© 1996 American Heart Association, Inc.
Articles |
1-Adrenergic Activation Inhibits ß-Adrenergic–Stimulated Unitary Ca2+ Currents in Cardiac Ventricular Myocytes
the Cardiology Division, Department of Medicine, Veterans Administration Medical Center and State University of New York, Health Science Center, Brooklyn.
Correspondence to Dr Mohamed Boutjdir, Cardiology Division (IIIA), VA Medical Center, 800 Poly Place, Brooklyn, NY 11209. E-mail boutjdir.mohamed@brooklyn.va.gov.
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Abstract |
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We have previously shown that whole-cell L-type Ca2+ current that was stimulated through ß-adrenergic receptors was negatively modulated by
1-adrenergic activation. In the present study, we investigated the kinetic basis of this modulation at the single-channel level in adult rat ventricular myocytes using Ba2+ as the charge carrier. Unitary current sweeps were evoked by 300-ms depolarizing pulses to 0 mV, from a holding potential of -50 mV at 0.5 Hz. During control conditions, the ensemble-averaged current amplitude was 0.18±0.01 pA (n=7). To achieve ß-adrenergic stimulation (ß effect), cells were superfused with norepinephrine (10 µmol/L) in the presence of prazosin (10 µmol/L), an 1-adrenergic blocker. ß-Adrenergic stimulation enhanced ensemble-averaged current (from 0.18±0.01 to 0.75±0.04 pA, P<.05, n=7), increased the open-time constants, and decreased the closed-time constants. To activate 1-receptors while maintaining the ß-adrenergic stimulation, cells were superfused with norepinephrine alone (1+ß effects). 1-Adrenergic activation reduced ensemble-averaged current (from 0.75±0.04 to 0.41±0.03 pA, P<.05, n=7), decreased open-time constants, and increased closed-time constants. 1-Adrenergic activation also inhibited ensemble-averaged currents stimulated by a low concentration (10 µmol/L) of 8-bromo-cAMP but not by (-)Bay K 8644 (1 µmol/L). Calphostin C (1 µmol/L), a specific inhibitor of protein kinase C, attenuated 1-adrenergic inhibition on ß-adrenergic–stimulated unitary currents. We conclude that 1-adrenergic activation exerts an inhibitory effect on ß-adrenergic–stimulated unitary Ba2+ current at the single-channel level. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the Ca2+-channel activity and the reduction in whole-cell Ca2+ current previously reported. This inhibition is in part mediated through the protein kinase C pathway.
Key Words: receptor • norepinephrine • phosphorylation • prazosin • protein kinase C
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Introduction |
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Calcium channels play a vital role in several functions of the cardiovascular system. They carry a significant amount of depolarizing current necessary for the propagation of cardiac action potentials, play a major role in the excitation-contraction coupling, and are sensitive to modulation by neurotransmitters.1 Sympathoadrenergic regulation of cardiac Ca2+ channels by NE is known to involve both ß- and
-adrenergic receptors.1 In this regard, we have previously reported that 1-adrenergic activation exerted an inhibitory effect on whole-cell L-type Ca2+ current, which had been stimulated through ß-adrenergic receptors in adult rat ventricular myocytes.2 To characterize the kinetic basis of this inhibition in the same animal model, we investigated the interaction of 1- and ß-adrenergic receptors on unitary Ba2+ currents at the single-channel level. |
Materials and Methods |
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Isolation of Cardiac Myocytes
Cardiac myocytes were obtained from hearts of Wistar rats (200 to 250 g) by enzymatic dissociation according to the method of Wittenberg et al,3 with some modifications.4 5 The heart was perfused at 37°C with a HEPES-buffered solution containing (mmol/L) NaCl 117, KCl 5.7, NaHCO3 4.4, NaH2PO4 1.5, MgCl2 1.7, HEPES 20, glucose 11, creatine 10, and taurine 20, along with 21 mU/mL insulin, and gassed with 100% O2. The pH was adjusted to 7.4 with NaOH. After 5 minutes of wash to eliminate the remaining blood in the heart, the heart was then perfused with fresh buffer mixed with 1.5 mg/mL collagenase type A or B (Boehringer Mannheim Corp) and 20 µmol/L Ca2+ for 40 to 50 minutes. First, both atria were removed, and then the ventricles were cut off and gently dissociated with forceps in the same solution without collagenase. Cells were suspended in Petri dishes containing HEPES buffer with 1 mmol/L CaCl2 and 0.5% BSA (pH 7.4). After incubation for 30 minutes, a small aliquot of the medium containing single cells was transferred to a 1-mL chamber mounted on the stage of an inverted microscope (TMS, Nikon Inc). Rod-shaped noncontracting cells with clear striations were used for the cell-attached single-channel recordings. In the present and prior studies,2 rat ventricular myocytes were chosen because (1) there is a high density of
