigma. Blockers for vasopressinergic receptors (OPC-21268¹³ and OPC-31260¹⁴ ) were gifts from Otsuka Pharmaceutical Co. OPC-21268 was dissolved in dimethyl sulfoxide (DMSO), and OPC-31260 was dissolved into distilled water to form a 10 mmol/L stock solution. Isoquinolinesulfonamide protein kinase inhibitors (H7¹⁵ and H89¹⁶ ) were obtained from Seikagaku Kogyo Co. H7 was dissolved in distilled water, and H89 was dissolved in DMSO to form a 10 mmol/L stock solution. Phorbol 12-myristate 13-acetate (PMA) and 4α-phorbol 12,13-didecanoate (4αPDD) were obtained from Sigma. They were dissolved in DMSO to form a 1 mmol/L stock solution.

Effects of AVP on Ca²⁺ Channel Activity

Application of AVP to the bath solution caused an increase in Ca²⁺ channel activity in almost all the patches examined. Fig 1A⇓ shows current tracings from a single-channel patch in the control condition and during the application of 100 nmol/L AVP. AVP increased the number of channel openings, with their duration moderately increased. Current levels for channel open state, however, were not discernibly affected. At +20 mV, it was 1.17±0.10 pA in the control condition and 1.12±0.11 pA (n=6) after AVP application. Thus, AVP increased the averaged current amplitude (shown in the bottom tracing) through enhanced Po. The temporal profile of Po from this patch is shown in Fig 1B⇓. It should be noted that effects produced by AVP took place slowly, and usually ≈5 to 10 minutes was required before the new steady state level was achieved. The increase in Po was parallel with the number of channel openings with long duration (≥5 ms).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Effect of arginine vasopressin (AVP) on L-type Ca²⁺ channel currents. A, Unitary Ca²⁺ channel current in the control condition (a) and during AVP application (b). Six consecutive sweeps are shown. Voltage-clamp pulses (shown on the top) were delivered at 1 Hz. Averaged current tracings on the bottom were obtained from 250 idealized recordings for each condition. B, Temporal profile of channel open probability (Po) during the 20-minute observation period (a). Each bar indicates averaged Po over five consecutive sweeps, including 0 for null sweeps. The numbers of sweeps containing long openings (≥5ms) are shown in the same way (b). The AVP application period is indicated by the line on the top.

Fig 2⇓ depicts the results of kinetic analysis from this patch. AVP increased the proportion of sweeps with channel activity (availability)¹⁷ from 46.0% in the control condition to 65.6% after the application. However, this effect was only partially responsible for AVP-induced potentiation. In a of Fig 2⇓, each sweep was characterized by its Po during depolarization and longest open time (Tmax) observed in the corresponding sweep.¹⁸ The plot of Po versus Tmax indicated that AVP shifted the distribution of sweeps toward increased Po and prolonged Tmax. This was associated with changes in both open and closed time distributions (b and c of Fig 2⇓). With the fitting range up to 10 ms, open time distributions during AVP application required two exponential components for the fit. The shorter time constant during AVP application was similar to the time constant in the control state; the fit assumed the presence of a single-exponential component.¹⁹ The induction or potentiation of an extraexponential component during AVP application was a consistent finding in an additional seven patches, including multichannel patches from which open time constants could be analyzed reliably because of the paucity of stacked channel openings (see also legend to Fig 3⇓). We then tried to evaluate the amount of charges carried by long openings induced by AVP. Because the total charge carried during each event was calculated as open duration times open current amplitude, we calculated the sum of durations of the events belonging to individual bins. These values after multiplication by current amplitude were superimposed on open time histograms (thick lines in b of Fig 2⇓). According to this calculation, channel openings whose duration exceeded 5 ms carried 5.5% of the total charge in the control state. After AVP application, long openings (≥5 ms) carried 13.8% of the total charges. We can also see that although the enhancement of long openings was one of the prominent findings after AVP application, an increase in the number of short openings (which dominated in the control state) also significantly contributed to the increase in overall Po. The latter effect should be explained in terms of the changes in closed time distribution (c of Fig 2⇓). They were fitted by the sum of two exponentials both in the control condition and after AVP application. In the present study, the effect of AVP was to shorten the longer time constant. This component corresponds to the duration of gaps between bursts.¹⁹ Similar findings were confirmed in three single-channel patches.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Changes in kinetic behavior during arginine vasopressin (AVP)–induced potentiation. Results are shown for the control condition (A) and during AVP application (B). Plot a indicates the distribution of sweeps with channel activity in the Po-Tmax plane (where Po is open probability and Tmax is longest open time), according to the method proposed by Yue et al¹⁸ ; b and c, open and closed time distributions, respectively. After AVP application, frequency distribution of open time was the sum of two exponential terms. Numbers indicate fitted time constants in milliseconds. In b, the thick lines indicate the product of open current amplitude and the sum of open durations of events belonging to each bin. Events exceeding 10 ms were lumped together and shown at the right end. Calibration bars on the right (]) are for 100 pA×ms (=1×10⁻¹³ coulomb) for both control and AVP application.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Effect of V1 blocker on arginine vasopressin (AVP)–induced potentiation. A, Temporal profiles of open probability (NPo, where N is the number of functional channels and Po is the probability of the channel opening) during an ≈1-hour observation period. Each bar indicates averaged NPo over 10 consecutive sweeps, including 0 for null sweeps. Period for application of OPC-21268 and AVP are indicated by lines on the top. B, Original current tracings during the period indicated by arrows in panel A. Small inverted triangles indicate the beginning and the end of 180-ms depolarizing pulses to 0 mV. Open time constants obtained from data sets a, b, and c were 0.31, 0.26, and 0.27 ms, respectively. Open time distribution from d required two exponential components for the fit (0.28 and 0.73 ms). The patch contained two Ca²⁺ channels, as judged from the maximum number of stacked openings.

