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Biomedical subjects

S E Mayer

Publications and source records attributed to S E Mayer.

At least 19 recordsLinked to original sources
PubMed1979

Interconversion between multiple glucose 6-phosphate-dependent forms of glycogen synthase in intact adipose tissue.

We have tested the hypothesis that interconversion between multiple glucose-6-P-dependent forms of glycogen synthase helps regulate glycogen synthesis in adipose tissue. Our results indicate that interconversion of glycogen synthase in adipose tissue involves primarily dependent forms and that these interconversions were measured better by monitoring the activation constant (A0.5) for glucose-6-P than measuring the -: + glucose-6-P activity ratio. Insulin decreased and epinephrine increased the A0.5 for glucose-6-P without significant change in the activity ratio. Insulin consistently decreased the A0.5 in either the presence or absence of glucose, indicating that the insulin-promoted interconversion did not require increased hexose transport. Isoproterenol increased the A0.5 for glucose-6-P, while methoxamine was without effect, indicating beta receptors mediate adrenergic control of interconversion between glucose-6-P-dependent forms. The changes in the A0.5 produced by incubations with insulin or epinephrine were mutually reversible. We conclude that 1) glycogen synthesis in adipose tissue is catalyzed by multiple glucose-6-P-dependent forms of glycogen synthase, 2) hormones regulate glycogen metabolism by promoting reversible interconversions between these forms, and 3) there is no evidence that a glucose-6-P-independent form of glycogen synthase exists in intact adipose tissue.

Adipose Tissue↗
PubMed1979

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.

Adipose Tissue↗
PubMed1979

Depressed cyclic AMP levels in airways smooth muscle from asthmatic dogs.

We tested mongrel dogs by intradermal injection and tracheobronchial aerosol challenge with Ascaris suum antigen extract. All dogs were skin-test positive but could be segregated, on the basis of airways resistance measurements, into "asthmatic" (bronchoreactive) and "nonasthmatic" (nonbronchoreactive) groups. By using tracheal rings from these dogs, we measured the abilities of the beta-adrenergic agonist, isoproterenol, to relax tracheal smooth muscle contracted by methacholine and to cause cyclic AMP (cAMP) accumulation in the presence and absence of methacholine. The magnitude of relaxation induced by any concentration of isoproterenol was always less in the smooth muscle from "asthmatic" dogs. In the same tissues the concentrations of cAMP after in vitro equilibration, but prior to isoproterenol addition, were significantly less in the "asthmatic" than "nonasthmatic" samples. The accumulation of cAMP due to isoproterenol was similar in both groups for every dose of isoproterenol so that the initial difference between groups in cAMP concentration was maintained in an additive fashion over the entire dose-response curve. Total protein content of trachealis muscles from both groups of dogs was the same. We conclude that beta-adrenergically sensitive adenylate cyclase is not impaired in tracheal smooth muscle from "asthmatic" dogs; rather, the basal concentration of cAMP is depressed in "asthmatic" airway smooth muscle, and this difference is maintained throughout the isoproterenol dose-response curve. The depressed intracellular cAMP concentrations may be related to the decreased relaxation induced by isoproterenol in the "asthmatic" tracheal smooth muscle.

Animals↗
PubMed1979

Hormonally specific expression of cardiac protein kinase activity.

The relationship between the effects of isoproterenol and prostaglandin E(1) (PGE(1)) on contractile state, cyclic AMP accumulation, and the activation states of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37), phosphorylase kinase, glycogen synthase, and glycogen phosphorylase have been studied in the isolated perfused rat heart. Perfusion of hearts with isoproterenol (10 or 80 nM) caused enhancement of left ventricular dP/dt (P, pressure), increased intracellular cyclic AMP, increased the activation states of protein kinase, phosphorylase kinase, glycogen phosphorylase, and conversion of glycogen synthase to a less active form. PGE(1) (2 or 30 muM) increased cyclic AMP accumulation and activated protein kinase, but caused no detectable changes in dP/dt or the activation states of the protein kinase substrates involved in glycogen metabolism. Perfusion of hearts with either 10 nM isoproterenol or 30 muM PGE(1) produced comparable increases in cyclic AMP accumulation and protein kinase activity. Exposure of hearts to a combination of these agents caused additive effects on cyclic AMP content and protein kinase activity. However, values for phosphorylase kinase, glycogen phosphorylase, glycogen synthase, and dP/dt did not differ from those observed in the presence of 10 nM isoproterenol alone. The failure of PGE(1) to stimulate phosphorylation of protein kinase substrates was not due to an increase in phosphorylase phosphatase activity. We conclude that an increase in intracellular cyclic AMP and the subsequent activation of protein kinase are insufficient to change either the activities of phosphorylase kinase, glycogen phosphorylase, and glycogen synthase or the inotropic state of heart muscle.