1-adrenergic receptors (
8x104 1-adrenergic receptors per myocyte6 ) compared with other species, (2) there is an absence of T-type ICa,7 8 9 and (3) there is no evidence for an 2-receptor subtype based on radioligand binding with [3H]yohimbine.6
Solutions
The composition of the depolarizing solution to bathe the cells was (mmol/L) potassium glutamate 120, KCl 25, MgCl2 2, ATP 1, EGTA 2, and HEPES 10, pH 7.4 adjusted with KOH. The pipette solution contained (mmol/L) BaCl2 70, sucrose 110, and HEPES 10, pH 7.4 adjusted with tetraethylammonium hydroxide. The use of isotonic K+ in the bath served to approximate a zero resting potential, allowing estimation of transpatch potential.10 11 Potentials are given in absolute values. The high concentration of Ba2+ ions was used to maximize the signal-to-noise ratio and to eliminate K+ currents.12 The concentrations of NE and Pz were chosen on the basis of whole-cell data.2 Drug-containing solutions were superfused for at least 3 minutes to reach the steady state effect2 before measurements were made. Control experiments (n=4) in the absence of drugs showed no significant channel activity changes during time equivalent to drug exposure.
Recording Methods
Unitary currents were recorded from membrane patches in the cell-attached configuration13 with electrodes drawn from glass capillaries (Drummond Scientific Co) on a horizontal electrode puller (model P-87, Sutter Instrument Co). The fire-polished pipette electrodes had resistances of 2 to 10 M
and were connected to the input stage of a patch-clamp amplifier (List model EPC-7, Medical System Corp). Current signals were filtered at 1 kHz (eight-pole Bessel filter), digitized at 10 kHz, and stored in a computer (486/33 MHz). Pulse generation, data acquisition, and analysis of the signals were performed using pCLAMP software (v6.0.2, Axon Instruments Inc). Capacitive artifact was minimized by the use of a low level of solution in the pipette and by coating electrodes with insulating varnish (Sylgard, Dow Corning Corp). Seal resistance was between 10 and 50 G. Unitary currents were evoked by a 300-ms step depolarization to 0 mV from a holding potential of -50 mV every 0.5 Hz. For current-voltage relations, depolarizing pulses to -20 through 10 mV in 10-mV increments were applied at intervals of 2 s to determine the channel conductance and the apparent reversal potential. All experiments were performed at room temperature of 22°C to 24°C.
Data Analysis
Membrane currents were digitally recorded at a sampling frequency of 10 kHz and analyzed using pCLAMP software (v6.0.2, Axon Instruments Inc) and Origin (Microcal Origin v3.7, Microcal Software Inc). Analysis of unitary currents was performed after digital subtraction of capacitive and leakage currents by averaging records without channel openings and subtracting the average from each record in the series. Channel opening and closing transitions were detected as crossings of a threshold level set halfway between closed and open current levels. Measurements of open and closed times were made at 0 mV, and patches exhibiting more than one open-channel current level were not included in the analysis. This was based on the absence of multiple conductance levels in the sweep. The ensemble mean current was obtained by averaging all subtracted current records of the series. po was determined by integrating over each sweep, and the probability P was defined as the average po for all the sweeps from seven patches. Average results are presented as mean±SEM. Student's t test for paired data was used to compare control conditions with drug interventions. The linear regression method was used to obtain the extrapolated reversal potential from the current-voltage curve. A value of P<.05 was considered statistically significant.
Drugs
The source of all the chemicals used in the present study was Sigma Chemical Co, except for NE, which was from Winthrop Pharmaceuticals, Pz, which was kindly provided by Pfizer Laboratory Division, and calphostin C, which was from Calbiochem.
