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M L Entman

Publications and source records attributed to M L Entman.

At least 127 records · Page 7Linked to original sources
PubMed1985

Nucleotide specificity of cardiac sarcoplasmic reticulum. GTP-induced calcium accumulation and GTPase activity.

We previously demonstrated that the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium and does not support the translocation of calcium and oxalate into the vesicular space. In response to GTP, however, calcium is accumulated into a compartment which is sensitive to pH and ionophore. In the present paper, we further explored the relationship between GTP hydrolysis and GTP-induced calcium accumulation. Both ATP- and GTP-induced calcium accumulation were prevented by the sulfhydryl reagent, N-ethylmaleimide (NEM; I50 = 0.2 mM). In contrast, the sensitivity of NTP hydrolysis to NEM differed markedly; GTPase activity was not affected by NEM, whereas ATPase activity was markedly inhibited. Conversely, although the GTPase was noncompetitively inhibited by the ATP analogue, adenylyl imidodiphosphate (Ki = 8 microM), and was competitively inhibited by the GTP analogue, guanylyl imidodiphosphate (Ki = 60 microM), GTP-induced calcium accumulation was not affected by the NTP analogues at any concentration. Therefore, the GTP-dependent accumulation of calcium into the pH- and ionophore-sensitive compartment of cardiac SR may not require GTP hydrolysis but may be dependent on GTP binding. The previously reported noncompetitive inhibition of the GTPase by ATP was also observed when the calcium-dependent hydrolysis of ATP was prevented by NEM (Ki = 1.2 microM). Along with the noncompetitive inhibition of the GTPase by adenylyl imidodiphosphate, the inhibition of the GTP by ATP in the presence of NEM suggests that ATP binding may be involved in the observed inhibition. The Ki for the noncompetitive inhibition of GTPase activity is compatible with ATP binding to the high affinity catalytic site of the ATPase. Thus, although GTP-induced calcium accumulation differs somewhat from ATP-dependent calcium translocation, the similarities between the two processes (i.e. similar time courses and sensitivity to pH, ionophore, and sulfhydryl modification) suggest that they may be related in some manner.

Adenosine Triphosphate↗
PubMed1985

Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting.

Highly purified dog heart sarcolemmal membranes, with a content of approximately 5 pmol of muscarinic acetylcholine receptor (mAChR)/mg of protein, were analyzed for mAChR-mediated inhibition of adenylyl cyclase and ligand binding in the absence and the presence of guanine nucleotides. Adenylyl cyclase was found to be coupled to the mAChR, being attenuated approximately 30% in a GTP-dependent manner. Direct binding studies, using 3H-labeled oxotremorine M, showed high affinity binding (apparent KD = 10 nM) that was reduced on nucleotide addition. Dose-response curves for GDP, GTP, and guanyl-5'-yl imidodiphosphate showed them to be equipotent. On the basis of pirenzepine binding, only one type of mAChR, commonly referred to as M2, was detected. Direct binding of [3H]quinuclidinyl benzilate [( 3H]QNB) uncovered 50% more binding sites than 150 nM 3H-labeled oxotremorine M; addition of guanine nucleotides uncovered the existence of positive cooperativity in the binding of [3H]QNB. Agonist displacement curves of [3H]QNB binding, without and with guanine nucleotides, extended over several orders of magnitude, which is inconsistent with single site competitive kinetics. The results and their analysis by computer-assisted curve fitting indicated that the data are well fitted by a model in which a receptor is at least bivalent and exists in two states: one with and the other without cooperativity between its sites, with guanine nucleotides decreasing both the degree of cooperativity between the sites and the proportion of the receptor that is in the cooperative form. Since the guanine nucleotide effect is mediated by the Ni coupling protein, it is suggested that direct binding detects R'Ni complexes (cooperative), R"NiG complexes (cooperative but distinct from R'Ni), and R0 complexes (non-cooperative and unaffected by Ni or NiG), where R = mAChR, Ni = the inhibitory regulatory component of adenylyl cyclase unaffected by guanine nucleotide, and NiG = Ni affected by guanine nucleotide (G).

Adenylyl Cyclases↗
PubMed1985

Cytochemical studies of a glycogen-sarcoplasmic reticulum complex.

