Lawrence A. Menahan

Diltiazem in myocardial recovery from global ischemia and reperfusion

Abstract Effects of diltiazem on global myocardial ischemia and reperfusion have been examined in the isolated perfused guinea-pig heart. Diltiazem (8 × 10−7 m to 2.5 × 10−5 m ) produced negative inotropic effects in nonischemic hearts. Hearts treated with diltiazem during low-flow ischemia which lasted 45 min followed by a 30 min reperfusion period showed significantly greater recovery of contractility. However, left ventricular end diastolic pressure remained elevated when compared to preischemic measurements. Diltiazem treatment resulted in significant amelioration of the increased wet wt/dry wt ratio and increased ATP and creatine phosphate levels during reperfusion. However, these parameters remained altered compared to nonischemic hearts. Pyruvate dehydrogenase in its active form (PDHa) was significantly decreased by ischemia. Diltiazem treatment partially alleviated the decrease in PDHa. These results suggest that diltiazem provides significant protection of myocardial function during ischemia and reperfusion.

Comparison of carbohydrate and lipid metabolism in mice and rats during fasting.

1. Swiss albino mice and Sprague-Dawley derived rats, 6-8 weeks in age were compared in the fed condition and after a fast of 24 and 48 hr. 2. Plasma glucose was significantly higher in mice than rats in the fed state but this difference disappeared after the 48 hr fast. 3. In the fed condition, plasma FFA and glycerol levels were significantly higher in mice than rats yet did not change during fasting. With starvation, there was a progressive increase in plasma FFA in the rat but no change in plasma glycerol. 4. The content of triacylglycerols in the plasma and liver was comparable in mice and rats in the fed state. Plasma triacylglycerols declined to a similar extent in both species during a fast of 48 hr. In mice, hepatic triacylglycerols increased markedly during fasting but declined in rats. 5. Heart triacylglycerols were higher in mice than rats in the fed state and increased still further during a fast of 48 hr during which time little change was observed in the rat. Myocardial glycogen was comparable in mice and rats in the fed state. With fasting, cardiac glycogen content decreased in the mouse but remained unchanged in the rat. 6. Distinct differences in certain of the parameters of carbohydrate and lipid metabolism were found with mice and rats in the fed and fasted condition.

Subcellular localization of marker enzymes, lipase and triglyceride in rat heart.

Abstract Subcellular fractions containing lipase and triglyceride, prepared from rat hearts homogenized in 0.25 m sucrose, were characterized by the use of marker enzymes. In addition to the usual mitochondrial and microsomal pellets, an additional fraction was sedimented between 8000 g × 10 min and 13 000 g × 10 min. Assays for N -acetyl-β-glucosaminidase, β-glucuronidase and cathepsin D indicated that the relative specific activity (R.S.A.) of these enzymes in this fraction was two to three times higher than any of the other subcellular fractions. Since these hydrolytic enzymes are “marker” enzymes for lysosomes in other tissues, the results indicate that it is possible to separate rat heart homogenate into several fractions, one of which has the characteristics of the light mitochondria or lysosomes. Several other hydrolytic enzymes (acid phosphatase, α-glucosidase and β-galactosidase) yielded R.S.A. values which indicated that there was no enrichment of their activity in the lysosomal fraction. The R.S.A. patterns of 5′-nucleotidase and alkaline phosphatase indicated that the plasma membranes (sarcolemma) were sedimenting with the microsomal fraction. This latter fraction exhibited a high R.S.A. of rotenone-insensitive NADH cytochrome- c reductase. Measurement of lipase activity and triglyceride content of the subcellular fractions indicated a relative enrichment of both of these in the lysosomal fraction. Although the R.S.A. of lipase was high in both the lysosomal and microsomal fractions, addition of serum in the assay reduced the R.S.A. of the lipase in the microsomal fraction without altering the high R.S.A. of the lysosomal pellet. Thus, a fraction has been separated from rat heart homogenates with lysosomal characteristics. Associated with this fraction was also an enrichment of triglyceride content and lipase activity, which may be important for turnover of the former in the heart cell.

