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Large conductance cardiac chloride channels can be observed only in membrane of neonatal rat myocytes
J Mol Cell Cardiol 24 (Supplement VI) (1992) 130
DIFFERENTIAL EFFECTS OF (-)BayK 8644 ON NON-PHOSPHORYLATED AND PHOSPHORYLATED CARDIAC Ca CURRENT. RBmy Hanf and Rodolphe Fischmeister. INSERM U-241, Bit. 443. Universitt! Paris&d, F-91405 Orsay, France. The effects of the dihvdroovridine (DHP) agonist (-)BavK 8644 (BavK) on the L-tvoe ,. Ca channel current (I& were studied in frog &t&lar my&y&. cyclic .iMG-dependent phosphorylation of Ca channels was obtained by inrracellular perfusion of 5 pM CAMP. At 0 mV membrane potential (V,), external application of BayK enhanced peak amplitude of basal I,, and phosphorylated Ic, (Ica P) with different efficacies but with equal potencies (E(&, = 4*2 nM and 6+2 nM, (Em,, = 166i: 17% and 5.5+3% [n=5], respectively) respectively). In both cases, the binding site for BayK appeared not to be modified by V, since ECso was unchanged at -30, 0 and +30 mV. On the other hand, I&, depended on V, since current-voltage and inactivation curves were shifted = 10 mV towards more negative potentials for both Ic, and ICa,P. Surprisingly, the changes induced by BayK on the kinetics of I,, inactivation were diametrically opposite on I,, and I,, p at all V,. For instance, at negative potentials, BayK accelerated inactivation of Ic, p while it slowed dbwn inactivation of I,,. Consequently, the changes in thk inactivation kinetics of I,, p col$nsated for the changes in peak amplitude. This was further confirmed hy measuring the total amount’of Ca’+ ions entering the cell through basal (Qc,) or phosphorylated Ca channels (Q ca,p) as the time intqral of Ic, and I,-, p, respectively. While BayK increased Qca in a strongly voltage-dependent manner, the effect of the DHP’ on Qca P was independent of V,,: 1 PM BayK increased QCa,P by 74+21%, 101+ 1 I % and 70+ 10% (n=S) at -30, 0 aid +30 mV, respectively. An attractive hypothesis to explain our data is that BayK interacts with the Ca?+-mediated inactivation process of Ca channels in a manner that is strongly altered by CAMP-dependent phosphorylation. (Supported by a grant from Bayer-Pharma)
131
LARGE CONDUCTANCE CARDIAC CHLORIDE CHANNELS CAN BE OBSERVED ONLY IN MEMBRANE OF NEONATAL RAT MYOCYTES. Alain Coulombe, Thierry Lefevre and Edouard Coraboeuf. Laboratoire de Physiologie Cellulaire, (URA CNRS 1121), Universite de Paris-Sud, 91405 Orsay Cedex, France. Large conductance ( = 400 pS) chloride (LC-type Cl] spontaneous channel activity have been observed in membranes of cultured ventricular cells of neonatal rat (Coulombe et al. 1987, Eur. Biophys. J. 14:155-162). Here the LC-type Cl channel was studied in freshly dissociated cardiac myocytes. In normal conditions, LC-type Cl channel activity was rarely seen either in cell-attached or inside-out configurations, whatever the age of rat. However it was observed if the patch was formed on membrane blebs. Application of a hypotonic medium (150 mOsm) to intact (without blebs) neonatal rat myocytes also initiated the channel activity, after long and variable delays (30 s to 3 min). Whereas activity was never observed in rats older than two weeks. The channe! could not be activated by stretch, cytochalasine D (3 ,uM), colchicine (50 PM) or trypsin (5 mg/ml). The fact that the channel exhibits activity when membranes are distended and/or detached from internal structures suggests that the disruption from, or the reorganization at the level of, the cytoskeleton is a triggering event in the channel activation.