Enzymatically active cardiac sarcoplasmic reticulum (SR) fractions contain glycogen. Previous biochemical and morphological studies indicate that the glycogen particles are membrane associated. In the present study, further evidence for membrane-associated glycogen particles in these cardiac SR fractions is presented: (1) morphological parameters, (2) enzymatic digestion by glucoamylase and alpha-amylase and (3) cytochemical staining by two different methods. Dense granules comparable in size (20-30 nm diameter), electron density and substructure to glycogen particles observed in intact cardiac muscle and in glycogen preparations isolated from skeletal muscle were seen. Most of these glycogen particles were removed by amylase digestion except for glycogen particles closely adhering to vesicle membranes. Two different cytochemical techniques (bismuth subnitrate and silver proteinate) revealed a positive reaction product over the glycogen particles. These findings provide further support for the biochemical finding of a structured enzyme complex involving the SR, glycogenolytic enzymes and glycogen.

Animals↗
PubMed1985

Creatine kinase and phosphorylase in cardiac lymph: coronary occlusion and reperfusion.

Cardiac lymph, collected from conscious dogs, was monitored for glycogen phosphorylase and creatine kinase (CK) enzymatic activity during control state, circumflex coronary artery (CFX) occlusion, and reperfusion. CFX occlusions, lasting for intervals as short as 10 min, initiated a release of phosphorylase and CK into the cardiac lymph, which was immediately observed during reperfusion of the ischemic tissue. Blood plasma levels did not appear for several hours. In the absence of reperfusion, the appearance of enzymes in cardiac lymph was delayed and peaked later. Glycogen phosphorylase and CK entered the lymph in greater quantities with reperfusion as the length of occlusion was increased. Histological examination of multiple sections of the reperfused hearts showed infarcts in hearts where CFX occlusions lasted 20 min or longer; occlusions of 10-15 min showed evidence of cell injury and death in two hearts and no definable infarct in the majority. Ischemic intervals of short duration release functionally active glycogen phosphorylase and CK, which reflect changes in myocardial cell egress of macromolecules and/or cell death.

Animals↗
PubMed1985

Protection of canine cardiac mitochondrial function by verapamil-cardioplegia during ischemic arrest.

Hemodynamic and mitochondrial function recover following 60 minutes of ischemic arrest and reperfusion in hearts pretreated with verapamil. The present study was carried out to determine whether verapamil prevents the onset of mitochondrial oxidative impairment after 60 minutes of ischemic arrest without reperfusion. Two preparations of mitochondria isolated following Polytron homogenization and subsequent treatment of the myofibrillar pellet with Nagarse were examined for phosphorylating respiration. The Polytron mitochondria were more sensitive to ischemic arrest than were the Nagarse mitochondria with either glutamate-malate (57% vs. 22% inhibition), succinate (+ rotenone) (41% vs. 14% inhibition), or palmitoylcarnitine (57% vs. 27% inhibition) as respiratory substrates. Verapamil pretreatment significantly increased oxidation of all substrates by the subsequently isolated Polytron mitochondria, but only succinate-supported respiration returned to control levels. In contrast, the small amount of respiratory inhibition exhibited by the Nagarse mitochondria after ischemic arrest was insensitive to verapamil pretreatment. We conclude that the Polytron preparation of mitochondria is more susceptible to ischemia than the Nagarse mitochondria, and this susceptibility correlates with a striking sensitivity to verapamil protection. In general, oxidation of NADH-linked substrates, including palmitoylcarnitine, is more affected by ischemic arrest than succinate, and only oxidation of the latter substrate is totally protected by verapamil. The beneficial action of verapamil on mitochondrial function occurs prior to reperfusion. The data suggest that alterations in calcium homeostasis occur during the ischemic period, as well as in the subsequent reperfusion period.

Animals↗
PubMed1985

Selective accumulation of the first component of complement and leukocytes in ischemic canine heart muscle. A possible initiator of an extra myocardial mechanism of ischemic injury.

Myocardial concentrations of C1q, a subunit of the first component of complement, were measured 5-120 minutes after ligation of a coronary artery in dogs injected with 125I-labeled human C1q and 131I-labeled human albumin. The 131I-labeled human serum albumin was used as a plasma protein marker. Ischemic regions of myocardium were defined by measuring regional myocardial blood flow by the reference sample method at intervals after coronary artery occlusion. Significant accumulations of 125I-C1q were demonstrated in the ischemic myocardium after coronary artery occlusions lasting 45 minutes. Some localization of C1q in ischemic myocardium was observed after a 15-minute occlusion, but the accumulations of C1q achieved in this case were not statistically significant. After coronary artery occlusions lasting greater than or equal to 45 minutes, left ventricular concentrations of C1q correlated reciprocally with regional myocardial blood flow. Moreover, high concentrations of C1q persisted in formerly ischemic segments after reperfusion. Radiolabeled neutrophils also accumulated selectively in ischemic segments relatively rich in C1q. It is suggested that complement activation may initiate the neutrophil-dependent portion of ischemic injury, delineated in recent years, that is associated with free radical release by phagocytic cells.

