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Am J Physiol Cell Physiol 273: C1666-C1672, 1997;
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Vol. 273, Issue 5, C1666-C1672, November 1997

Modulation of cardiac Ca2+ channels by isoproterenol studied in transgenic mice with altered SR Ca2+ content

Hidenori Sako1, Stuart A. Green2, Evangelia G. Kranias1, and Atsuko Yatani1

Departments of 1 Pharmacology and Cell Biophysics and 2 Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Phospholamban (PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR) Ca2+ uptake and attenuation of the cardiac contractile responses to beta -adrenergic agonists. In the present study, we compared the effects of isoproterenol (Iso) on the Ca2+ currents (ICa) of ventricular myocytes isolated from wild-type (WT) and PLB knockout (PLB-KO) mice. Current density and voltage dependence of ICa were similar between WT and PLB-KO cells. However, ICa recorded from PLB-KO myocytes had significantly faster decay kinetics. Iso increased ICa amplitude in both groups in a dose-dependent manner (50% effective concentration, 57.1 nM). Iso did not alter the rate of ICa inactivation in WT cells but significantly prolonged the rate of inactivation in PLB-KO cells. When Ba2+ was used as the charge carrier, Iso slowed the decay of the current in both WT and PLB-KO cells. Depletion of SR Ca2+ by ryanodine also slowed the rate of inactivation of ICa, and subsequent application of Iso further reduced the inactivation rate of both groups. These results suggest that enhanced Ca2+ release from the SR offsets the slowing effects of beta -adrenergic receptor stimulation on the rate of inactivation of ICa.

beta -adrenergic agonist; phospholamban; patch clamp; cardiac myocytes; mouse heart; sarcoplasmic reticulum

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IN CARDIAC MUSCLE, Ca2+ influx through the voltage-dependent L-type Ca2+ channel (CaCh) is closely related to the initiation, maintenance, and modulation of contractility by catecholamines. Sympathetic stimulation results in enhancement of both contraction and heart rate (8, 15), and modulation is clearly dependent on activation of beta -adrenergic receptors (beta -ARs). It is well known that beta -AR-mediated regulation occurs through the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) pathway. PKA activation results in the phosphorylation of several intracellular proteins, including CaCh (9, 15) and the regulatory protein of sarcoplasmic reticulum (SR) Ca2+-adenosinetriphosphatase (ATPase), phospholamban (PLB) in the SR membrane (11). Under basal conditions, dephosphorylated PLB is an inhibitor of SR Ca2+-ATPase. Phosphorylation of PB by PKA relieves this inhibition by increasing the affinity of the SR Ca2+-ATPase for Ca2+. These changes in SR Ca2+-ATPase during beta -AR stimulation correlate with the increased rate of rise and fall of contraction (7, 18). Indeed, recent studies have demonstrated that the PLB knockout (PLB-KO) mouse heart showed significantly increased basal contractile properties similar to those occurring with beta -AR stimulation (13). Moreover, ventricular myocytes isolated from the PLB-KO mouse heart exhibited a much smaller Ca2+ transient response to the beta -AR agonist isoproterenol (Iso) compared with wild-type (WT) controls (23).

The effects of beta -AR agonists on the intrinsic properties of CaCh currents have been extensively studied (15). Whole cell CaCh currents are markedly increased by Iso, and studies performed over the course of 20 years have strongly implicated that the beta -AR-mediated regulation of the channel occurs via PKA-mediated phosphorylation. Nevertheless, despite the extensive study of this pathway, the role of SR Ca2+ release in modulating CaCh activity during beta -AR stimulation in intact myocytes still remains unclear, in large degree because of the difficulty in separating the effects of SR Ca2+ release from those that are the result of direct agonist-promoted phosphorylation of CaCh. Thus, in agreement with single-channel measurements (16, 21), it has been reported that stimulation of beta -AR markedly slows down the inactivation kinetics of whole cell Ba2+ currents (IBa) during depolarization (3, 20). On the other hand, when the physiologically permeant ion Ca2+ was used as the charge carrier, beta -AR agonists did not slow the decay of Ca2+ current (ICa) during depolarization (15).

