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Role of external [Ca2+] in the inotropic and metabolic effects of phorbol esters
J Mol
Cell
P152
ALTERED INTRACELLULAR Ca*+ SIGN-G CAUSES SKELETAL MUSCLE CONTRACTILE DYSFUNCTIONINCHRONICHEARTFAlLURE Cynthia L. Perreault, Hugo Gonzalez-Serratos, Sheldon E. Litwin, Xinhui Sun, Clara Franzini-Armstrong, James P. Morgan. Harvard Medical School, Boston, MA 02215; Biophysics, Univ. Maryland, School of Medicine, Baltimore, MD 21201; Anatomy, Univ. Pennsylvania, School of Medicine, Philadelphia, PA 19104. To determine if chronic heart failure (HF) leads to functional or structural alterations of skeletal muscle, we compared intracellular Ca’+ (Ca2+,) signalling, contractility and the rate of fatigue development together with electron microscopy (EM) in rats with myocardial infarction-induced HF versus controls (C). Bundles of 100-200 cells were dissected from the extensor digitorum longus muscle and were either loaded with aequorin or fixed for EM. Ca’+, transients during twitches and tetani and the rate of fatigue development during repetitive tetanic (75Hz) stimulation were compared. EM revealed a lack of cellular atrophy in skeletal muscles from HF rats: however, muscles from HF rats exhibited prolonged CZX*+~transients and twitches, depressed peak and maximal tension (PT) and peak Ca*+, during twitches and tetani and a faster rate of fatigue development. Twitches (0.33Hz) Tetani /75Hz) Time to Fatigue wcaz+li PICa2+1, 5oaPrc~P+ m m I!x.m% C
Cardiol
2.8-cO.7
24 (Supplement
0.7kO.l
III)
(1992)
10.722.4
1.s+o.3
66Ok168
36lk49
HF
1.4kO.2’ 0.4*0.1* 5.3*1.4* 0.9+0.2* 286-c61* 131%26* PT (g/mm?; PICaz’li (PM); Fatigue (sec)=time to 50% force reduction; [Ca2+],=2.5mM, *peO.O.5 vs. C. In HF rats CZI*+~ signalling and contractility is altered in skeletal muscle. HF may be a condition characterized by generalized striated muscle dysfunction.
P153
TBX BFFBCT OF CBBOBIC DIABBTBS ON EDCLXOTIDXTRIPI~OS~IBE (BTPase)ACTIVITTABD PASSlXiBCALCIDMBIItDIWIBABAT BDCISAX -PB m FBACTIOl? Bram Rarajiawan. James S.C. Gilchrist, John C. Docherty, Grant N. Pierce. Div. Cardiovasc. Sci.. St. Boniface Hospital Research Centre, Winnipeg, Canada RZH 2A6 Protein synthesis during diabetes is markedly depressed. The nuclear NTPase regulates peptide and mRNA transport across the nuclear membrane. Ca2+ interactions with the nuclei may also influence gene expression. Thus, we hypothesized that the alteration in rotein metabolism during diabetes may be caused 4+ by changes in NTPase activity and Ca binding by the nuclei. Nuclei were isolated from hepatic tissue after 8 weeks of diabetes. The nuclei appeared structurally intact, contained little cellular debris, stained strongly with toluidine blue and exhibited negligible marker enzyme activies for other membrane fractions. Nuclear NTPase activity was not different between the diabetic and control samples. The ATP and Mg2+ dependence of the NTPase activit was also %5Ca2+ similar between the two groups. We could not detect high affinity binding sites in the nuclear membrane fraction. Both nuclei and nuclear membrane Ca2+ binding was unaffected by the diabetic condition of the animals. Our results demonstrate that the alteration in protein metabolism in rats during chronic experimental diabetes does not involve a change in NTPase activity or the Ca2+ binding capacity of hepatic nuclei. Supported by MRC.
