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Phenylisopropyladenosine (PIA) inhibits catecholamine-stimulated adenylate cyclase activity in adult rat ventricular myocyte membranes
j Mol Cell Cardiol 19 (Supplement IV) (1987)
121
THE BEE VENOM APAMIN, DECREASES Isi AND INCREASES K + CURRENT IN SINGLE HEART CELL. G. Bkaily, M. Benabderrazik, Y. Yamamoto, D. Jacques, A. Sculptoreanu, N. Sperelakis. Department of Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Qu~bec, Canada, JIH 5N4 and Department of Physiology and Biophysics, University of Cincinnati, Cincinnati, Ohio, U.S.A. Apamin, a2bee venom polypeptide, was recently reported to block the naturallyoccurring Ca slow action potentials (APs) at low concentrations in cultured cell reaggregates from chick hearts (3). Quinidine reversed the apamin blockade of the slow APs. Using the whole-cell voltage clamp technique in single ventricular cells from chick embryo, apamin wa~ found to decrease the slow inward current (Ca 2+, Ba 2+) and to increase an outward K current. Washout of apamin with Tyrode solution did not reverse. -8the blockade of Ica and IK', however, I almost completely recovered. Quinidlne (iO M) slightly reversed the decreased IBa, greatly reversed the blockade of I Ca Ba and fully reversed the increased I~ produced by apamin. The present data confirm our previous results on the blockade or the slow APs by apamin, and show that apamin increases a K + current in heart cells and quinidine antagonizes this effect of apamin on I K. Therefore, apamin has other potent effects on heart cells in addition to the reported block of the TEA-insensitive Ca2+-activated K + conductance channels in skeletal muscle. Supported by grants from MRCC (MT-9816, ME-9788) and QHF to G. Bkaily who is a scholar of the CHF. Also supported by HL-31942 to N. Sperelakis.
122 125IODOPINDOLOL BINDING IN POSTNATAL PRIMARY MYOCARDIAL CELL CULTURES, MYOCYTE SUSPENSIONS, AND WHOLE HEART MEMBRANE PREPARATIONS. Allison A. Welder, Tina Machu, Richard E. Wilcox, *June Bradlaw, and Daniel Acosta. Deptartment of Pharmacology, College of Pharmacy, The University of Texas, Austin, Texas 78712 and *Food and Drug Administration, Washington, D.C. 20204. Primary myocardial cell cultures and myocardial cell suspensions represent two isolated, denervated, intact cell models for investigating cellular and subcelluar physiological and biochemical properties of the postnatal myocardium. Studies were performed to characterize beta-receptor number and affinity in primary myocardial cell cultures, freshly isolated myocytes in suspension, and whole heart crude membrane preparations obtained from 3-5 day old Sprague-Dawley rats. All three myocardial preparations had saturable beta-adrener~ic binding sites as measured with ten concentrations of the radioligand, 125I-iodopindolol (r25I-IPIN), ranging from 5.6 to 454 pM. The suspensions had a significantly (p < 0.05) lower Bma x (42 5:6 fmol/mg protein) than the membranes and cultures (77 + 8 and 95 5:10 fmol/mg protein, respectively). The K D of the cultures (218 5:2 pM) was significantly (p < 0.05) higher than that for the suspensions (107 5: I pM) and membranes ( 93 5:7 pM). Scatchard plots were linear for all three myocardial preparations, indicating a single class of binding sites. These data suggest the suspensions are injured in the isolation procedure and the cultures have a lower binding affinity when compared to the membranes and suspensions. (Supported by FDA contract #223-86-2109).
123
PHENYLISOPROPYLADENOSINE (PIA) INHIBITS CATECHOLAMINE-STIMULATED ADENYLATE CYCLASE ACTIVITY IN ADULT RAT VENTRICULAR MYOCYTE MEMBRANES. S.G. Macdonald, F.D. Romano, J.G. Dobson, Jr. Dept. of Physiology, Univ. of Mass. Mad. Sch. Worcester, MA 01605. Adenosine (ADO) is thought to attenuate catecholamine mediated increases in contractility and glyeogenolysis by inhibiting the stimulation of adenylate cyelase (AC) via an R i receptor. The aim of this study was to examine the effects of PIA, an ADO analog, on AC activity in membranes prepared from primary cultured adult rat ventricular myocytes to ascertain whether the antl-adrenergic actions of ADO are independent of other cell types present in the heart. After three hours of incubation rod-shaped cells were harvested and membranes prepared at 4Oc in HEPEB buffer. AC was assayed using a deoxyATP system to prevent the formation of endogenous ADO. Isoproterenol (ISO, 1 nM to i00 ~M) stimulated AC activity from a basal value of 3.4 pmollminlmg to a maximum of i0.i pmollmlnlmg. In the presence of PIA (i ~M), ISO stimulated AC from a basal of 1.4 pmol/mlnlmg to a maximum of 4.2 pmol/min/mg. PIA attenuated the magnitude of ISO-stimulated AC activity by 57% without significantly affecting the ECs0 (310 nM and 230 nM for control and PIA treated respectively), The inhibition by PIA was attenuated by theophylline (i0 ~M) suggesting that the actions of PIA are mediated by an R i receptor. These data indicate that ADO attenuation of myocardial eatecholsmine-stimulated AC occurs in ventricular myocytes and is independent of other cell types present in the heart. (Supported by PH~ grants HL-22828 and HL-36964)