Regarding the latter, inactivation of cardiac CaCh has been postulated to be regulated by two mechanisms: one depends on membrane potential (voltage-dependent inactivation), and the other depends on Ca2+ entry (Ca2+-dependent inactivation). When Ba2+ is used as the permeant ion, the role of Ca2+-dependent inactivation is greatly minimized (12). It is therefore possible that the lack of agreement regarding the effects of Iso on the inactivation kinetics between ICa and IBa could be related to the Ca2+-dependent inactivation process of the channel. In support of this hypothesis, we have recently demonstrated that CaCh inactivation of mouse ventricular myocytes is controlled by membrane potential as well as by Ca2+-dependent inactivation that is regulated locally by the Ca2+ released from the SR in response to Ca2+ entry through the CaCh (14). Consistent with this idea, myocytes from PLB-KO mouse hearts with an enhanced SR Ca2+ uptake and content (6, 13) exhibited a significant increase in the contribution of the Ca2+-dependent inactivation component without a change in current density or voltage dependence of the channel compared with WT myocytes (14).

In the present study, we sought to delineate the relative contribution of beta -AR agonist-promoted effects on CaCh modulation and SR Ca2+ release to overall CaCh activation and inactivation. The PLB-KO mouse model is ideal to isolate the effects of SR Ca2+ release on inactivation kinetics of ICa during beta -adrenergic stimulation because PLB ablation is associated with significant attenuation of the normal increase in Ca2+ release from the SR in response to Iso (13, 23). Our data show that although the sensitivity of ICa to Iso was similar between WT and PLB-KO ventricular myocytes, there was a profound difference in the effects on the inactivation kinetics of ICa. Iso produced a marked prolongation of the decay of ICa in PLB-KO but not in WT. In contrast, Iso slowed the inactivation kinetics of both WT and PLB-KO cells when Ba2+ was the charge carrier. Furthermore, when SR Ca2+ was depleted by ryanodine, Iso prolonged the decay of ICa in both WT and PLB-KO cells. These results indicate that, under physiological conditions, increases in both Ca2+ entry and SR Ca2+ release induced by beta -AR stimulation can work synergistically and offset the direct slowing effects of beta -agonists on ICa decay.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Generation of PLB-deficient mice. The PLB locus was disrupted in embryonic stem cells, and PLB-deficient mice were generated as described previously (13). Adult mice ~2-4 mo of age were used in the present study.

Preparation of myocytes. Single ventricular myocytes were isolated from the hearts of WT and PLB-KO mice with use of a modified version of a method described previously (14). Briefly, the heart was perfused with Ca2+-free Tyrode solution containing collagenase type I (Worthington; 0.5 mg/ml) and bovine serum albumin (1 mg/ml) for 30-40 min by the Langendorff method at 37°C. At the end of the perfusion period, the heart was removed, passed through 200-µm nylon mesh, and centrifuged for 3 min at 100 g. The cells were stored in low-Cl-, high-K+ medium at room temperature (20-21°C). All experiments were performed at room temperature.

Electrophysiology. We recorded whole cell currents using previously described patch-clamp techniques (14, 25). To measure CaCh currents, depolarizing pulses were applied every 10 s from a holding potential of -50 mV unless otherwise stated. Under these conditions, there was no evidence of low-threshold T-type CaCh currents. We measured the membrane capacitance of the cells using voltage ramps of 0.8 V/s from a holding potential of -50 mV. PLB ablation did not alter cell capacitances [113 ± 7 pF (n = 17) for WT and 129 ± 11 pF (n = 13) for PLB-KO cells]. The patch pipettes had a resistance of 2 MOmega or less. The experimental chamber (0.2 ml) was placed on a microscope stage, and external solution changes were made with the use of a modified Y tube technique (24). The external solution contained (in mM): 2 CaCl2 or 2 BaCl2, 1 MgCl2, 135 tetraethyl ammonium chloride, 5 4-aminopyridine, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3. The pipette solution was (in mM): 100 cesium aspartate, 20 CsCl, 1 MgCl2, 2 MgATP, 0.5 GTP, 5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 5 HEPES, pH 7.3. In some experiments, EGTA was replaced with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). These solutions provided isolation of CaCh currents from other membrane currents; Na+ and K+ channel currents were completely eliminated. The omission of Na+ from external and internal solutions also excludes Ca2+ flux through the Na+/Ca2+ exchanger (22).