The effects of AVP were persistent during continuous application for >20 minutes of the observation period (n=4) and were reversible on washout. This time course was gradual similar to the onset of potentiation. In three patches, the time periods around 15 minutes were required for the channel activity to return to the control levels.

Involvement of a Vasopressinergic Receptor

The effectiveness of bath-applied AVP (without direct access from the pipette solution to the channel molecule) and the slow onset of potentiation suggest that the AVP effect is mediated by intracellular signaling systems. AVP is known to exert its effects through two different types of receptors.^{20 21 22} Therefore, we tested whether AVP-induced potentiation was affected during the blockade of vasopressinergic receptors. Fig 3⇑ shows a test for the effect of OPC-21268,¹³ a specific blocker of V₁ receptor. As seen in b of Fig 3⇑, OPC-21268 (8 μmol/L, 20 times the IC₅₀ for V₁ receptor blockade) itself had no effect on channel activity. When AVP was added in the presence of OPC-21268, it failed to potentiate the activity of Ca²⁺ channels (c of Fig 3⇑). This failure should be explained by the blockade of specific receptor on the cell membrane, because AVP-induced potentiation appeared when OPC-21268 was washed out (d of Fig 3⇑). Similar results were obtained in three additional patches. On the other hand, OPC-31260,¹⁴ a specific blocker of the V₂ receptor, failed to suppress AVP-induced potentiation of Ca²⁺ channels even at the dose of 5 μmol/L (>1000 times the IC₅₀ for V₂ receptor blockade) in four of four cases tested. The AVP-induced changes in NPo during receptor blockade were summarized in Fig 4⇓. The significance of the AVP effect was evaluated by taking the ratio of channel open probability (NPo during AVP application/NPo in the control condition) as an index. The values were 2.92±1.43 (n=15) without blockers (left panel, P<.05), 1.05±0.22 (n=5) with OPC-21268 (middle panel, P=NS), and 2.67±0.97 (n=4) with OPC-31260 (right panel, P<.05).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Effects of vasopressinergic receptor blockade on arginine vasopressin (AVP)–induced potentiation. Summaries for AVP-induced changes in open probability (NPo, where N is the number of functional channels and Po is the probability of the channel opening) are as follows: left panel, without receptor blockade; middle panel, V1 receptor blockade by OPC-21268 (8 μmol/L); and right panel, V2 receptor blockade by OPC-31260 (5 μmol/L). NPo values are the average of at least 250 consecutive sweeps and presented without correction for the numbers of channels in the patch. Different symbols indicate different test potentials (○, 0 mV; ▵, +10 mV; and •, +20 mV).

