Willem C. Hülsmann

Inhibition of (Na++K+)-stimulated ATPase of heart by fatty acids

(Na++K+)-stimulated ATPase of a plasma membrane fraction from guinea-pig heart is inhibited by fatty acids (octanoate and oleate tested). This inhibition is also observed when ATP is replaced by p-nitrophenylphosphate. The inhibition by fatty acids is competitive with K+. These results are in agreement with the earlier experiments conducted by Ahmed and Thomas [1] on brain microsomes. It is suggested that inhibition of (Na++K+)-stimulated ATPase may contribute to the arrhythmogenic action of high levels of fatty acids observed under pathological conditions.

Metabolic aspects of isolated cells from rat small intestinal epithelium

Abstract 1. 1. Epithelial cells from the small intestine of the rat were isolated according to mechanical procedures as described by S jostrand 1 and H arrison and W ebster 2,3, respectively. The two methods were compared with regard to yield and integrity of the cells obtained. 2. 2. While the cells in the suspension prepared by the method of S jostrand showed signs of appreciable morphological and biochemical damage, the cells harvested according to the principle of H arrison and W ebster appeared essentially intact and displayed a glyocolytic activity, in the absence of added cofactors, of the same order as intestinal mucosa in situ. 3. 3. The cells isolated by the second method were homogenized and the homogenate subjected to differential centrifugation. A mitochondrial preparation was obtained which showed intact oxidative phosphorylation. The stimulation of respiration by the addition of ADP was small. The mitochondrial preparation had ATPase activity, stimulated by Mg2+ or 2,4-dinitrophenol. 4. 4. Analysis of adenine nucleotide concentrations in intestinal mucosa revealed a high AMP+ADP to ATP concentration ratio. This finding could explain the high rate of aerobic glycolysis of rat small intestinal epithelium.

The effect of lipid intermediates on Ca2+ and Na+ permeability and (Na+ + K+)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia

The effect of fatty acid and acylcarnitine on Ca2+ and Na+ transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na+ gradient-induced Ca2+ uptake. Half-maximal reduction was obtained at 15 microM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca2+ when added to Ca2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca2+. A rapid release of Ca2+ was also observed if Ca2+ was taken up by action of the Ca2+ pump. The (Ca2+ + Mg2+)-ATPase, which most likely drives this active Ca2+ uptake, was 90% increased by 50 microM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 microM did not affect the ouabain-sensitive (Na+ + K+) -ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca2+ release and to inhibit (Na+ + K+)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 microM) or oleate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na+ + K+)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.

Carnitine requirement of vascular endothelial and smooth muscle cells in imminent ischemia

Vascular endothelial and -smooth muscle cells have been shown to use fatty acids as substrates for oxidative phosphorylation. Endothelial cells are more vulnerable to oxidative stress than muscle cells and are prone to loose carnitine early during hypoperfusion. This has been suggested by two observations. The first is that incubation of isolated endothelial cells in a low carnitine medium leads to oleate oxidation, dependent upon carnitine addition, whereas smooth muscle cells do not depend on carnitine addition duringin vitro incubation, although aminocarnitine, a specific inner-membrane carnitine palmitoyltransferase inhibitor, inhibits fatty acid oxidation. The second observation is that rat hearts labeledin vivo with14C-carnitine loose, as paced Langendorff heart, only 4% of their carnitine in 20 min perfusion, following 60 min global ischemia. The carnitine released had a much higher specific radioactivity than the carnitine that was not released. It indicates compartmentation of carnitine in heart. As earlier and presently discussed work shows endothelial vulnerability, it is to be expected that this cell type may become carnitine deficient during pacing and ischemia. Endothelial incompetence in flow regulation could be delaved by the presence of carnitine and fatty acids in pre-ischemia. It is speculated how activated fatty acids could protect endothelium.