In the initial series of experiments, we also dialyzed the myocytes with lower concentrations of Ca2+ chelating buffer using varying amounts of EGTA (0, 0.1, and 2 mM) to minimize interference with Ca2+ signaling from the SR. In such myocytes, ICa decay was indeed faster compared with myocytes dialyzed with 5 mM EGTA. However, under these conditions, ICa exhibited accelerated "run down" and in many cases, activation of cell contraction occurred in the initial periods of cell dialysis, thus rendering stable recordings difficult. We therefore performed experiments in the presence of 5 mM EGTA, because we have previously shown that Ca2+-dependent inactivation properties can be reliably measured under these experimental conditions (14).

Mean values ± SE are given in the text. Comparisons between conditions were evaluated with the use of Student's t-test, with significance imparted at the P < 0.05 level.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of Iso on CaCh currents. Potentiation of ICa in mouse myocytes was examined with various concentrations of Iso. Figure 1 shows typical examples of the effects of Iso (100 nM) on ICa obtained from WT (A) and PLB-KO (B) myocytes. The traces show ICa activated at different membrane potentials in the absence (Fig. 1A and B, a) and presence (Fig. 1A and B, b) of Iso. Peak ICa amplitude, normalized relative to cell capacitance (pA/pF) as a function of voltage (I-V relationships) before and after exposure to Iso, was also plotted (Fig. 1A and B, c). The mean current density and the I-V relationships were similar between the two groups. Perfusion of Iso increased the current amplitude at all test potentials measured and also shifted the mean I-V relationships toward more negative potentials. Application of Iso (100 nM) resulted in an increase in the amplitude of ICa elicited at +10 mV by 77 ± 12% (n = 17) in WT and 80 ± 12% (n = 13) in PLB-KO cells. The shift in the I-V relationship was -8.8 ± 1.6 and -5.7 ± 1.1 mV for WT and PLB-KO cells, respectively.


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  		Fig. 1.   Effects of isoproterenol (Iso, 100 nM) on whole cell Ca2+ current (ICa) recorded in wild-type (WT, A) and phospholamban knockout (PLB-KO, B) cells. Traces show currents recorded from a holding potential of -50 mV to indicated test potentials in absence (a) and presence (b) of Iso. Times to one-half decay (T1/2, at +10 mV) in control and presence of Iso were 24.3 and 23.5 ms in WT cells and 10.1 and 16.5 ms in PLB-KO cells. Voltage dependence of peak ICa in absence (open circles) and presence (filled circles) of Iso are plotted in c. ICa was measured and normalized to cell capacitance to give current densities (pA/pF). Data points are means ± SE of WT (n = 17) and PLB-KO (n = 13) cells.

Inactivation of ICa was faster and more pronounced in PLB-KO cells (Fig. 1B, a) than in WT cells (Fig. 1A, a). In most experiments (such as that shown in Fig. 1A, b), the inactivation kinetics of ICa in WT myocytes were not significantly altered by Iso exposure. Table 1 lists inactivation kinetic data at which ICa reached maximum value (+10 mM) for WT and PLB-KO cells. In WT cells, the time to one-half decay (T1/2) was 19.7 ± 1.5 ms in the control and 19.2 ± 1.8 ms in the presence of Iso (n = 16). In contrast, Iso markedly prolonged the decay of ICa in PLB-KO cells (Fig. 1B, b), and the T1/2 increased from 9.2 ± 0.5 to 16.3 ± 2.1 ms (n = 15).

                              
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  		Table 1.   Kinetics of ICa inactivation in WT and PLB-KO mouse myocytes before and after application of 100 nM isoproterenol

Analysis of cumulative concentration-response effects of Iso on peak ICa revealed that both WT and PLB-KO cells showed a similar 50% effective concentration of 57.1 nM (Fig. 2B). For both cells, an increase in ICa was detectable with 10 nM Iso and reached a maximum at 1 µM (Fig. 2A). The maximum increases in ICa amplitude were also similar: 2.1 ± 0.3-fold (n = 12) and 2.2 ± 0.2-fold (n = 14) for WT and PLB-KO cells, respectively.