Effects on [Ca²⁺]_i

Effects of AVP through V₁ receptor activation in- clude mobilization of intracellular Ca²⁺ in several tissues.^{5 6 20 23 24} Because limited increase in [Ca²⁺]_i is known to stimulate the activity of Ca²⁺ channels,^{11 25} we examined whether potentiation by AVP is related to changes in [Ca²⁺]_i in our experimental conditions. Fig 5⇓ shows the results of simultaneous measurement of channel activity and the fura 2 signals. AVP increased NPo by a factor of ≈2 in this patch. The ratio of fura 2 signals (R340/380), however, was not detectably influenced. Similar disparity between NPo and R340/380 was confirmed in two additional patches.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Effects of arginine vasopressin (AVP) on [Ca²⁺]_i and channel open probability (NPo, where N is the number of functional channels and Po is the probability of the channel opening). Currents were obtained from fura 2–loaded myocytes. Upper panel shows the fluorescence ratio (R340/380) averaged over five sweeps and indicates [Ca²⁺]_i level. Absolute values of [Ca²⁺]_i shown on the left should be taken only as an approximation.¹¹ In the lower panel, each bar indicates averaged NPo over five sweeps. The AVP application period is indicated by the line on the top.

Effects of Protein Kinase Inhibitors on AVP-Induced Potentiation

Ca²⁺ channels in muscles and neurons are under the modulation of channel phosphorylation cycles by protein kinases,²⁶ whose activities are specifically affected by various types of receptor stimulation. We observed that AVP-induced potentiation took place via the V₁ vasopressinergic receptor but not via the V₂ receptor, which is coupled to cAMP-dependent protein kinase (PKA) activation. This raises the possibility that protein kinase C (PKC) might be involved as a possible signal transduction pathway. Therefore, we examined whether AVP-induced potentiation was affected when kinase activity was suppressed. Fig 6⇓ shows the effect of AVP in the continued presence of H7,¹⁵ a membrane-permeant kinase inhibitor with broad spectrum, on AVP-induced potentiation. After the bath-application of H7 (100 μmol/L), NPo usually began to decline after ≈10 minutes and then settled to ≈50% of the control value. This was presumably due to the change in the basal phosphorylation level of the Ca²⁺ channels.²⁶ When myocytes were incubated with H7 for 60 minutes, AVP failed to potentiate channel activity with the averaged NPo ratio (AVP/control) of 0.93±0.17 (n=7). We then examined the effect of H89,¹⁶ a membrane-permeant kinase inhibitor with high sensitivity for PKA (Fig 7⇓). As described below, we confirmed the efficacy of H89 under our experimental conditions through the response to 8-bromo-cAMP. When myocytes were incubated by H89 for 60 minutes, bath application of 1 mmol/L 8-bromo-cAMP increased the channel activity with an averaged NPo ratio (AVP/control in the continued presence of H89) of 1.89±0.89 (n=4). This factor, however, was only approximately half of the value without intervention (3.52±1.37, n=5), with a significant difference (P<.05). We then compared the effects of AVP with and without H89 treatment. When applied after incubation by H89, AVP still exerted upregulation of the channel activity. AVP/control amounted to 2.52±1.34 (n=5), without a significant difference compared with the control condition (see Fig 4⇑, left panel). This was associated with the changes in kinetic behavior that were observed in the absence of PKA suppression, such as the increase in the number of sweeps with long openings (b of Fig 1B⇑).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

Effect of protein kinase inhibitor H7 on arginine vasopressin (AVP)–induced potentiation. A, Temporal profiles of open probability (NPo, where N is the number of functional channels and Po is the probability of the channel opening) during an ≈40-minute observation period, at a test potential of +10 mV. Each bar indicates averaged NPo over five sweeps. Periods for application of H7 and AVP are indicated by the lines on the top. B, Summary of changes in NPo. AVP was ineffective in the continued presence of H7. Different symbols indicate different test potentials (○, 0 mV; ▵, +10 mV; and •, +20 mV).

• Download figure
• Open in new tab
• Download powerpoint

Figure 7.

Effect of protein kinase inhibitor H89. A, Effect of H89 on arginine vasopressin (AVP)–induced potentiation. Temporal profiles of open probability (NPo, where N is the number of functional channels and Po is the probability of the channel opening) during a 40-minute observation period are presented (test potential, +10 mV). Each bar indicates averaged NPo over five sweeps (a) and the average number of sweeps with long openings (exceeding 5 ms) (b). Data were obtained from myocytes treated with H89 for 60 minutes. The AVP application period is indicated by the line on the top. B, Summary of the changes in NPo for H89-treated myocytes. H89 failed to suppress AVP-induced potentiation. Different symbols indicate different test potentials (○, 0 mV; •, +20 mV).