132
ADAPTATION FUNCTIONAL Univcrsid
OF RAT HEART TO CHRONIC HYPOXIA : AN ELECTROPHYSI~LOGICAL AND STUDY. Paul KREHER. Christiane KERIEL, Pierre CUCHJZT. Physiologic Cell&ire Cardiaque,
Joseph Fourier, B.P.53X, F38041-Grenoble
Cedex.
Our aim was to obtain chronically hypoxic rats and to explore the electrophysiology and function of their hearts. Adaptation to normobaric hypoxia was obtained by an exposure of the rats to 10% 02 : a mixture of nitrogen, extracted from air, at&pressurized air is injected into a chamber at the rate of 12 Vmin : a pas outlet allows a normal uressure and a constant Fi02. Rats live there for 1.2 or 3 weeks. In 3 weeks .-a-~---~-the hematocrit increases frok 0.42 + 0.02 (mean + SD, n=8) to 0.65 rt 0.0.5 (n=8). Hearts are then perfused in norrnoxia (PO2 = 660 mmHg) in a Langendorff open system with a 10 ml/min constant flow (glucose 1 lmM, Ca++ 2.4 mM, 37°C). Left ventricular (LV) pressure is measured with a latex balloon : the LV of chronically hypoxic rats exhibits similar performances (LV developed pressure, dP/dt max) to
con trof LV. On the action potentials of hearts of hypoxic rats, we observe a lengthening
of all
rcnolarization ohases : this is evident on the right ventri& (RV) as soon as the 1st wk and is enhanced at -Gi 2 and 3 ; it’appears on the LV from the 2nz wk and at wk 3’the lengthening is as important as for the RV. At the end of the 50 min perfusion, hearts are separated into RV, septum and LV, and weighed. The RV increases from 0.064 + 0.012 g/lOOg body weight for controls to 0.103 + 0.011 after 3 wk of hypoxia, whereas there is no hypertrophy for the LV. Thus, action potential lengthening does not seem to bk-related to hypertrophy, but $&haps to a humoral or neural factor that remains to be determined. S.48
DIFFERENTIAL EFFECTS OF (-)BayK 8644 ON NON-PHOSPHORYLATED AND PHOSPHORYLATED CARDIAC Ca CURRENT. RBmy Hanf and Rodolphe Fischmeister. INSERM U-241, Bit. 443. Universitt! Paris&d, F-91405 Orsay, France. The effects of the dihvdroovridine (DHP) agonist (-)BavK 8644 (BavK) on the L-tvoe ,. Ca channel current (I& were studied in frog &t&lar my&y&. cyclic .iMG-dependent phosphorylation of Ca channels was obtained by inrracellular perfusion of 5 pM CAMP. At 0 mV membrane potential (V,), external application of BayK enhanced peak amplitude of basal I,, and phosphorylated Ic, (Ica P) with different efficacies but with equal potencies (E(&, = 4*2 nM and 6+2 nM, (Em,, = 166i: 17% and 5.5+3% [n=5], respectively) respectively). In both cases, the binding site for BayK appeared not to be modified by V, since ECso was unchanged at -30, 0 and +30 mV. On the other hand, I&, depended on V, since current-voltage and inactivation curves were shifted = 10 mV towards more negative potentials for both Ic, and ICa,P. Surprisingly, the changes induced by BayK on the kinetics of I,, inactivation were diametrically opposite on I,, and I,, p at all V,. For instance, at negative potentials, BayK accelerated inactivation of Ic, p while it slowed dbwn inactivation of I,,. Consequently, the changes in thk inactivation kinetics of I,, p col$nsated for the changes in peak amplitude. This was further confirmed hy measuring the total amount’of Ca’+ ions entering the cell through basal (Qc,) or phosphorylated Ca channels (Q ca,p) as the time intqral of Ic, and I,-, p, respectively. While BayK increased Qca in a strongly voltage-dependent manner, the effect of the DHP’ on Qca P was independent of V,,: 1 PM BayK increased QCa,P by 74+21%, 101+ 1 I % and 70+ 10% (n=S) at -30, 0 aid +30 mV, respectively. An attractive hypothesis to explain our data is that BayK interacts with the Ca?+-mediated inactivation process of Ca channels in a manner that is strongly altered by CAMP-dependent phosphorylation. (Supported by a grant from Bayer-Pharma)