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121
THE BEE VENOM APAMIN, DECREASES Isi AND INCREASES K + CURRENT IN SINGLE HEART CELL. G. Bkaily, M. Benabderrazik, Y. Yamamoto, D. Jacques, A. Sculptoreanu, N. Sperelakis. Department of Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Qu~bec, Canada, JIH 5N4 and Department of Physiology and Biophysics, University of Cincinnati, Cincinnati, Ohio, U.S.A. Apamin, a2bee venom polypeptide, was recently reported to block the naturallyoccurring Ca slow action potentials (APs) at low concentrations in cultured cell reaggregates from chick hearts (3). Quinidine reversed the apamin blockade of the slow APs. Using the whole-cell voltage clamp technique in single ventricular cells from chick embryo, apamin wa~ found to decrease the slow inward current (Ca 2+, Ba 2+) and to increase an outward K current. Washout of apamin with Tyrode solution did not reverse. -8the blockade of Ica and IK', however, I almost completely recovered. Quinidlne (iO M) slightly reversed the decreased IBa, greatly reversed the blockade of I Ca Ba and fully reversed the increased I~ produced by apamin. The present data confirm our previous results on the blockade or the slow APs by apamin, and show that apamin increases a K + current in heart cells and quinidine antagonizes this effect of apamin on I K. Therefore, apamin has other potent effects on heart cells in addition to the reported block of the TEA-insensitive Ca2+-activated K + conductance channels in skeletal muscle. Supported by grants from MRCC (MT-9816, ME-9788) and QHF to G. Bkaily who is a scholar of the CHF. Also supported by HL-31942 to N. Sperelakis.
122 125IODOPINDOLOL BINDING IN POSTNATAL PRIMARY MYOCARDIAL CELL CULTURES, MYOCYTE SUSPENSIONS, AND WHOLE HEART MEMBRANE PREPARATIONS. Allison A. Welder, Tina Machu, Richard E. Wilcox, *June Bradlaw, and Daniel Acosta. Deptartment of Pharmacology, College of Pharmacy, The University of Texas, Austin, Texas 78712 and *Food and Drug Administration, Washington, D.C. 20204. Primary myocardial cell cultures and myocardial cell suspensions represent two isolated, denervated, intact cell models for investigating cellular and subcelluar physiological and biochemical properties of the postnatal myocardium. Studies were performed to characterize beta-receptor number and affinity in primary myocardial cell cultures, freshly isolated myocytes in suspension, and whole heart crude membrane preparations obtained from 3-5 day old Sprague-Dawley rats. All three myocardial preparations had saturable beta-adrener~ic binding sites as measured with ten concentrations of the radioligand, 125I-iodopindolol (r25I-IPIN), ranging from 5.6 to 454 pM. The suspensions had a significantly (p < 0.05) lower Bma x (42 5:6 fmol/mg protein) than the membranes and cultures (77 + 8 and 95 5:10 fmol/mg protein, respectively). The K D of the cultures (218 5:2 pM) was significantly (p < 0.05) higher than that for the suspensions (107 5: I pM) and membranes ( 93 5:7 pM). Scatchard plots were linear for all three myocardial preparations, indicating a single class of binding sites. These data suggest the suspensions are injured in the isolation procedure and the cultures have a lower binding affinity when compared to the membranes and suspensions. (Supported by FDA contract #223-86-2109).
123
PHENYLISOPROPYLADENOSINE (PIA) INHIBITS CATECHOLAMINE-STIMULATED ADENYLATE CYCLASE ACTIVITY IN ADULT RAT VENTRICULAR MYOCYTE MEMBRANES. S.G. Macdonald, F.D. Romano, J.G. Dobson, Jr. Dept. of Physiology, Univ. of Mass. Mad. Sch. Worcester, MA 01605. Adenosine (ADO) is thought to attenuate catecholamine mediated increases in contractility and glyeogenolysis by inhibiting the stimulation of adenylate cyelase (AC) via an R i receptor. The aim of this study was to examine the effects of PIA, an ADO analog, on AC activity in membranes prepared from primary cultured adult rat ventricular myocytes to ascertain whether the antl-adrenergic actions of ADO are independent of other cell types present in the heart. After three hours of incubation rod-shaped cells were harvested and membranes prepared at 4Oc in HEPEB buffer. AC was assayed using a deoxyATP system to prevent the formation of endogenous ADO. Isoproterenol (ISO, 1 nM to i00 ~M) stimulated AC activity from a basal value of 3.4 pmollminlmg to a maximum of i0.i pmollmlnlmg. In the presence of PIA (i ~M), ISO stimulated AC from a basal of 1.4 pmol/mlnlmg to a maximum of 4.2 pmol/min/mg. PIA attenuated the magnitude of ISO-stimulated AC activity by 57% without significantly affecting the ECs0 (310 nM and 230 nM for control and PIA treated respectively), The inhibition by PIA was attenuated by theophylline (i0 ~M) suggesting that the actions of PIA are mediated by an R i receptor. These data indicate that ADO attenuation of myocardial eatecholsmine-stimulated AC occurs in ventricular myocytes and is independent of other cell types present in the heart. (Supported by PH~ grants HL-22828 and HL-36964)
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