Effects of Phorbol Esters on AVP-Induced Potentiation

We then tested whether AVP could induce the potentiation of Ca²⁺ channels when applied after the activation of PKC by phorbol esters. In guinea pig myocytes at a dose of 100 nmol/L, PMA caused transient increases in channel activity in three of five cases. When cell-attached recordings were carried out from the myocytes incubated with PMA for 60 minutes, however, the channel activity was in a stable condition in the continued presence of PMA. Fig 8⇓ shows the effect of AVP after myocytes were treated with PMA and 4αPDD (a phorbol ester that is ineffective in the activation of PKC). After incubation by PMA (100 nmol/L) for 60 minutes, AVP failed to potentiate channel activity with AVP/control of 1.14±0.11 (n=4). This should be related to antecedent PKC activation by PMA, because 4αPDD failed to suppress AVP-induced potentiation with AVP/control of 2.04±0.38 (n=5).

• Download figure
• Open in new tab
• Download powerpoint

Figure 8.

Effect of phorbol esters on arginine vasopressin (AVP)–induced potentiation. A, Temporal profiles of open probability (NPo, where N is the number of functional channels and Po is the probability of the channel opening) from myocytes incubated with phorbol 12-myristate 13-acetate (TPA [PMA], 100 nmol/L) for 60 minutes (test potential, +10 mV). Each bar indicates averaged NPo over five sweeps. The TPA and AVP application periods are indicated by the lines on the top. Changes in NPo are summarized on the right. AVP was ineffective in the continued presence of TPA. B, Temporal profiles of NPo from myocytes incubated with 4α-phorbol 12,13-didecanoate (4-α PDD, 100 nmol/L) for 60 minutes (test potential, +10 mV). Each bar indicates averaged NPo over five sweeps. The 4-α PDD and AVP application periods are indicated by the lines on the top. Changes in NPo are summarized on the right. AVP was effective in spite of the continued presence of 4-α PDD. Different symbols indicate different test potentials (○, 0 mV; ▵, +10 mV).

Mechanisms of AVP-Induced Potentiation

The abolition of AVP-induced potentiation by a specific V₁ blocker, OPC-21268 (Fig 3⇑), indicates that the cardiac effect of AVP takes place through V₁ receptor stimulation. This is in agreement with previous observations using a different type of V₁ blocker, d(CH₂)₅Tyr(Me)AVP.^{1 21} This agent has been reported to suppress various types of AVP-induced effects in the heart.^{2 4 6 27}

Although the V₂ receptor is coupled to increased adenylate cyclase activity, leading to the activation of PKA, the V₁ receptor exerts its effect through phosphatidylinositol hydrolysis, leading to the mobilization of intracellular Ca²⁺ and the activation of PKC.^{6 20 21} Between two pathways evoked by V₁ stimulation, changes in [Ca²⁺]_i might be related to the stimulatory effects on Ca²⁺ channels. This is because AVP is known to increase [Ca²⁺]_i in several tissues,^{6 20 23 24} and moderately increased [Ca²⁺]_i is reported to potentiate Ca²⁺ channels in smooth muscle²⁸ and cardiac myocytes.^{11 25} In our recent study using an almost identical experimental setting,¹¹ the activity of Ca²⁺ channels was potentiated when [Ca²⁺]_i exceeded approximately two times the resting level. AVP-induced potentiation, however, was observed without significant changes in [Ca²⁺]_i, as measured by fura 2 fluorescence (Fig 5⇑). Thus, cellular processes following the changes in [Ca²⁺]_i appear to be not responsible for the AVP-induced potentiation described in the present study. Intracellular alkalinization through the stimulation of Na⁺-H⁺ exchanger might be assumed to be another candidate responsible for the potentiation.²⁹ However, this effect was not expected, because our bath solution contained no Na⁺ for countertransport.

On the other hand, the suppressive effect of H7 (Fig 6⇑) indicated the involvement of channel phosphorylation cycles in AVP-dependent potentiation. However, contribution by PKA²⁶ was not likely, because V₂ blocker (Fig 4⇑) and PKA inhibitor H89 (Fig 7⇑) consistently failed to suppress the potentiation. On the other hand, a role for PKC is supported by the observation that AVP failed to potentiate channel activity in the continued presence of PMA, a PKC activator (Fig 8⇑). Thus, our results are consistent with the view that PKC activation is at least partially responsible for the AVP-induced potentiation.