Animals↗
PubMed1979

Effects of ischemia on tissue metabolites in red (slow) and white (fast) skeletal muscle of the chicken.

Brief periods of ischemia have been shown to produce marked reactive hyperemia in both red (slow) and white (fast) skeletal muscle. However, evidence is lacking for specific vasodilator metabolites which are rapidly produced in ischemic skeletal muscle. The present study examined the effects of 1 and 3 minutes of ischemia on creatine phosphate (CrP), adenine nucleotide metabolism, and anaerobic glycolysis in red anterior (ALD) and white posterior latissimus dorsi (PLD) muscles of the chicken. Tissue metabolite concentrations were determined from perchloric acid or trichloroacetic acid extracts using enzymatic assay or high pressure liquid chromatography. CrP or adenine nucleotides were not significantly altered in either muscle following 1 or 3 minutes of ischemia. However, adenosine increased by 611% in the ALD at 1 minute. Following 3 minutes of ischemia, adenosine concentrations were elevated by 439% and 201% in the ALD and PLD, respectively. The PLD showed the greatest increases in inosine and IMP. Inorganic phosphate increased by 67% and lactate increased by 142% in the ALD at 3 minutes. The PLD, which is reported to have a high anaerobic glycolytic capacity, showed no increase in lactate. These results support the hypothesis that adenosine may be a mediator of akeletal muscle reactive hyperemia following short periods of ischemia.

Adenosine↗
PubMed1977

Metabolism of adenine nucleotides in the cultured fetal mouse heart.

Intact beating fetal mouse hearts in organ culture were deprived of oxygen and glucose for up to 4 h, resulting in loss of beating, an 80% fall in ATP, reduction of energy charge from 0.85 to 0.48, and doubling of total nucleoside concentration. Radiolabeled adenine nucleotides were degraded to hypoxanthine and inosine, which were lost from the hearts into the medium during the deprivation period. Adenosine and adenine also appeared in the medium when adenosine deaminase was inhibited. After 24 h of O2 and glucose resupply, ATP returned to 60% of control, and energy charge rose to 0.76. Labeled nucleosides and bases remaining in the heart or exogenous labeled adenine were utilized to resynthesize ATP. [14C]glycine was rapidly taken up by recovering hearts but was not used for de novo adenine nucleotide synthesis. Ability to recover ATP and spontaneous contraction appear related to residual nucleotide and nucleoside content rather than to energy charge.

Adenine Nucleotides↗
PubMed1977

Contractile and biochemical responses of cardiac and skeletal muscle to isoproterenol covalently linked to glass beads.

The effects of (-)-isoproterenol covalently linked to glass beads on inotropic state, cyclic AMP concentration, and phosphorylase b to a conversion was studied in cat and guinea pig papillary muscles. Biochemical responses also were measured in mouse diaphragm sections. In cat papillary muscles under normal conditions and in guinea pig muscles partly depolarized with 22 mM K+ an increased inotropic state and phosphorylase activation could be dissociated from the formation of cyclic AMP. This contrasts with close correlation reported between isometric tension and cyclic AMP concentration in guinea pig papillary muscles exposed to varying concentrations of isoproterenol dissolved in the bath medium. The cyclic nucleotide did increase in guinea pig papillary muscles exposed to a freshly prepared batch of isoproterenol beads and in the mouse diaphragms. The type and age of the bead preparation had marked effects on all responses that were measured and on the rate of leakage of biologically active products from the beads. Nevertheless, experimental conditions could be obtained under which the initiation of the contractile and biochemical responses was probably limited to intense stimulation of receptors at or near the points of contact between beads and muscle and not due to gross leakage into the bath. The results indicate that cyclic AMP is probably involved in the initiation but not the propagation of the inotropic response of papillary muscles exposed to isoproterenol glass beads.

Animals↗
PubMed1977

Electrophysiological responses of cardiac muscle to isoproterenol covalently linked to glass beads.