PI54
ROLE OF EXTERNAL [Ca*‘] IN THE INOTROPIC AND METABOLIC EFFECTS OF PHORBOL ESTERS Morris Karmazyn, James Haist. Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario N6A 5Cl Canada. Our study examined the effects of altering [Ca2+]. on functional and metabolic effects of phorbol 1Zmyristate 13-acetate (PMA, 10”M to 10e6M) and phorbol 12,13-dibutyrate (PDBu, 10“*M to IU’M) in Langendorff-perfused rat hearts. Under normal [Ca*+], (1.25 mM) 1o”‘M PMA produced a sustained elevation in contractile force whereas in the presence of elevated [Ca’+]. (2.5 mM) only a negative inotropic effect was observed. The positive inotropic effect was also reversed by increasing PMA concentrations such that a rapid (> 90%) loss in function was seen with 10m6M irrespective of [Ca*+],. The effect of PDBu was generally identical to that seen with PMA except that, in the presence of normal [Ca”+]., the positive inotropic effect was maintained with both of the two lowest concentrations (10.“M and 1Cr’OM) employed. However, a negative inotropic influence was seen when identical concentrations of PDBu were examined under conditions of elevated [Ca*+],. Only the highest concentration of either phorbol significantly depressed high energy phosphate contents, an effect not influenced by [Ca’+].. Furthermore, phorbol ester-induced elevations in resting tension as well as coronary constriction were unaffected by [Cat+] Our studies demonstrate complex concentration-dependent effects of phorbol esters and suggest that the myokHrdial contractile response to low concentrations of these compounds is strongly influenced by [Ca”+],. (Supported by MRC of Canada) S.56
Cell
P152
ALTERED INTRACELLULAR Ca*+ SIGN-G CAUSES SKELETAL MUSCLE CONTRACTILE DYSFUNCTIONINCHRONICHEARTFAlLURE Cynthia L. Perreault, Hugo Gonzalez-Serratos, Sheldon E. Litwin, Xinhui Sun, Clara Franzini-Armstrong, James P. Morgan. Harvard Medical School, Boston, MA 02215; Biophysics, Univ. Maryland, School of Medicine, Baltimore, MD 21201; Anatomy, Univ. Pennsylvania, School of Medicine, Philadelphia, PA 19104. To determine if chronic heart failure (HF) leads to functional or structural alterations of skeletal muscle, we compared intracellular Ca’+ (Ca2+,) signalling, contractility and the rate of fatigue development together with electron microscopy (EM) in rats with myocardial infarction-induced HF versus controls (C). Bundles of 100-200 cells were dissected from the extensor digitorum longus muscle and were either loaded with aequorin or fixed for EM. Ca’+, transients during twitches and tetani and the rate of fatigue development during repetitive tetanic (75Hz) stimulation were compared. EM revealed a lack of cellular atrophy in skeletal muscles from HF rats: however, muscles from HF rats exhibited prolonged CZX*+~transients and twitches, depressed peak and maximal tension (PT) and peak Ca*+, during twitches and tetani and a faster rate of fatigue development. Twitches (0.33Hz) Tetani /75Hz) Time to Fatigue wcaz+li PICa2+1, 5oaPrc~P+ m m I!x.m% C
Cardiol
2.8-cO.7
24 (Supplement
0.7kO.l
III)
(1992)
10.722.4
1.s+o.3
66Ok168
36lk49
HF
1.4kO.2’ 0.4*0.1* 5.3*1.4* 0.9+0.2* 286-c61* 131%26* PT (g/mm?; PICaz’li (PM); Fatigue (sec)=time to 50% force reduction; [Ca2+],=2.5mM, *peO.O.5 vs. C. In HF rats CZI*+~ signalling and contractility is altered in skeletal muscle. HF may be a condition characterized by generalized striated muscle dysfunction.
P153
TBX BFFBCT OF CBBOBIC DIABBTBS ON EDCLXOTIDXTRIPI~OS~IBE (BTPase)ACTIVITTABD PASSlXiBCALCIDMBIItDIWIBABAT BDCISAX -PB m FBACTIOl? Bram Rarajiawan. James S.C. Gilchrist, John C. Docherty, Grant N. Pierce. Div. Cardiovasc. Sci.. St. Boniface Hospital Research Centre, Winnipeg, Canada RZH 2A6 Protein synthesis during diabetes is markedly depressed. The nuclear NTPase regulates peptide and mRNA transport across the nuclear membrane. Ca2+ interactions with the nuclei may also influence gene expression. Thus, we hypothesized that the alteration in rotein metabolism during diabetes may be caused 4+ by changes in NTPase activity and Ca binding by the nuclei. Nuclei were isolated from hepatic tissue after 8 weeks of diabetes. The nuclei appeared structurally intact, contained little cellular debris, stained strongly with toluidine blue and exhibited negligible marker enzyme activies for other membrane fractions. Nuclear NTPase activity was not different between the diabetic and control samples. The ATP and Mg2+ dependence of the NTPase activit was also %5Ca2+ similar between the two groups. We could not detect high affinity binding sites in the nuclear membrane fraction. Both nuclei and nuclear membrane Ca2+ binding was unaffected by the diabetic condition of the animals. Our results demonstrate that the alteration in protein metabolism in rats during chronic experimental diabetes does not involve a change in NTPase activity or the Ca2+ binding capacity of hepatic nuclei. Supported by MRC.
PI54
ROLE OF EXTERNAL [Ca*‘] IN THE INOTROPIC AND METABOLIC EFFECTS OF PHORBOL ESTERS Morris Karmazyn, James Haist. Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario N6A 5Cl Canada. Our study examined the effects of altering [Ca2+]. on functional and metabolic effects of phorbol 1Zmyristate 13-acetate (PMA, 10”M to 10e6M) and phorbol 12,13-dibutyrate (PDBu, 10“*M to IU’M) in Langendorff-perfused rat hearts. Under normal [Ca*+], (1.25 mM) 1o”‘M PMA produced a sustained elevation in contractile force whereas in the presence of elevated [Ca’+]. (2.5 mM) only a negative inotropic effect was observed. The positive inotropic effect was also reversed by increasing PMA concentrations such that a rapid (> 90%) loss in function was seen with 10m6M irrespective of [Ca*+],. The effect of PDBu was generally identical to that seen with PMA except that, in the presence of normal [Ca”+]., the positive inotropic effect was maintained with both of the two lowest concentrations (10.“M and 1Cr’OM) employed. However, a negative inotropic influence was seen when identical concentrations of PDBu were examined under conditions of elevated [Ca*+],. Only the highest concentration of either phorbol significantly depressed high energy phosphate contents, an effect not influenced by [Ca’+].. Furthermore, phorbol ester-induced elevations in resting tension as well as coronary constriction were unaffected by [Cat+] Our studies demonstrate complex concentration-dependent effects of phorbol esters and suggest that the myokHrdial contractile response to low concentrations of these compounds is strongly influenced by [Ca”+],. (Supported by MRC of Canada) S.56