Animals↗
PubMed1984

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane.

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.

Animals↗
PubMed1984

Hepatic extraction of exogenous insulin in depancreatized conscious dogs.

Hepatic and mesenteric extraction of exogenous insulin and glucose appearance and clearance were compared in conscious depancreatized and normal dogs after intraportal or peripheral intravenous insulin infusion. Portal vein insulin levels were higher, whereas arterial insulin levels were lower after intraportal compared with intravenous peripheral infusion of insulin. During the intraportal infusion of 1 and 2 mU X kg-1 X min-1 insulin, 40 +/- 3% of the insulin presented to the liver was extracted by that organ in the diabetic dogs, similar to the value obtained in normal dogs (39 +/- 5%). Hepatic extraction of insulin after intravenous peripheral infusion of that hormone was similar in normal and diabetic dogs and was not significantly different from intraportal infusion. Mesenteric extraction of insulin in the diabetic dogs (13 +/- 2%) was similar to the 19 +/- 3% in the normal animals. The blood sugar changes were similar after both routes of insulin infusion. Suppression of glucose appearance in diabetic dogs was also similar during both routes of infusion. Glucose clearance during the peripheral intravenous infusion of insulin in diabetic dogs was greater than during intraportal insulin. These findings indicate that hepatic extraction of exogenous insulin was similar in normal and depancreatized dogs and was not influenced by the different infusion routes. Suppression of glucose appearance in diabetic dogs was similar after both routes despite different portal vein insulin levels. The peripheral action of intravenous peripheral infused insulin in diabetic dogs was greater than that of intraportal insulin reflecting the higher arterial insulin levels.

Animals↗
PubMed1984

Effects of portal and peripheral venous insulin infusion on glucose production and utilization in depancreatized, conscious dogs.

The relation between portal vein insulin concentrations and suppression of hepatic glucose production, as well as peripheral venous insulin level and increase of peripheral glucose utilization, was compared in conscious, depancreatized, diabetic dogs after infusion of insulin at 0.25 and 0.5 mU/kg/min into either the portal system or the peripheral circulation. Glucose appearance and clearance was measured using [3-3H]-glucose. Before infusion of insulin, portal vein insulin concentrations were undetectable. The intraportal infusion of insulin at 0.25 mU/kg/min increased portal vein insulin to 16 +/- 1 microU/ml, significantly higher than the arterial concentration (9 +/- 1 microU/ml). Infusion of the same amount of insulin into a peripheral vein raised peripheral insulin levels to 14 +/- 1 microU/ml and portal vein concentrations to 12 +/- 1 microU/ml. When 0.5 mU/kg/min of insulin was infused into the portal system, the portal vein insulin level was 28 +/- 2 microU/ml and significantly greater than the arterial concentration (16 +/- 1 microU/ml). After the same amount of insulin was infused into a peripheral vein, the arterial insulin level was higher than that of the portal vein (25 +/- 1 microU/ml versus 20 +/- 1 microU/ml, respectively). The ensuing hypoglycemia was greater after the 0.5 mU/kg/min infusion compared with the 0.25 mU/kg/min infusion. At each dose there was no significant difference between the peripheral venous or the portal route.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Adrenergic and serotonergic regulation of skeletal muscle metabolism in rat. I. The effects of adrenergic and serotonergic antagonists on the regulation of muscle amino acid release, glycogenolysis, and cyclic nucleotide levels.