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Results |
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Sensitivity of Unitary Currents to Dihydropyridines
To establish that unitary currents recorded from adult rat ventricular myocytes are carried through L-type Ca2+ channels, we investigated their sensitivity to dihydropyridines. Fig 1
illustrates the effects of 2 µmol/L nisoldipine (an L-type Ca2+ channel antagonist) and 1 µmol/L (-)Bay K 8644 (an L-type Ca2+-channel agonist) on unitary Ba2+ currents. Currents were recorded in response to a 300-ms depolarization to 0 mV from a holding potential of -50 mV. Currents were blocked by nisoldipine added to the bath solution, stimulated by (-)Bay K 8644, and had an average unitary conductance of 27.7±0.7 pS (n=5). In addition, the membrane was held at -50 mV, and prior studies7 8 9 could not detect the presence of T-type Ca2+ current in adult rat ventricular myocytes. Therefore, we consider that these unitary currents are carried through L-type Ca2+ channels.
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Unitary Currents and Ensemble Current Analysis
Fig 2A shows 10 consecutive current tracings of the L-type Ca2+ channel unitary currents (with Ba2+ as a charge carrier) evoked by 300-ms depolarization to 0 mV from a holding potential of -50 mV. Current tracings recorded in control conditions showed 35% of channel activity corresponding to 71 sweeps with channel activity out of 200 depolarizing pulses. The tracings were composed of completely blank sweeps termed "mode 0" and a more conventional form of activity termed "mode 1."14 Tracings with channel activity had a unitary current amplitude of 1.53 pA. The macroscopic currents obtained by the ensemble average of single-channel recordings showed an averaged amplitude of 0.18±0.01 pA (n=7). Little or no inactivation occurred during the 300-ms voltage-step duration as expected with Ba2+ as a charge carrier (Fig 2A, bottom).
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To achieve a ß-adrenergic stimulation, we used the physiological neurotransmitter NE (10 µmol/L) in the presence of Pz (10 µmol/L), an 1-adrenergic blocker. Superfusion of cells with NE+Pz (ß effect) resulted in a pronounced increase in the channel activity. Of 200 current sweeps, we observed 131 active sweeps corresponding to a 66% overall increase in channel activity (Fig 2B
), indicating an increase in the proportion of nonblank sweeps. The ensemble-averaged currents were significantly enhanced, from 0.18±0.01 pA during the control condition to 0.75±0.04 pA after steady state application of NE+Pz (P<.05, n=7). Fig 2B, bottom, shows such an effect. Examination of individual sweeps showed more active sweeps with the pattern of long opening ("mode 2") and fewer silent sweeps. Fig 3A illustrates sweep-to-sweep variations in channel opening behavior as po, and Fig 3B shows the averaged po values of all sweeps from seven patches as the probability P. ß-Adrenergic stimulation significantly increased the number of active sweeps and the probability P from 0.11±0.01 to 0.54±0.03 (P<.05, n=7).
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To achieve 1-adrenergic activation while maintaining the ß-adrenergic stimulation, we superfused cells with NE alone (1 and ß effects). This activation reduced overall stimulated channel activity to 58% (115 active sweeps of 200 sweeps), resulting in the reduction in ensemble-averaged current from 0.75±0.04 to 0.41±0.03 pA (P<.05, n=7). Fig 2C illustrates such effects. Examination of Fig 3 shows that 1-adrenergic activation also reduced the number of active sweeps and reduced the probability P from 0.54±0.03 to 0.29±0.02 (P<.05, n=7).
Kinetics Analysis
Individual openings and closings of the channel were investigated in seven patches, and the means of all open and closed times were calculated for each patch containing a single channel. In the control conditions, both the open- and closed-time histograms were best fitted by two exponentials. The average 
of and os values were 0.56±0.04 and 6.46±0.98 ms, respectively. cf and cs values were 1.05±0.16 and 11.87±2.12 ms, respectively. ß-Adrenergic stimulation resulted in an increase in of and os to 0.82±0.05 and 10.44±1.93 ms (P<.05, n=7) and a decrease in cf and cs to 0.53±0.08 and 5.75±1.38 ms (P<.05, n=7), respectively. 1-Adrenergic activation caused a decrease in of and os to 0.75±0.07 and 8.44±1.57 ms (P<.05, n=7) and an increase in cf and cs to 0.78±0.06 and 8.57±1.27 ms (P<.05, n=7), respectively. It appears from these data that 1-adrenergic activation makes the channel spend less time open and more time closed compared with ß-adrenergic stimulation. Open- and closed-time histogram analysis shown in Fig 4 illustrates such effects.