We investigated the effects of isoproterenol aryl glass beads on the electrical properties of cardiac muscle and related these to our previous results concerning biochemical and contractile effects (Ingebretsen et al., Circ, Rs., 40: 474-484, 1977). Beads (10-15) were placed near one end to guinea pig papillary muscles mounted horizontally in a bath perfused with Krebs-Henseleit solution at 30 degrees C and stimulated at 0.2 Hz. The beads produced increased tension and elevation and slight lengthening of the plateau potential when [k+]o = 3.8 mM. After depolarization to a resting potential of -49 mV with [K+]o = 22 mM, isoproterenol beads restored contraction to a comparable extent as occurred with 10(-8) M soluble drug. During field stimulation, action potentials were initiated at the site of bead application and spread decrementally. When beads were placed distal to the site of point stimulation, virtually no excitation could be obtained from cells in the vicinity of the beads. When they were placed close to the stimulating electrode, the beads increased excitability and typical slow action potentials spread to the other end of the muscle. These potentials had the characteristics associated with the slow inward Ca2+ current. The slow channel blocker, D-600, blocked responses to isoproterenol beads. Tetrodotoxin caused responses similar to those obtained with K+ depolarization. The beads probably act by stimulating only a small fraction of the papillary muscle catecholamine receptors. Spread of action potentials from these sites and propagated tension depend on Ca2+ influx, but the nature of an intermediate messenger involved in the propagation of contractions is unknown.

Action Potentials↗
PubMed1976

The role of cyclic adenosine 3', 5'-monophosphate and calcium in the regulation of contractility and glycogen phosphorylase activity in guinea pig papillary muscle.

We studied the relationships between the positive inotropic effects of isoproterenol, increased frequency of contraction or paired electrical stimulation, and cyclic AMP concentration and phosphorylase activity in isolated guinea pig papillary muscles. The minimum concentration of isoproterenol (10 nM) that augmented isometric force development increased cyclic AMP concentration. However 100 nM isoproterenol was required to increase the phosphorylase activity ratio (-AMP/+AMP) from 0.15 +/- 0.03 to 0.25 +/- 0.03. After addition of 1 muM isoproterenol to the bath, cyclic AMP increased within 0.5 minute from 0.58 +/- 0.03 to 1.04 +/- 0.13 mol/kg (wet weight), peak contractile force was elevated 2-fold at 1 minute, and the phosphorylase activity ratio rose to 0.40 +/- 0.02 in 4 minutes. Although an increase in contraction frequency (6/min to 36/ min) and paired stimulation produced more than a 3-fold increase in peak contractile force, there were no changes in cyclic AMP and phosphorylase activity. The cyclic AMP concentration during diastole was 0.60 +/- 0.04 and in midsystole, 0.55 +/- 0.03 mumol/kg. Anoxia increased the phosphorylase activity ratio from 0.19 +/-0.02 to 0.41 +/- 0.04 without elevation of cyclic AMP concentration. Removal of Ca2+ from the bathing medium prevented active force development and the anoxic increase in phosphorylase activity, but did not prevent the isoproterenol-induced increase in cyclic AMP and phosphorylase. These results suggest that cyclic AMP is a factor in the catecholamine-induced enhancement of inotropic state. However, it does not appear to play a role in the maintained augmentation of inotropic state produced by increased contraction frequency and paired stimulation, nor does the concentration of the cycle nucleotide appear to vary during the contraction cycle or during anoxia. Extracellular Ca2+ is required for contraction, the positive inotropic aciton of catecholamines and phosphorylase b to a conversion by anoxia.

Animals↗
PubMed1976

Stimulation of glycogenolysis by beta adrenergic agonists in skeletal muscle of mice with the phosphorylase kinase deficiency mutation (I strain).