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  		Fig. 2.   Concentration-dependent effects of Iso on ICa in WT and PLB-KO cells. A: currents recorded from PLB-KO cell before (a) and after superfusion with 10 nM (b), 100 nM (c), and 1 µM (d) Iso are shown. Holding potential was -50 mV, and test pulse was +10 mV. B: concentration-response curve of Iso for ICa in WT and PLB-KO cells. In each cell, relative increase of current amplitude (+10 mV) at different Iso concentrations was obtained by normalizing to value produced by drug (1 µM). Solid line is a one-to-one binding model with a 50% effective concentration of 57.1 nM. Data are means ± SE of 6-14 cells. cont, Control.

Effects of Iso on CaCh current decay. To further examine the effects of Iso on CaCh currents, we analyzed the effects of Iso on the inactivation kinetics, using Ca2+ or Ba2+ as the charge carrier.

Decay of ICa in both WT and PLB-KO myocytes was fitted by the sum of the two (fast and slow) exponentials. Consistent with our previous findings (14), the fast time constant of inactivation (tau f) was significantly smaller in PLB-KO cells than in WT cells, whereas the slow time constant of inactivation (tau s) was comparable between the two groups (Fig. 3 and Table 1). The relative magnitude of fast inactivation tau f [A<SUB>&tgr;<SUB>f</SUB></SUB>/(A<SUB>&tgr;<SUB>f</SUB></SUB> + A<SUB>&tgr;<SUB>s</SUB></SUB>), where A is amplitude] was also considerably larger in PLB-KO cells compared with WT cells (Table 1).


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  		Fig. 3.   Influence of Iso (100 nM) on rate of inactivation of ICa during depolarizing voltage pulse in WT (A) and PLB-KO (B) cells. Traces show currents recorded before (a) and after (b) superfusion with Iso during depolarizing steps to +10 mV from holding potential of -50 mV. Current traces in control and after Iso are scaled and superimposed to compare inactivation time course in c. Currents were fitted to sum of 2 exponentials (solid lines in c). WT cell (A, c): control fast time constant of inactivation (tau f) = 13.2 ms, slow time constant of inactivation (tau s) = 67.3 ms, and relative proportion of tau f = 44.9%; Iso tau f = 11.1 ms, tau s = 56.8 ms, and relative proportion of tau f = 39.2%. PLB-KO cell (B, c): control tau f = 8.0 ms, tau s = 80.4 ms, and relative proportion of tau f = 73.4%; Iso tau f = 8.7 ms, tau s = 67.3 ms, and relative proportion of tau f = 52.1%.

In WT cells, although the values did not reach statistical significance, Iso showed a general trend of speeding up the inactivation time course (Fig. 3A). In contrast, in PLB-KO cells, Iso markedly slowed the inactivation kinetics of ICa (Fig. 3B). The results summarized in Table 1 suggest that Iso produced a significant decrease in the relative proportion of tau f from 72.9 to 51.8% without affecting the value of tau f or tau s. Note that the profound differences in inactivation kinetics of ICa between WT and PLB-KO cells were abolished during Iso stimulation.

In Ba2+ solution, the current decay was well fitted by a single exponential in both WT and PLB-KO cells (Fig. 4). No difference was observed in the time course of inactivation of IBa between WT and PLB-KO cells. Addition of Iso resulted in a significant increase in the peak current amplitude and slowed the decay of IBa inactivation to a similar extent in both WT and PLB-KO cells (Fig. 4). Iso slowed the time constant from 90.3 ± 3.9 to 108.3 ± 8.4 ms (n = 5, P < 0.05) in WT cells and from 98.7 ± 14.0 to 115.6 ± 18.4 ms in PLB-KO cells (n = 5, P < 0.05). These results suggest that Iso slows CaCh current decay in the absence of a Ca2+-dependent inactivation component in mouse ventricular myocytes.


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  		Fig. 4.   Influence of Iso (100 nM) on rate of inactivation of currents recorded in presence of Ba2+ in external solution during depolarizing voltage pulse in WT (A) and PLB-KO (B) cells. Traces show currents recorded before (a) and after (b) superfusion with Iso during depolarizing steps to 0 mV from holding potential of -50 mV. Current traces in control and after Iso are scaled and superimposed to compare inactivation time course in c. Currents were fitted to single exponential (solid lines in c). WT cell (A, c), tau  = 97.9 ms (control) and tau  = 113.1 ms (Iso). PLB-KO cell (B, c), tau  = 98.2 ms (control) and tau  = 107.1 ms (Iso).