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Current-Voltage Relations and Conductance
Channel conductance was measured from the slope of the relationship between single-channel current amplitude and membrane potential during step depolarizations between -20 and 10 mV. Unitary current amplitudes varied with applied potentials, the current-voltage relationship being linear with an average channel conductance of 27.7±0.7 pS (n=5). The extrapolated reversal potential was 56.5 mV. Neither ß- nor 1-adrenergic activation affected the channel conductance 26.8±0.6 pS (n=5) and 27.0±0.5 pS (n=5), respectively. Fig 5 shows unitary current tracings at different voltages (Fig 5A) and current-voltage relations (Fig 5C) during control, ß-adrenergic stimulation (NE+Pz), and 1-adrenergic activation (NE). Fig 5B shows control amplitude histograms fitted with gaussian curves at different voltages.
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, mean±SEM, from 15 traces for each potential), that of NE is 27.5 pS (
, mean±SEM, from 20 traces for each potential), and that of NE+Pz is 27.1 pS (
, mean±SEM, from 20 traces for each potential). Similar results were observed in four other patches. The conductances were yielded by the linear regression with a coefficient of .999. The extrapolated reversal potential was
1-Adrenergic Effect on 8-Bromo-cAMP– and Bay K 8644–Activated Unitary Currents
To bypass the ß-adrenergic adenylyl cyclase complex, we used the membrane-permeable 8-bromo-cAMP to stimulate unitary currents and then tested the effects of 1-adrenergic activation. When high concentrations15 of 8-bromo-cAMP (4, 1, and 0.200 mmol/L) were used, the expected increase in channel activity was obtained; however, 1-adrenergic activation failed to inhibit this activity. Fig 6 illustrates such effect. The ensemble-averaged currents and probability P were 0.55±0.03 pA and 0.36±0.02, respectively, during steady state application of 4 mmol/L 8-bromo-cAMP and 0.56±0.03 pA and 0.35±0.02, respectively, during 1-adrenergic stimulation achieved by cell superfusion with NE (10 µmol/L) plus esmolol (a ß-blocker, 10 µmol/L)2 (n=5, P=NS). Interestingly, when a low concentration16 of 8-bromo-cAMP (10 µmol/L) was used, 1-adrenergic activation inhibited unitary currents. Fig 7A shows 10 consecutive current tracings elicited by 300-ms depolarization to 0 mV from a holding potential of -50 mV in a cell that was exposed to 10 µmol/L 8-bromo-cAMP for 9 minutes. Fig 7B illustrates the effect of 1-adrenergic activation for 8 minutes. 1-Adrenergic activation significantly inhibited ensemble-averaged currents and probability P from 0.31±0.02 pA and 0.23±0.03, respectively, to 0.20±0.03 pA and 0.15±0.02, respectively (P<.05, n=7).
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On the other hand, when Ca2+ channels were directly stimulated by the dihydropyridine (-)Bay K 8644 (1 µmol/L), 1-adrenergic activation had no significant effect on channel activity (Fig 8). The ensemble-averaged currents and probability P were 0.53±0.03 pA and 0.32±0.03, respectively, during (-)Bay K 8644 and 0.56±0.03 pA and 0.35±0.02, respectively, during 1-adrenergic activation (P=NS, n=5).
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Possible Role of PKC in 1- and ß-Adrenergic Interaction With Ca2+ Channels
To test whether PKC plays a role in the 1-adrenergic inhibitory effect on ß-adrenergic–stimulated unitary currents, we performed experiments in the presence of the highly specific PKC inhibitor17 calphostin C. Since the IC50 for PKC inhibition by calphostin C is 50 nmol/L,17 we used 1 µmol/L to ensure maximum inhibition. Fig 9 shows unitary currents, ensemble-averaged currents, and po during ß-adrenergic stimulation (Fig 9A), ß-adrenergic stimulation plus calphostin C (Fig 9B), and ß-adrenergic stimulation plus calphostin C plus 1-adrenergic activation (Fig 9C). Calphostin C had no significant effect on ß-adrenergic–stimulated ensemble-averaged currents and probability P (from 0.51±0.03 pA and 0.32±0.02, respectively, to 0.53±0.02 pA and 0.33±0.03, respectively; P=NS, n=7). However, calphostin C significantly attenuated the 1-adrenergic effect, thus reducing ensemble-averaged currents and probability P from 0.53±0.02 pA and 0.33±0.03, respectively, to 0.40±0.03 pA and 0.24±0.02, respectively (P<.05, n=7). The 1-adrenergic inhibitory effect on ß-adrenergic–stimulated ensemble-averaged currents in the presence of calphostin C was 24.5% compared with 45% in its absence (Fig 2).