The biochemical mechanisms of serotonergic and adrenergic action on skeletal muscle cyclic nucleotide, glycogen, and amino acid metabolism have been investigated in intact rat epitrochlaris skeletal muscle preparations. Endogenous catecholamine levels in these preparations were 28.6 +/- 2.1 pg/mg of muscle. Release of these catecholamines by tyramine produced a 25% inhibition of alanine and glutamine release. Pretreatment of animals in vivo with 6-hydroxydopamine depleted catecholamine content by 85%. On incubation, preparations from these pretreated animals showed no effect of tyramine on amino acid metabolism. Serotonin (10(-5) M) and epinephrine (10(-5) M) inhibited alanine and glutamine release equally in preparations from 6-hydroxydopamine-pretreated as compared to control rats. Adrenergic antagonists such as dl-propranolol (10(-8)-10(-6) M), oxprenolol (10(-8)-10(-6) M), and practolol (10(-6)-10(-4) M) blocked equally the inhibition of alanine and glutamine release, prevented the stimulations of muscle cAMP levels, phosphosphorylase a formation, and the depletion of muscle glycogen produced by either epinephrine or serotonin. In contrast, serotonergic antagonists such as methysergide (10(-8)-10(-6) M) and cyproheptadine (10(-8)-10(-6) M) blocked the inhibition of alanine and glutamine release, the stimulations of muscle cAMP levels and phosphorylase a formation, and the decreased muscle glycogen content effected by serotonin but not by epinephrine. Incubation of muscles with both epinephrine and serotonin together produced additive stimulation of muscle cAMP levels, but not of the inhibition of alanine and glutamine release. These data indicate that the action of these agonists on skeletal muscle protein and amino acid, glycogen, and cyclic nucleotide metabolism proceeds directly via separate and discrete serotonergic and adrenergic receptor-adenylyl cyclase mechanisms in skeletal muscle.

Alanine↗
PubMed1983

Serotonergic and adrenergic regulation of skeletal muscle metabolism in the rat. II. The use of [125I]iodolysergic acid diethylamide and [125I]iodopindolol as probes of sarcolemmal receptor function and specificity.

To evaluate serotonin receptor kinetics in skeletal muscle, we synthesized and developed 2-[125I]iodolysergic acid diethylamide [( 125I]iodoLSD) as a high affinity, high specific activity probe of serotonergic receptor function. The kinetics of binding of this probe and the profile of agonist and antagonist displacement have been compared to results obtained using [125I] iodopindolol as a probe for beta-adrenergic receptor binding. [125I]IodoLSD was prepared by chloramine-T iodination and purified by high pressure liquid chromatography. Fluorescence, ultraviolet, and nuclear magnetic resonance spectra as well as mass spectroscopy demonstrate that the iodinated compound is 2-iodoLSD. This probe bound in a concentration-dependent fashion to sarcolemma in a manner consistent with existence of a single population of specific receptors having a Kd of 1.46 nM and an abundancy of 47 fmol/mg of protein. Half-maximal binding of the probe occurred within 1.0 min and equilibrium binding was observed at 8.0 min. The apparent t 1/2 for the probe-receptor complex was 40 s; the Kd calculated from kinetic data was 1.69 nM. The IC50 for displacement of iodoLSD was 330 +/- 185 nM for methysergide, 757 +/- 309 nM for cyproheptadine, and 2,570 +/- 1,390 nM for serotonin. Adrenergic antagonists such as l-propranolol and oxprenolol also displaced [125I]iodoLSD, but did so with IC50 values of 17,800 +/- 5,100 and 23,300 +/- 5,500 nM, respectively. Using [125I]iodopindolol as a receptor probe, we found the order of potency for adrenergic antagonists to be l-propranolol greater than oxprenolol much much greater than practolol. Serotonergic antagonists, although effective in displacing [125I]iodopindolol, did so only at very high concentrations. These results are consistent with the existence of separate and discrete D-serotonergic and beta 2-adrenergic receptors in skeletal muscle and the finding that adrenergic agonists and antagonists may interact with low affinities with the serotonergic receptor, but that serotonergic agonists and antagonists interact poorly, if at all, with the beta 2-adrenergic receptor.

Animals↗
PubMed1983

Anion effects on in vitro sarcoplasmic reticulum function. Co-transport of anions with calcium.

