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Contractile protein synthesis and myofibrillar organization in cultured rabbit cardiac myocytes
J Mol
p64
Cell
Cardiol
GLUTAMJNE
24 (Supplement
CATABOLISM
III)
BY HEART
(1992)
MUSCLE:
PROPERTIES
OF PHOSPHATE-ACTIVATED
GLUTAhtlNASE
David Nelson*, William Rumsey+ and Maria Erecinska* Department of Pharmacology*, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; Bristol-Myers Squibb Pharmaceutical Research Institute+, New Brunswick NJ 08903. Rat heart homogenates and isolated mitochondria catablize glutamine in the presence of rotenone, an inhibitor of the respiratory chain. The reaction is markedly stimulated by phosphate, inhibited by glutamate and modestly affected by ammonia. Glutamine breakdown is enhanced by lactate, malate, citrate and ATP and essentially blocked by 1 mM Nethylmaleimide. chloride (30-50 mM) is a potent inhibitor, particularly at physiologic pH. The latter effect is not mediated via changes in osmolality since at equivalent concentration, mannitol haa only a small influence. In both preparations, the Km for glutamine is about 4 mM in the presence of either 50 or 100 mM phosphate and at pH 7.3 or 8.0 . The Vmax is about 3fold greater in mitochondria than in homogenates and, in both systems, larger at higher anion concentrations and at the more alkaline pH. Titrations with phosphate as tbe sole anion yield sigmoid curves with a Hill coefficient of about 2. Addition of a constant [KCI] (100 mhl) decreases sigmoidicity and the value of the Hill coefficient. A comparison of pH dependencies of glntaminase activity in 100 mM phosphate (no other buffer present) and 100 mM sulfate (buffered with 20 mM his-MOPS) suggests that HPO& is the more powerful activator of the enzyme than H2PO4-. Subcellular distribution of activity is the same as that of a mitochondrial marker, cytochrome c oxidase. Properties of glutaminase with respect to its dependence on the concentrations of phosphate, glutamine and H+ are the same in intact and broken mitochondria and very similar to those in homogenates. However, the specific activity of the enzyme is considerably smaller in frozen-thawed than in intact organelles. Thus, heart mitochondria possess a kidney-type, phosphate-activated glutaminase that is associated with the mitochontial inner membrane and, in a functional state, exists as a dimer. The association/dissociation cycle may be an important mechanism that modulates enzyme activity. Under physiologic conditions several ions: phosphate, chloride, ciirate and ATP, may conhibute to the regulation of glutaminase activity.
P65
CONTRACTILE PROTEIN SYNTHESIS AND MYOFIBRILLAR ORGANIZATION IN CULTURED RABBIT CARDIAC MYOCYTES. Robert S. Decker, Melissa G. Cook, Monica Behuke-Barclay, Marlene L. Decker and Allen M. Samarel*. Department of Medicine, Northwestern University Medical School, Chicago IL 60611, and *Loyola University Medical School, Maywood, IL 60153 USA. Considerable controversy revolves around the relative contribution of mechanical activity and neurohumoral agents in regulating actin (A) and myosin heavy chain (MHC) synthesis in the heart. Quiescent rabbit myoeytes were cultured for even days and then exposed to cc and p adrenergic agonists for 48 hours and radiolableled with [ $ Hj leucine during the last 4 hours of exposure to the adrenergic agonists. Fractional rates (KS) of total protein (‘IF) synthesis and A and MHC synthesis were monitored in these preparations. In non-beating myocytes, the K for TP is only 72% of values derived from intact rabbit heart, whereas KS for A and MHC is only iO% and lo%, respectively. cx and/or p adrenergic agonists restore TF’ KS to that observed in viva but only modestly elevates A and MHC synthesis. Under such conditions SDS-PAGE gels reveal that a significant depletion of contractile proteins develop in these non-beating myocytes, regardless of whether adrenergic agonists are present. Parallel double label immunofluorescence distribution of actin and myosin further illustrates that neither norepinephrine, isoproterenol or phenylephrine alters the progressive disruption of the contractile apparatus that develops in these non-beating heart cells. We conclude that in the absence of mechanical activity (ie., beating) neumhumoral agents are unable to significantly elevate the fractional rate of contractile protein synthesis and are not sufficient to maintain myofibtillar order.