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Discussion |
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The results of the present study showed that
1-adrenergic activation inhibited unitary currents through Ca2+ channels that were previously enhanced via ß-adrenergic pathways but not by direct channel activation with (-)Bay K 8644. These effects were attenuated by the highly specific inhibitor of PKC, calphostin C.17 1-Adrenergic activation consistently reduced the probability of the phosphorylated Ca2+ channel being open during step depolarizations but had no effect on channel conductance. This reflects both a shortening of the mean time during which the channel remained open and a lengthening of the mean time during which the channel remained closed. These observations indicate that 1-adrenergic activation altered the kinetics of opening and closing to favor shut states and are also consistent with the whole-cell data reported earlier by our group.2
Type of Ca2+ Channel Studied
Two types of Ca2+ currents (L- and T-type) have been described in the heart.1 The unitary currents recorded in the present study were enhanced by the dihydropyridine agonist (-)Bay K 8644, blocked by the dihydropyridine antagonist nisoldipine, and had a channel conductance of 27 pS, consistent with L-type Ca2+ channel characteristics. Similar findings at the single-channel level using adult rat ventricular myocytes were reported by Scamps et al.9
Comparison of Unitary Current Properties With Others
The single-channel conductance of the L-type Ca2+ channel obtained under our experimental conditions (70 mmol/L Ba2+ pipette solution) is 27 pS, close to that reported (25 pS) by Kokubun et al18 and Reuter et al19 in rat ventricular myocytes with relatively comparable experimental settings. The unitary current amplitude recorded at 0 mV was 1.5 pA, similar with that reported by Kokubun et al18 (1.5 pA) and Scamps et al9 (1.2 pA) in rat ventricular myocytes. The behavior of L-type Ca2+ channels in cardiac tissue has been modeled with two closed states.12 The data presented here are also consistent with such models, since two exponential components could be fitted to the distributions of closed times. The distribution of open times was also best fit by two exponentials, consistent with the possibility of two open states. Tohse and Sperelakis20 21 have reported the occurrence of long opening under control conditions requiring two to three exponential fits for open times in embryonic chick heart cells. On the other hand, others12 14 19 have described only one exponential fit for open times, in the absence of drugs such as Bay K 8644, in adult ventricular myocytes. This discrepancy is likely due to the presence, under our experimental conditions, of more frequent long openings (18% of all sweeps accounted for a high po >0.5, and 7% accounted for a po >0.7) or mode-2 behavior described by Hess et al.14 The question now is what could have promoted mode-2 behavior?
Conditions that can promote mode-2 behavior have been reviewed and reported.1 These are dihydropyridine agonists, ß-adrenergic stimulation, pulsing to high positive potentials, and low frequency of stimulation. We did not use Bay K 8644 except for experiments shown in Figs 1 and 8. In addition, separate chambers, reservoirs, and tubing were used for Bay K 8644 and nisoldipine experiments. ß-Adrenergic stimulation was performed only after control recording was finished. The depolarization step used was 0 mV and was not preceded by high-amplitude prepulse. The stimulation frequency was 0.5 Hz. Therefore, none of the above conditions could account for this long opening mode, which could reflect the intrinsic behavior of Ca2+ channels in adult rat ventricular myocytes (similar long openings have been observed in adult rat ventricular cells by Dr D. Yue, unpublished data).
1-Adrenergic Inhibition of Stimulated Single Ca2+ Channels
Since we2 and others22 23 have established that 1-adrenergic activation has no substantial effect on basal ICa (ie, without prior ß-adrenergic stimulation) using the whole-cell patch-clamp technique, we did not examine the 1-adrenergic effects on basal unitary currents.
The results of the present study show that the fraction of silent traces of ß-adrenergic–stimulated Ca2+ channels was increased subsequent to 1-adrenergic activation. The appearance of these long shut periods is often attributed to shifting between modes of channel opening and closing.12 According to this analysis, part of the decrease in probability of the channel being open would be the result of 1-adrenergic activation, leading Ca2+ channels to spend more time in mode 0. It is apparent from the histograms that during 1-adrenergic activation there was a decrease in the proportion of short open times and an increase in the proportion of long closed times.
1-Adrenergic activation is known to result in activation of a phospholipase C that hydrolyzes phosphatidylinositol 4,5-diphosphate and generates inositol 1,4,5-tris-phosphate24 and diacylglycerol.25 In isolated cardiac myocytes, phosphoinositide metabolism studies showed that activation of 1-receptors causes increased turnover of phosphatidylinositol and increases in inositol phosphates.26 Inositol 1,4,5-tris-phosphate was reported to cause the release of an intracellular pool of Ca2+ in skeletal27 and cardiac28 myocytes, although this was not found in another study of cardiac myocytes.29 Diacylglycerol activates PKC, which can phosphorylate Ca2+ channels.25 Much of the data available about the diacylglycerol/PKC regulation of Ca2+ channels seems inconclusive; an increase,30 no effect,31 and an increase followed by a decrease32 33 have been reported.