Effects of Iso on ICa decay after depletion of SR Ca2+ by ryanodine or in the presence of BAPTA. To examine whether the difference in effects of Iso on the rate of inactivation of ICa between WT and PLB-KO cells was a result of the contribution of the Ca2+ released from the SR, the SR Ca2+ content was depleted by ryanodine and the effects of Iso were tested. As shown in Fig. 5, after the application of ryanodine (10 µM), the rate of ICa inactivation was significantly reduced in both WT and PLB-KO cells. The T1/2 of inactivation in the presence of ryanodine was not significantly different in WT cells and PLB-KO cells [35.7 ± 1.9 ms (n = 5) and 30.0 ± 1.6 ms (n = 5), respectively]. Subsequent addition of Iso (100 nM) enhanced ICa and produced additional slowing of inactivation in both cell types, with the inactivation T1/2 significantly increased to 51.9 ± 4.9 ms (n = 5) in WT and 49.0 ± 5.3 ms (n = 5) in PLB-KO cells. These results support the view that the differences between WT and PLB-KO myocytes in the effects of Iso on ICa inactivation are dependent on the SR Ca2+ content.


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  		Fig. 5.   Effects of Iso (100 nM) on inactivation of ICa after SR Ca2+ depletion by ryanodine (10 µM) recorded from WT (A) or PLB-KO (B) cells during depolarizing steps to +10 mV from holding potential of -50 mV. Current after application of ryanodine and subsequent addition of Iso (ryanodine + Iso) were scaled to same peak current amplitude recorded before (control) to compare wave form. T1/2 for control, ryanodine, and ryanodine + Iso were 18.1, 35.1, and 48.1 ms in WT cell and 9.3, 28.2, and 52.7 ms in PLB-KO cells, respectively.

In addition, to explore the effects of intracellular Ca2+ buffering on the inactivation kinetics of ICa, myocytes were dialyzed with the faster Ca2+ chelator BAPTA (10 mM) through patch pipettes. Inactivation rates in the presence of BAPTA were significantly slower compared with cells dialyzed with 5 mM EGTA (Table 1). The T1/2 was 44.2 ± 2.4 ms (n = 10) and 44.4 ± 3.4 ms (n = 6) in WT and PLB-KO myocytes, respectively. Subsequent addition of Iso (100 nM) enhanced ICa and produced additional slowing of inactivation in both groups, with an increase in T1/2 to 59.5 ± 6.4 ms (n = 10) in WT and 62.8 ± 6.7 ms (n = 6) in PLB-KO cells. When taken together with the EGTA data, it is clear that Iso indeed slows down the inactivation kinetics of ICa as the buffer capacity and the speed of Ca2+ buffering is increased in the experimental system. Furthermore, the difference in inactivation rate between WT and PLB-KO cells is reduced under these conditions. However, it should be noted that with a large concentration of intracellular Ca2+ chelators, the ability of the SR to reaccumulate and release Ca2+ may be compromised (1, 19).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The modulation of CaCh activity by beta -AR agonists has been extensively studied in many cardiac cell types, but little is known about the effects of beta -AR stimulation on this channel in adult mouse ventricular myocytes despite the potential importance of the transgenic mouse approaches in the functional regulation of beta -AR (4, 17). Earlier studies (13) showed that ventricular myocytes isolated from the PLB-KO mouse exhibited a markedly enhanced basal contractility compared with WT cells. Furthermore, the response of transient Ca2+ to Iso stimulation was significantly reduced in PLB-KO cells (23). In cardiac myocytes, the positive inotropic action of beta -AR is caused by increased Ca2+ influx through the CaCh and by enhanced SR Ca2+ uptake (5). However, it has remained unclear whether the difference in the response to Iso between WT and PLB-KO cells was caused at the SR level or if other mechanisms, perhaps acting at the level of the CaCh, were involved.