Calcium dependency of hormone stimulated lipolysis in the perfused rat heart.

Drug and hormone stimulated triglyceride mobilization in the Langendorff perfused rat heart in vitro was estimated by an automated assay of perfusate glycerol. The disappearance of heart triglyceride-glycerol during perfusion of hearts with isoproterenol in Krebs-Henseleit buffer (5.5 m m glucose, 2.5 m m Ca 2+ ) agreed closely with the perfusate glycerol output. At perfusate [Ca 2+ ] of 1.2 m m or below, bolus injections of isoproterenol and glucagon failed to increase glycerol output over baseline. Raising the perfusate [Ca 2+ ] from 1.2 m m to 1.8 m m resulted in a restoration of the glycerol response following injection of isoproterenol or glucagon. Determination of protein kinase activity ratios after injection of lipolytic doses of glucagon and isoproterenol demonstrated that these agents were able to elevate the protein kinase activity ratio at all [Ca 2+ ] tested, including those at which an increase in glycerol release was not observed. These results suggest that an increase in cyclic AMP dependent protein kinase activity, following injection of glucagon or isoproterenol, does not lead to an increase in heart triglyceride mobilization unless a threshold perfusate [Ca 2+ ] is present.

Inhibition of hormonal stimulation of lipolysis in perfused rat heart by ketone bodies

Abstract The interaction of ketone bodies, acetate and octanoate with isoproterenol-stimulated lipolysis (glycerol output) was studied in the Langendorff perfused rat heart. Increasing perfusate concentrations of β-hydroxybutyrate or acetoacetate progressively and completely inhibited the isoproterenol-augmented glycerol output of the perfused rat heart. Likewise, acetate and octanoate suppressed the lipolytic response to isoproterenol but the maximal inhibition by these latter compounds amounted to approximately 50% of the isoproterenol effect. Isoproterenol elevated the protein kinase activity ratio, an index of heart cyclic AMP, at all concentrations of ketone bodies studied, including those at which a complete inhibition of the isoproterenol-stimulated glycerol release occurred. Thus, ketone bodies have the ability to inhibit isoproterenol-stimulated lipolysis in the isolated perfused rat heart even in the presence of elevated protein kinase ratios.

Effect of insulin in vitro on the isolated, perfused alloxan-diabetic rat liver

SummaryWithdrawal of exogenous insulin and a subsequent fast (24 h) of alloxan diabetic rats stimulated rates of gluconeogenesis, ureogenesis, ketogenesis, and amino acid release by in situ perfused livers when compared to those from normal, fasted rats. The contribution of liver glycogen to the high rates of gluconeogenesis observed with the diabetic liver could be excluded. Perfusate lactate concentrations remained constant during the period when the elevated rate of gluconeogenesis was observed with diabetic liver. Addition of insulin as a bolus (750 mU) and continuous infusion (12.5 mU/min) to the perfusion medium of diabetic livers resulted in constant perfusate levels of glucose, urea and α-amino nitrogen indicating a suppression of the catabolic processes present in the fasted, diabetic liver. The rate of ketogenesis was also slowed by insulin to about half the rate prior to addition of the hormone. These data indicate that insulin has an immediate anti-catabolic effect in the perfused, diabetic liver.

Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

Abstract The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.