In isolated sarcoplasmic reticulum vesicles, calcium-chelating but non-calcium-precipitating dicarboxylates, such as maleate and succinate, stimulated ATP-dependent Ca2+ accumulation and its ensuring spontaneous Ca2+ accumulation and its ensuring spontaneous Ca2+ release, and Ca2+-dependent ATPase activity (Chu, A., Tate, C. A., Bick, R. J., Van Winkle, W. B., and Entman, M. L. (1983) J. Biol. Chem. 258, 1656-1664). We further examined the effect of dicarboxylates on enzyme turnover. The anionic buffer maleate enhanced the rate of rapid acyl phosphoenzyme hydrolysis compared to that in the zwitterionic buffer piperazine-N,N'-bis(2-ethanesulfonic acid) but had no effect on the phosphoenzyme formation. The presence of a calcium-precipitating anion, oxalate, or a Ca2+ ionophore, A23187, eliminated the differences observed in the phosphoenzyme decay between the two buffers, but accelerated the rate of decay. Furthermore, the catalytic activity of the purified Ca2+-dependent ATPase was not affected by maleate, whether oxalate was present or not. [14C]Succinate was transported into the sarcoplasmic reticulum in a manner which was dependent on Ca2+ transport, and occurred over a similar time course as Ca2+ accumulation/release. The net succinate uptake was equivalent to the amount of succinate-stimulated Ca2+ accumulation. Rapid efflux of both [14C]succinate and 45Ca2+ was induced by A23187, whereas the efflux induced by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid was slower and less compared to A23187. Succinate accumulation exhibited saturation kinetics with positive cooperativity (Km congruent to 20 mM; Hill coefficient = 1.70). When maleate and succinate were both present, they were equipotent, and had an additive stimulatory effect on peak 45Ca2+ accumulation at low concentrations. Maleate was a competitive inhibitor of succinate accumulation (Ki approximately equal to 17 mM; Hill coefficient = 1.75). KCl in the presence or absence of valinomycin did not influence succinate accumulation or release. The data suggest that succinate accumulation is Ca2+-dependent, but occurs at a saturable, divalent, anion-specific site. While this carrier or channel requires Ca2+ transport, it may be controlled by additional factors as well.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Nucleotide triphosphate utilization by cardiac and skeletal muscle sarcoplasmic reticulum. Further evidence for an alternative substrate hydrolysis cycle and the effect of calcium NTPase purification.

It was previously observed that the hydrolysis of GTP by cardiac sarcoplasmic reticulum (SR) (in contrast to skeletal muscle SR: (a) was identical in rate with total ATP hydrolysis; (b) gave a similar nonlinear substrate response; (c) was not Ca2+ dependent; and (d) stimulated Ca2+ accumulation but not Ca2+ translocation. Evidence was presented that both the ATPase and GTPase are effected by the same enzyme and represent different hydrolysis cycles (Van Winkle, W. B., Tate, C. A., Bick, R. J., and Entman, M. L. (1981) J. Biol. Chem. 256, 2268-2274). In the present paper, we report that purification of the NTPase from both muscle sources resulted in an alteration in the NTP concentration response compatible with a single high affinity binding site for ATP only in cardiac SR and for both substrates in skeletal muscle SR. As is the case with native skeletal muscle SR, purified skeletal muscle NTPase hydrolyzed GTP in a manner qualitatively similar to ATP (but with no Ca2+-independent NTPase) but with reduced velocity. In contrast, there was no GTPase activity or Ca2+-independent "basic" ATPase activity in the purified cardiac NTPase. Inclusion of oxalate or the ionophore, A23187, in assays with cardiac SR and ATP as the substrate increased the total ATPase activity but had no effect on GTPase activity. Furthermore, the nucleotide-dependent uptake of oxalate by cardiac SR was only apparent with ATP and not with GTP. In the presence of Ca2+, ATP was a potent inhibitor (noncompetitive, Ki of 2-5 microM) of GTPase activity, whereas it was a weaker competitive inhibitor in the absence of Ca2+. We suggest that GTPase and basic ATPase represent similar alternative enzyme cycles for the CaATPase enzyme that are inhibited by the presence of ATP plus Ca2+ but are rendered inactive during the purification of cardiac NTPase.

Adenosine Triphosphate↗
PubMed1983

Alteration of sarcoplasmic reticulum after denervation of chicken pectoralis muscle.

To determine the neural influence on the function of the sarcoplasmic reticulum (SR) of fast-twitch skeletal muscle, the superior pectoralis muscle of adult chicken was denervated, and the SR was isolated at 20 days post-denervation. The isolated SR was probably derived from the longitudinal SR and was relatively free of contaminants. The protein profile of the SR was quantitatively changed after denervation with an increase in the M55 and 30000-mol.wt. proteins relative to the Ca2+-ATPase. Ca2+-dependent ATPase activity and phosphoenzyme formation were lower in the denervated-muscle SR; however, the enzyme catalytic-centre activity was similar to the control value. The decrease in Ca2+-ATPase activity in denervated-muscle SR was accompanied by a lower Ca2+ accumulation so that the relationship between Ca2+ accumulation and Ca2+-dependent ATPase activity was well maintained in the SR from denervated muscle. The data imply that denervation may result in a diminution of functional Ca2+ pump sites. Evidence is presented, though, which suggests that denervation affects a single class of Ca2+-binding sites of the Ca2+-ATPase, resulting in a lower affinity for Ca2+.