The mechanism by which beta adrenergic agonist stimulate glycogenolysis in intact skeletal muscle was investigated in mice with the phosphorylase kinase deficiency mutation (I strain). Although extracts of I strain diaphragm muscle had only 3.7% of the phosphorylase kinase activity found in extracts of the control strain (C57BL), incubation of I strain hemidiaphragms in Krebs-Ringer bicarbonate buffer with either isoproterenol or epinephrine resulted in a stimulation of the rate of glycogenolysis. In C57BL diaphragms, the EC50 values for isoproterenol and epinephrine were 2 and 14 nM, respectively. With I strain diaphragms, dl-isoproterenol or l-epinephrine stimulated glycogenolysis as a linear function of the log of the drug concentration with no apparent plateau of response up to concentrations of 30 to 40 mugM. For each 10-fold increase in drug concentration, isoproterenol and epinephrine stimulated glycogenolysis in I strain muscles an additional 0.37 to 0.42 mg/g/hr, a slope in the concentration-response relationship of 0.17 and 0.37, respectively, of that measured in C57BL diaphragms at concentrations around the EC50. The highest glycogenolytic response measured in I strain hemidiaphragms (at 40 mugM isoproterenol) was 80% of the maximal catecholamine-stimulated glycogenolysis in C57BL diaphragms. Both 4 nM and 4 mugM isoproterenol, in a concentration-dependent manner, stimulated phosphorylase b to a conversion in I and C57BL diaphragms and increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) concentrations. The glycogenolytic response to 10.1 nM dl-isoproterenol in both I and C57BL diaphragms was blocked by 34 nM l-propranolol but not by 34 nM d-propranolol. The response to 4 mugM isoproterenol was enhanced by the cyclic nucleotide phosphodiesterase inhibitors papaverine (27 mugM) or dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 3 mugM). From the results of these studies, we conclude: 1) Catecholamines stimulate glycogenolysis in skeletal muscle of I mice, as in C57BL mice, by interacting with the beta adrenergic receptor, thereby increasing tissue cyclic AMP concentrations and stimulating phosphorylase b to a conversion. 2) alternative hypotheses for the mechanism of the catecholamine-stimulated decrease in glycogen concentration in I skeletal muscle-inhibition of glycogen synthesis, hyposia and 5'-AMP stimulation of phosphorylase b activity-have been ruled out. 3) the activity of the mutant phosphorylase kinase, although it is only 3.7% of that in extracts of C57BL muscle, is sufficient to produce phosphorylase b to a conversion and thereby account for the glycogenolytic response of I strain muscle to catecholamines.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1975

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.

Adenosine Monophosphate↗
PubMed1975

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. II. Glycogen synthase.

Glycogen synthase from swine adipose tissue was purified to apparent homogeneity using ethanol precipitation, DEAE chromatography, and affinity chromatography utilizing glucosamine 6-phosphate as the ligand. The purified enzyme migrated as a single protein component during electrophoresis on polyacrylamide gels at pH 7.3 although some protein failed to enter the running gel. Enzyme incubated with sodium dodecyl sulfate (SDS) migrated as one component (mol wt similar to 90,000) on SDS-polyacrylamide gel electrophoresis. The enzyme was relatively unstable at all stages of the purification procedure, but stability was increased in the presence of glucose 6-phosphate, UDPG, or glycerol. The isoelectric point of the purified enzyme and of enzyme activity in crude homogenates was pH 4.8. The sedimentation coefficient of the enzyme in crude homogenates was 8.5 S. The pH-activity profile showed an optimum at pH 7.8 in the absence of glucose 6-phosphate but no definable optimum between pH 7.0 and 9.2 in its presence. The Km of glycogen synthase I for UDPG was 250 muM in the absence and 37 muM in the presence of glucose 6-phosphate; the K-a for glucose 6-phosphate was 18 mu-M. The K-m of glycogen synthase D for UDPG was 130 mu-M in the presence of glucose 6-phosphate; the Ka for glucose 6-phosphate was 1 mM. The anions sulfate and phosphate activated the enzyme when assays were performed in the absence of glucose 6-phosphate. Fluoride produced activation of enzyme assayed either in the presence or in the absence of glucose 6-phosphate.

Adipose Tissue↗
PubMed1975

Phosphorylase kinase mediating the effects of cyclic AMP in muscle.

In the classic view of the control of phosphorylase b to a conversion by catecholamines, cyclic AMP acts as the second messenger stimulating the activity of cyclic AMP-dependent protein kinase to covalently modify phosphorylase kinase. Phosphorylation of phosphorylase kinase converts this enzyme form with a nonactivated to an activated form with a markedly higher activity at pH 7. There is now considerable evidence that the activity of phospphorylase kinase is also regulated by changeds in the Ca-2+ concentration. The activity of both nonactivated and activated phosphorylase kinase is stimulated by Ca-2+ in the range of concentrations that have been reported to occur in the sacroplasm of contracting muscle, with the activated pphosphorylase kinase having a lower K-alpha for Ca-2+. Thus there are at leaset two mechanisms for the regulation of phosphorylase kinase activity in muscle. These mechanisms may act independently or in concert in controlling glycogenolysis stimulated by catecholamines, anoxia, or tetanic electrical stimulation...

Adenosine Triphosphate↗