A comparison of direct enzymatic determination of glycogen in liver and heart

Abstract Several enzymatic methods for the measurement of glycogen have been developed including degradation by diazyme (1) or amylo-α-1,4-α-1,6-glucosidase (2–6) followed by determination of glucose with glucose oxidase (1–3,6) or hexokinase and glucose-6-P- dehydrogenase (4,5). Bueding and Hawkins (7) developed a method for the microdetermination of glycogen using phosphorylase and amylo-α-1,4-α-1,6-glucosidase which was later modified so that commercially available enzymes: phosphrylase a, P - glucomutase, and glucose-6-P-dehydrogenase could be used (8). In determining the glycogen content of tissue samples, homogenization in perchloric acid (4,5) or heating tissue in 0.03 n HCl (9) have been included prior to theenzymatic analysis. Yet, it has also been reported that glycogen can be deermined directly in very dilute homogenates of liver or muscle in water (3). Likewise, direct enzymatic procedure for the detemrination of glycogen in homogenates of liver in water by degrading glycogen to glucose by treament with amylo-α-1,4-α-1,6-glucosidase has yielded higher values for glycogen content than those for tissue digestion with KOH, precipitation of glycogen with ethanol, and finally acid hydrolysis (6). In the present report, the feasibility direct enzymatic determination of glycogen in homogenates of liver with amylo-α-1,4-α-1,6-glucosidase has been reaffirmed. However, glycogen content of heart tissue determined by the same enzymatic procedure directly in homogenates in water was substantially lower than that obtained by extraction with KOH, glycogen precipitation with ethanol, and then enzymatic analysis.

Delineation of membrane-bound phosphatase activities in normal and leukemic thymocytes

Abstract The activities of the various phosphatases on the plasma membrane of normal and leukemic mouse thymocytes were delineated using the specific inhibitors, α, β-methylene ADP, which inhibits 5′-nucleotidase while not affecting nonspecific phosphatases, (−)-p-bromotetramisole, which inhibits nonspecific alkaline phosphatase, and the competitive substrate p-nitrophenylphosphate. The Km for AMP and the Ki for the inhibition of AMPase by α, β-methylene ADP at pH 7.5 were 13 and 38 μM, respectively. The activity also shows substrate inhibition at this pH. 30% of the AMPase activity could not be inhibited, and an Eadie-Hofstee plot shows mixed inhibition. Inability of the combined use of α, β-methylene ADP and (−)-p-bromotetramisole to totally suppress activity indicates the presence of a third phosphatase enzyme. Subsequent analysis at various pH values indicates that this third enzyme has an activity peak in the acid range. Using β-glycerophosphate as a substrate, 81% of the activity at pH 9.5 is due to alkaline phosphatase and none is due to 5′-nucleotidase. Likewise virtually none of the β-glycerophosphatase activity at pH 7.5 is due to alkaline phosphatase. With both Swiss albino and AKR mice, activity of 5′-nucleotidase increased markedly in adult male mice when compared to younger immature animals whereas alkaline phosphatase showed little change. In leukemic AKR mice, 5′-nucleotidase activity was sharply lower in the cells of thymomas when compared to nonleukemic AKR mice of comparable age. Alkaline phosphatase activity in the thymus of leukemic AKR mice was either increased markedly or remained unchanged. Thus the specificity of three distinct phosphatase activities in thymocytes are clearly defined and monitored during developmental and pathological alterations of the cell: (1) 5′-nucleotidase, (2) alkaline phosphatase, and (3) a third phosphatase with a lower pH optimum.

A radioisotopic method for the determination of acetoacetyl coenzyme a with 3-hydroxy-3-methylglutaryl coenzyme a synthase

Abstract A simple and sensitive assay for the quantitative determination of acetoacetyl-CoA (AcAc-CoA) in liver and heart is described. The method is based on incorporation of [ 14 C]acetyl-CoA into acid-stable nonvolatile material in the presence of avian HMG-CoA synthase. The specificity of this procedure for the measurement of AcAc-CoA was demonstrated by pretreating tissue extracts with 3-hydroxyacyl-CoA dehydrogenase or CoA transferase from Escherichia coli to deplete. AcAc-CoA prior to assay. Acid-stable nonvolatile 14 C activity measured in the assay was proportional to the amount of tissue extract added. Satisfactory recovery of AcAc-CoA added at the initial extraction step further validated this procedure. This radioactive assay for acetoacetyl-CoA using a highly purified avian 3-hydroxy-3-methylglutaryl-CoA synthase has the advantages of both extreme specificity for AcAc-CoA as substrate and high sensitivity, facilitating the determination of this metabolite under a variety of physiological conditions.