The possibility exists that the above second messengers are in part responsible for the inhibitory effect described. This is further supported by the fact that NE applied in the solution bathing cardiac ventricular cells can initiate cytoplasmic messengers that gain access to the region of Ca2+ channels located beneath the cell-attached micropipette to decrease the probability that the channel will be open during step depolarizations. The cell-attached configuration isolates Ca2+ channels in the patch from any direct interaction with the channel. Therefore, it is unlikely that any direct receptor–G protein–Ca2+ channel interaction scheme would be involved in the observed inhibition but, rather, that it results from diffusible second-messenger effects. Our findings show that PKC reduced the 1 effect on ß-adrenergic–stimulated ensemble-averaged currents from 45% to 24%, indicating the involvement, at least in part, of the PKC pathway in the observed inhibitory effect. This is further supported by whole-cell data, where PKC inhibition also reduced the phorbol ester (phorbol 12-myristate 13-acetate) effect on isoproterenol-stimulated ICa (Reference 34 and Z.-H. Zhang, J.A. Johnson, N. El-Sherif, D. Mochly-Rosen, M. Boutjdir, unpublished data, 1996). However, the fact that PKC inhibition did not completely prevent the 1 effect suggests that other pathways cannot be totally ruled out.
Danziger et al35 studied the interactive 1- and ß-adrenergic actions of NE on the extent and velocity of shortening and contraction in rat cardiac myocytes. Interestingly, they found that 1-adrenergic effects were significant in inhibiting ß-adrenergic stimulation. They suggested that the actions of NE on the cardiac myocyte may be attributed to an intrinsic feedback mechanism. A possible mechanism could be that 1-adrenergic activation of cAMP-dependent phosphodiesterase6 leads to a lowered concentration of cAMP.6 36 The present 8-bromo-cAMP experiments (low dose) excluded this possibility, since 8-bromo-cAMP is a poor substrate for phosphodiesterases. Alternatively, it is possible that the effect could be, in part, mediated by a direct interaction between guanine nucleotide–binding G protein(s) and adenylyl cyclase,37 38 since 1-adrenergic activation reduced ensemble-averaged currents stimulated by low concentrations of 8-bromo-cAMP by 35% instead of 45% when the channel was stimulated through the ß-receptor–adenylyl cyclase complex.
When Ca2+ channels were directly enhanced by (-)Bay K 8644, 1-adrenergic activation had no effect on channel activity. This could imply that for the 1-adrenergic effect to be seen, the channel had to be enhanced through the protein kinase A-dependent phosphorylation process.
Comparison of Single-Channel Findings With Whole-Cell Data
In a preliminary report, we showed that 1-adrenergic activation decreased the whole-cell L-type ICa when it was enhanced by ß-adrenergic stimulation or by forskolin (an adenylyl cyclase activator) in rat ventricular myocytes.2 However, 1-adrenergic receptor activation did not inhibit ICa when this current was increased by an intracellular application of 25 to 200 µmol/L cAMP.2 This lack of inhibition could be attributed to the use of saturating concentrations16 of cAMP (>20 to 30 µmol/L). This is further supported by the present results, which showed that the 1 effect was also absent when high concentrations of 8-bromo-cAMP (>200 µmol/L) were used.
Nevertheless, the observed alterations in single-channel activity would be expected to lead to a reduction in whole-cell ICa and to further establish the existence of the negative regulatory feedback mechanism between 1- and ß-adrenergic receptors vis-a-vis Ca2+ channels. This feedback control is essential for understanding the complex regulation of Ca2+ channels not only in physiological settings but also in pathological settings, such as in dilated cardiomyopathic human hearts39 and in myocardial ischemia and reperfusion,40 where alterations in 1-adrenergic receptor density have been demonstrated.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by Veterans Administration Medical Research Funds. We would like to thank Drs D. Mochly-Rosen, M. Restivo, D. Qin, H. Zhang, and E. Caref for their invaluable scientific discussion and advice. We also thank the animal laboratory staff and N. Stergiopoulos for their assistance.
Received September 26, 1995; accepted April 17, 1996.
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