131
LARGE CONDUCTANCE CARDIAC CHLORIDE CHANNELS CAN BE OBSERVED ONLY IN MEMBRANE OF NEONATAL RAT MYOCYTES. Alain Coulombe, Thierry Lefevre and Edouard Coraboeuf. Laboratoire de Physiologie Cellulaire, (URA CNRS 1121), Universite de Paris-Sud, 91405 Orsay Cedex, France. Large conductance ( = 400 pS) chloride (LC-type Cl] spontaneous channel activity have been observed in membranes of cultured ventricular cells of neonatal rat (Coulombe et al. 1987, Eur. Biophys. J. 14:155-162). Here the LC-type Cl channel was studied in freshly dissociated cardiac myocytes. In normal conditions, LC-type Cl channel activity was rarely seen either in cell-attached or inside-out configurations, whatever the age of rat. However it was observed if the patch was formed on membrane blebs. Application of a hypotonic medium (150 mOsm) to intact (without blebs) neonatal rat myocytes also initiated the channel activity, after long and variable delays (30 s to 3 min). Whereas activity was never observed in rats older than two weeks. The channe! could not be activated by stretch, cytochalasine D (3 ,uM), colchicine (50 PM) or trypsin (5 mg/ml). The fact that the channel exhibits activity when membranes are distended and/or detached from internal structures suggests that the disruption from, or the reorganization at the level of, the cytoskeleton is a triggering event in the channel activation.
132
ADAPTATION FUNCTIONAL Univcrsid
OF RAT HEART TO CHRONIC HYPOXIA : AN ELECTROPHYSI~LOGICAL AND STUDY. Paul KREHER. Christiane KERIEL, Pierre CUCHJZT. Physiologic Cell&ire Cardiaque,
Joseph Fourier, B.P.53X, F38041-Grenoble
Cedex.
Our aim was to obtain chronically hypoxic rats and to explore the electrophysiology and function of their hearts. Adaptation to normobaric hypoxia was obtained by an exposure of the rats to 10% 02 : a mixture of nitrogen, extracted from air, at&pressurized air is injected into a chamber at the rate of 12 Vmin : a pas outlet allows a normal uressure and a constant Fi02. Rats live there for 1.2 or 3 weeks. In 3 weeks .-a-~---~-the hematocrit increases frok 0.42 + 0.02 (mean + SD, n=8) to 0.65 rt 0.0.5 (n=8). Hearts are then perfused in norrnoxia (PO2 = 660 mmHg) in a Langendorff open system with a 10 ml/min constant flow (glucose 1 lmM, Ca++ 2.4 mM, 37°C). Left ventricular (LV) pressure is measured with a latex balloon : the LV of chronically hypoxic rats exhibits similar performances (LV developed pressure, dP/dt max) to
con trof LV. On the action potentials of hearts of hypoxic rats, we observe a lengthening
of all
rcnolarization ohases : this is evident on the right ventri& (RV) as soon as the 1st wk and is enhanced at -Gi 2 and 3 ; it’appears on the LV from the 2nz wk and at wk 3’the lengthening is as important as for the RV. At the end of the 50 min perfusion, hearts are separated into RV, septum and LV, and weighed. The RV increases from 0.064 + 0.012 g/lOOg body weight for controls to 0.103 + 0.011 after 3 wk of hypoxia, whereas there is no hypertrophy for the LV. Thus, action potential lengthening does not seem to bk-related to hypertrophy, but $&haps to a humoral or neural factor that remains to be determined. S.48