Adenosine Triphosphatases↗
PubMed1983

Anion effects on in vitro sarcoplasmic reticulum function. The relationship between anions and calcium flux.

Isolated sarcoplasmic reticulum vesicles exhibited different functional characteristics in the presence of zwitterionic as compared to anionic buffers. In the absence of oxalate, dicarboxylic anions (e.g. maleate, succinate) in a dose-dependent manner enhanced ATP-supported Ca2+ accumulation, the ensuing spontaneous Ca2+ release, and Ca2+-dependent ATPase activity compared to zwitterionic buffers (e.g. piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Hepes). This was not attributed to ionic strength and osmotic effects. The additional anion-dependent Ca2+ accumulation was linked to augmented Ca2+-dependent ATPase activity, and both could be induced by the addition of anion at any time during Ca2+ accumulation as long as ATP was present. Since the initial Ca2+ accumulation rates and acyl phosphoenzyme formation were the same between the two buffer classes, and the presence of either oxalate (a Ca2+-precipitating anion) or A23187 (a Ca2+ ionophore) abolished differences in Ca2+-dependent ATPase activity between the two buffer classes, it is likely that conditions favoring high intravesicular Ca2+ concentration allow the expression of the observed effect of the anions. Initial spontaneous Ca2+ release in the presence of maleate was not caused by ATP depletion, and it was virtually absent in Pipes buffer. The rate of spontaneous release was also stimulated in a dose-dependent manner by the dicarboxylic anions, with the time of release being related to the time of anion addition and not ATP addition. A later, more rapid release phase in either maleate or Pipes buffer corresponded to ATP depletion, and could be duplicated at any time in the Ca2+ accumulation/release cycle by the addition of an ATP trap. With an ATP-regenerating system present or with very high ATP concentrations, the maximal peak Ca2+ accumulation in Pipes buffer could approach that in maleate buffer. The data suggest that dicarboxylic anions stimulate the filling of a Ca2+ compartment from which spontaneous Ca2+ release occurs.

Animals↗
PubMed1983

Psychological stress activates phosphorylase in the heart of the conscious pig without increasing heart rate and blood pressure.

The present study uses a technique that enables the collection of multiple freeze-biopsy samples from the myocardium of the conscious pig (i.e., through a thoracic window). This technique enables sequential analysis of the metabolic state of the myocardium during different behavioral conditions. The results demonstrate that with daily adaptations to an unfamiliar environment (i.e., stress reduction), the phosphorylase activation ratio (phosphorylase a/total phosphorylase) in the quiescent pig declines steadily from approximately 80% to 30% (r = -0.91, P less than 0.01). This decline occurs with both the mean resting heart rate and left ventricular blood pressure remaining constant. The decline is seen within individual subjects during the whole adaptation sequence as well as between subjects whose samples were taken either early or late in the adaptation series. The dissociation of hemodynamic functional and metabolic activation in the unadapted, psychologically stressed pig may be associated with the occurrence of increased vulnerability of the ischemic heart to ventricular fibrillation, a phenomenon previously observed under the same behavioral conditions.

Animals↗
PubMed1983

Doppler measurement of myocardial thickening with a single epicardial transducer.

To eliminate the need for intramyocardial transducers in measuring regional left ventricular (LV) function we have developed a pulsed Doppler technique utilizing a single epicardial transducer. Wall thickening is evaluated by digitally integrating the velocity of myocardial layers passing through the sample volume located at a selected depth. Thickening fraction (TF) can then be estimated by dividing the systolic excursion by the sample volume depth. The Doppler method was compared with the transit-time method in three acute dogs by placing the 4-mm-diameter epicardial Doppler transducer over a 2-mm-diameter endocardial crystal tunneled through the LV wall. With the sample volume set to 1 mm less than the minimum LV thickness, simultaneous measurements of TF by the Doppler and transit-time methods showed good agreement (r = 0.95) during control, ischemia, volume overload, shock, and anoxia. In 28 chronically instrumented piglets signals were obtained for longer periods with Doppler transducers than with transit-time segment-length crystals. We conclude that the Doppler technique provides a high-fidelity wall thickening waveform and a good estimate of TF with minimal disturbance to the ventricle and that the technique is suitable for use in both acute and chronically instrumented animals.

Animals↗