In the present study, we found that potentiation of peak amplitude of ICa by Iso in PLB-KO cells was quantitatively similar to WT cells. However, under control conditions, PLB-KO cells showed a faster ICa inactivation rate compared with WT cells and Iso prolonged the rate of inactivation. In contrast, Iso did not significantly alter the rate of inactivation in WT cells. When Ca2+ was replaced by Ba2+ as the charge carrier, or, in the presence of ryanodine (which depletes SR Ca2+), Iso significantly slowed the decay of the currents in both WT and PLB-KO cells. These results suggest that Ca2+ released from the SR is indeed responsible for the differential effects of Iso on the inactivation kinetics of ICa between WT and PLB-KO cells.

Localized Ca2+ signaling between CaCh and SR Ca2+ has been proposed in cardiac myocytes (18). We have recently shown that, in mouse myocytes, inactivation kinetics of ICa exhibit two (fast and slow) components, similar to other mammalian species. Inactivation of ICa was controlled by the amount of Ca2+ released from the SR, because the decay of ICa was significantly slowed in the presence of ryanodine or when Ca2+ was replaced by Ba2+ (14). Consistent with this, PLB-KO cells with increased SR Ca2+ content had a significantly faster inactivation rate compared with WT cells (14). These results supported the hypothesis that the SR Ca2+ load is the major source of the regulator Ca2+ and that elevation of Ca2+ near the CaCh strongly modulates the inactivation kinetics of CaCh (1, 18, 19).

The involvement of Ca2+-dependent inactivation during beta -AR stimulation has long been proposed (15). For example, Iso increases whole cell current amplitude and markedly slows the decay of the current when Ba2+ is used as the charge carrier. This is in agreement with the single-channel measurements, in which Iso increases average Ba2+-carried current amplitude and slows down the time course of decay. This slowing has been suggested to reflect a change in the slow-gating kinetics (an increase in the proportion of nonblank sweep) caused by Iso (16). However, Iso does not slow the time course of ICa; if anything, it rather accelerates inactivation (15). This lack of agreement on the changes in inactivation kinetics has been suggested to result in part from the offsetting effects of Ca2+-dependent inactivation and Iso-promoted increases in ICa amplitude.

In this respect, we also found that, in WT mouse ventricular myocytes, Iso does not significantly alter the inactivation kinetics of ICa, but, in the absence of SR Ca2+ release, Iso significantly slowed the decay of the currents in both WT and PLB-KO cells. Because current densities and Iso-promoted current amplitudes between WT and PLB-KO cells were comparable, these results suggest that the SR Ca2+ load is the major factor in the regulation of ICa inactivation rate. In WT cells, Iso enhances Ca2+-dependent inactivation by increasing both ICa and SR Ca2+ load. Thus the slowing effect of Iso was offset by enhanced Ca2+-dependent inactivation. In contrast, in PLB-KO cells in which the SR Ca2+ content is already enhanced under basal conditions, Iso had little effect on the amount of SR Ca2+ release (23) and subsequent Ca2+-dependent inactivation. Consistent with this, recent studies (2, 10) have shown that the fraction of SR Ca2+ release activated by ICa is highly dependent on the SR Ca2+ load. Although our experimental conditions do not permit quantitative measurement, our results provide strong support for the idea that the enhanced Ca2+ content in the SR plays a significant role in the modulation of the time course of ICa during beta -AR stimulation. In the physiological context, this negative feedback of CaCh activity may provide a potentially important mechanism for regulation of Ca2+ overload, because catecholamines induce increases in both Ca2+ influx and SR Ca2+ load, leading to subsequent increased SR Ca2+ release during depolarization.

    ACKNOWLEDGEMENTS

This work was supported by a National Science Foundation Career Advancement Award (A. Yatani), National Institute of General Medical Sciences Grant GM-54169 (A. Yatani), and National Heart, Lung, and Blood Institute Grants HL-26507, HL-52318, and HL-22619 (E. G. Kranias).

    FOOTNOTES

Address for reprint requests: A. Yatani, Dept. of Pharmacology and Cell Biophysics, Univ. of Cincinnati College of Medicine, Cincinnati, OH 45267-0575.

Received 19 May 1997; accepted in final form 22 July 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1666-C1672
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