p66
PHYSIOLOGIC HYPERINSULINEMIA INHIBITS CANINE MYOCARDIAL PROTEIN DEGRADATION IN VIVO Lawrence H Young, Douglas M Dahl, Deborah Rauner, Eugene J Barrett. Deparhuent of Internal Medicine, Yale University School of Medicine, New. Haven, CT 06510, USA. To study the effect of insulin on heart protein turnover in vivo, we examined anesthetized dogs after either a 16 or 36 hr fast, and again during a hyperinsulinemic (2 mU/kg/min) euglycemic clamp with or without amino acid replacement or during a saline infusion. We measured heart phenylalanine (PHE) balance, and rates of protein synthesis and degradation using the extraction of )H-PHE and the dilution of its specific activity (HPLC analysis) across the heart at isotopic steady-state. After both a 16 hr (n= 19) and 36 hr fast (n= lo), there was a net negative heart PHE balance, -.52+9 nmollmin and -38_+9 respectively. Heart protein degradation was lower in the 36 hr fasted animals (81+13 vs 121212 nmol/min, p < 0.05). Heart PHE balance and rates of protein synthesis and degradation did not change during insulin infusion alone (n= lo), but plasma amino acids fell signficantly (40%). In animals receiving insulin and replacement amino acids (n= ll), PHE balance shifted from negative to neutral (-40~6 to 6215 nmollmin, p
p64
Cell
Cardiol
GLUTAMJNE
24 (Supplement
CATABOLISM
III)
BY HEART
(1992)
MUSCLE:
PROPERTIES
OF PHOSPHATE-ACTIVATED
GLUTAhtlNASE
David Nelson*, William Rumsey+ and Maria Erecinska* Department of Pharmacology*, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; Bristol-Myers Squibb Pharmaceutical Research Institute+, New Brunswick NJ 08903. Rat heart homogenates and isolated mitochondria catablize glutamine in the presence of rotenone, an inhibitor of the respiratory chain. The reaction is markedly stimulated by phosphate, inhibited by glutamate and modestly affected by ammonia. Glutamine breakdown is enhanced by lactate, malate, citrate and ATP and essentially blocked by 1 mM Nethylmaleimide. chloride (30-50 mM) is a potent inhibitor, particularly at physiologic pH. The latter effect is not mediated via changes in osmolality since at equivalent concentration, mannitol haa only a small influence. In both preparations, the Km for glutamine is about 4 mM in the presence of either 50 or 100 mM phosphate and at pH 7.3 or 8.0 . The Vmax is about 3fold greater in mitochondria than in homogenates and, in both systems, larger at higher anion concentrations and at the more alkaline pH. Titrations with phosphate as tbe sole anion yield sigmoid curves with a Hill coefficient of about 2. Addition of a constant [KCI] (100 mhl) decreases sigmoidicity and the value of the Hill coefficient. A comparison of pH dependencies of glntaminase activity in 100 mM phosphate (no other buffer present) and 100 mM sulfate (buffered with 20 mM his-MOPS) suggests that HPO& is the more powerful activator of the enzyme than H2PO4-. Subcellular distribution of activity is the same as that of a mitochondrial marker, cytochrome c oxidase. Properties of glutaminase with respect to its dependence on the concentrations of phosphate, glutamine and H+ are the same in intact and broken mitochondria and very similar to those in homogenates. However, the specific activity of the enzyme is considerably smaller in frozen-thawed than in intact organelles. Thus, heart mitochondria possess a kidney-type, phosphate-activated glutaminase that is associated with the mitochontial inner membrane and, in a functional state, exists as a dimer. The association/dissociation cycle may be an important mechanism that modulates enzyme activity. Under physiologic conditions several ions: phosphate, chloride, ciirate and ATP, may conhibute to the regulation of glutaminase activity.
P65
CONTRACTILE PROTEIN SYNTHESIS AND MYOFIBRILLAR ORGANIZATION IN CULTURED RABBIT CARDIAC MYOCYTES. Robert S. Decker, Melissa G. Cook, Monica Behuke-Barclay, Marlene L. Decker and Allen M. Samarel*. Department of Medicine, Northwestern University Medical School, Chicago IL 60611, and *Loyola University Medical School, Maywood, IL 60153 USA. Considerable controversy revolves around the relative contribution of mechanical activity and neurohumoral agents in regulating actin (A) and myosin heavy chain (MHC) synthesis in the heart. Quiescent rabbit myoeytes were cultured for even days and then exposed to cc and p adrenergic agonists for 48 hours and radiolableled with [ $ Hj leucine during the last 4 hours of exposure to the adrenergic agonists. Fractional rates (KS) of total protein (‘IF) synthesis and A and MHC synthesis were monitored in these preparations. In non-beating myocytes, the K for TP is only 72% of values derived from intact rabbit heart, whereas KS for A and MHC is only iO% and lo%, respectively. cx and/or p adrenergic agonists restore TF’ KS to that observed in viva but only modestly elevates A and MHC synthesis. Under such conditions SDS-PAGE gels reveal that a significant depletion of contractile proteins develop in these non-beating myocytes, regardless of whether adrenergic agonists are present. Parallel double label immunofluorescence distribution of actin and myosin further illustrates that neither norepinephrine, isoproterenol or phenylephrine alters the progressive disruption of the contractile apparatus that develops in these non-beating heart cells. We conclude that in the absence of mechanical activity (ie., beating) neumhumoral agents are unable to significantly elevate the fractional rate of contractile protein synthesis and are not sufficient to maintain myofibtillar order.
p66
PHYSIOLOGIC HYPERINSULINEMIA INHIBITS CANINE MYOCARDIAL PROTEIN DEGRADATION IN VIVO Lawrence H Young, Douglas M Dahl, Deborah Rauner, Eugene J Barrett. Deparhuent of Internal Medicine, Yale University School of Medicine, New. Haven, CT 06510, USA. To study the effect of insulin on heart protein turnover in vivo, we examined anesthetized dogs after either a 16 or 36 hr fast, and again during a hyperinsulinemic (2 mU/kg/min) euglycemic clamp with or without amino acid replacement or during a saline infusion. We measured heart phenylalanine (PHE) balance, and rates of protein synthesis and degradation using the extraction of )H-PHE and the dilution of its specific activity (HPLC analysis) across the heart at isotopic steady-state. After both a 16 hr (n= 19) and 36 hr fast (n= lo), there was a net negative heart PHE balance, -.52+9 nmollmin and -38_+9 respectively. Heart protein degradation was lower in the 36 hr fasted animals (81+13 vs 121212 nmol/min, p < 0.05). Heart PHE balance and rates of protein synthesis and degradation did not change during insulin infusion alone (n= lo), but plasma amino acids fell signficantly (40%). In animals receiving insulin and replacement amino acids (n= ll), PHE balance shifted from negative to neutral (-40~6 to 6215 nmollmin, p