Contribution by Intracellular Environment?

When compared with PKA-induced stimulation, PKC modulation of L-type Ca²⁺ channels has been only sporadically reported in cardiac preparations^{30 31 32} and therefore less well characterized. This might be partly explained if PKC modulation of Ca²⁺ channels is much more sensitive to the intracellular metabolic environment than is PKA modulation. In preliminary experiments, we investigated the effect of AVP by using whole-cell recordings. In contrast to cell-attached recordings, upregulation by AVP in this configuration was not constantly observed when either Ba²⁺ or Ca²⁺ was used as the charge carrier. This was probably because the intracellular milieu was largely replaced by the pipette solution. The dependence on the intracellular environment appears to be more prominent in the cases of L-type Ca²⁺ channels than other types of channels. For example, Walsh and Kass³³ observed that PKC activation by a phorbol ester modulated delayed rectifier K⁺ channels but not Ca²⁺ channels in whole-cell experiments.

The importance of the intracellular environment in maintaining Ca²⁺ channel activity is well recognized.²⁶ For example, single-channel activities always subside after patch excision. Whole-cell current amplitudes decline during the course of experiments. Although mechanisms for these observations were not yet fully clarified, it is generally ascribed to the loss of some important intracellular constituents for the modulation and functioning of Ca²⁺ channels. Therefore, it is reasonable that the response to external stimuli is diminished or even eliminated after the intracellular environment is replaced by artificial solutions, as encountered in whole-cell experiments. Examples include the response to thrombin,³⁴ the effect of PMA in rat ventricular myocytes,³² and the reduced sensitivity to isoprenaline.³⁵

Kinetic Behavior of Single-Channel Current During AVP Application

Besides increasing the number of channel openings (or shortened closed times), AVP moderately prolonged open time durations in the present study. Under the simplified assumption that open time distribution in the control state consisted of a single-exponential component, the effect of AVP could be interpreted as the induction or potentiation of another open state with a slightly prolonged time constant. This effect is qualitatively similar to the observations of Bonev and Isenberg⁸ regarding smooth muscle cells, where AVP induced another open state with long openings through unidentified mechanisms. In this case, however, the time constant of the new component amounted to 10 times the control value, similar to that observed in the presence of Bay K 8644. These results led them to conclude that AVP induced “mode 2” gatings in their preparations. On the other hand, the time constant of the new component in cardiac myocytes was not much different from the control value.

Since its introduction during the analysis of dihydropyridine effects,³⁶ the “mode” concept has been successfully applied to explain the complex behaviors of Ca²⁺ channel gating, including those during β-adrenergic stimulation¹⁸ and high-voltage–dependent stimulation.³⁷ In theory, it might be possible to assign the component induced by AVP as a new gating mode. This is because minimal requirements for the modal concept are well-defined sets of gating patterns, within which transitions are fast relative to conversions among sets. In practice, however, it appeared hopeless to analyze our data on the basis of modal theory. For example, without large differences in time constants for open time distri- butions, we could not follow different gating patterns on a sweep-to-sweep basis.

The kinetic behavior of cardiac L-type Ca²⁺ channels is highly complicated. There are observations that could not be well accommodated either in conventional multistate sequential schemes or even in mode concepts.^{37 38} Further studies, with special focus on biophysical aspects of channel behavior, will be required for a full understanding of gating processes.

Pathophysiological Implications

Effects of AVP on cardiovascular regulatory systems are multiple, ranging from vasoconstriction to increased sensitivity to vasoreflex or interaction with other humoral factors.¹ Therefore, although infusion of AVP is known to decrease cardiac performance in vivo, assessments on the “direct” cardiac effect of AVP might be variable, depending on experimental conditions.^{2 3 4} In the present study, cell-attached recordings from isolated myocytes in the absence of extracellular Ca²⁺ were chosen to make the experimental system as simple as possible. Our results indicate that AVP potentially is a positive inotropic agent when extramyocardial compensatory mechanisms are excluded.

It should be noted that the dose of AVP used in the present study (100 nmol/L or 40 μU/mL) was at a level obtainable during certain pathophysiological conditions described with elevated plasma vasopressin levels.^{1 39 40} Potentiation of cardiac L-type Ca²⁺ channels is a part of the multifaceted cardiovascular regulatory systems of AVP and should be taken into consideration when we assess various types of circulatory disturbances.