Cyclic nucleotide-, pyruvate- and hormone-induced changes in pyruvate kinase activity in isolated rat hepatocytes

Summary Incubation of isolated rat hepatocytes with glucagon (10−6 M), db-cAMP (0.1 mM) and db-cGMP (0.1 mM) causes a decrease in pyruvate kinase activity of 46, 49 and 34% respectively, when measured at 1 mM Mg2+free and suboptimal substrate (P-enolpyruvate) concentrations, while the Vmax is uninfluenced. An increase in activity (25%) is noticed when the cells are incubated with 1 mM pyruvate. The glucagon inactivated enzyme (Lb) shows a decreased affinity for the substrate P-enolpyruvate and for the allosteric activator Fru-1,6-P2 as compared to the activated form (La). The nature of the hormone and cyclic nucleotide-induced changes in pyruvate kinase is discussed. It is concluded that the P-enolpyruvate cycle is under comparable acute hormonal control as the FDPase-PFK cycle. Both cycles are linked by the common effector Fru-1,6-P2 making not only direct but also indirect hormonal control of pyruvate kinase flux possible.

Lipoprotein lipase from heart and liver: An immunological study

Summary Lipoprotein lipase activity was purified from rat postheparin serum. An antibody against this activity was raised. It was found that this antibody inhibits 95% of the postheparin lipoprotein lipase activity. The same inhibition was found in the perfusate of heparin-perfused rat liver. When measuring lipoprotein lipase activity in perfusates of rat hearts, perfused with heparin, no inhibition by this antibody was found. It is concluded that lipoprotein lipase activities from heart and liver are catalysed by different enzymes and that the heart enzyme contributes not more than 5% to the overall lipase activity in postheparin serum.

Effects of hormones, amino acids and specific inhibitors on rat heart heparin-releasable lipoprotein lipase and tissue neutral lipase activities during long-term perfusion

Rat hearts were perfused for long periods in the presence of 14C-labeled amino acids. From these hearts, postheparin-effluent and a tissue homogenate containing lipoprotein lipase and neutral lipase, respectively, were derived. Lipolytic activity and 14C-labeled protein in both preparations were characterized by affinity chromatography, immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Lipase activity and 14C-labeled protein co-eluted from heparin-Sepharose 4B at 1.2 M NaCl and were inhibited and precipitated by preincubation with anti-lipoprotein lipase gamma-globulins. Gel electrophoresis of both preparations showed the presence of 14C-labeled protein with a molecular weight of 35 000. These data strongly suggest similarity between lipoprotein lipase and neutral lipase and their possible precursor-product relationship and indicate that during perfusion continuous synthesis, secretion and vascular binding of lipase molecules occur. Cycloheximide perfusion induced a dramatic decrease of lipoprotein lipase and neutral lipase activity, indicating a half-life of less than 90 min for both enzymes. Tunicamycin present during perfusion also induced a drop in lipoprotein lipase and tissue neutral lipase activity, indicating that glycosylation is necessary for secretion of lipoprotein lipase. Long-term perfusion of rat hearts in the presence of norepinephrine, glucagon or tyrosine leads to reciprocal alterations in lipoprotein lipase and neutral lipase activities, i.e., lipoprotein lipase activity increased and neutral lipase activity decreased, whereas total lipase activity (lipoprotein lipase + neutral lipase) remained unaltered. During perfusion in the presence of insulin, no net change in lipase activities was observed. Also, insulin did not affect the glucagon-induced inverse effects on either lipase activity. The reciprocal changes in lipase activities occurring during norepinephrine perfusion were hampered by colchicine and propranolol, pointing towards beta-receptor and microtubular mediation of tissue lipase processing and endothelial binding. Our data suggest that the tissue flux and vascular binding of lipase protein may be important sites of hormonal regulation of lipoprotein lipase homeostasis.

Erucic acid-induced alteration of cardiac triglyceride hydrolysis

Male Wistar rats were fed for 3 or 10 days with high erucic acid rapeseed oil (HEAR) or trierucate (TE). These diets produced increased myocardial triglyceride (TG) levels. Cardiac lipid accumulation was related to basal-and hormone- (glucagon, norepinephrine) stimulated lipolysis, determined as glycerol release, which proved to be enhanced in isolated, perfused hearts from HEAR- and TE-fed rats. Endogenous TG levels in isolated hearts from rats fed the stock and the sunflowerseed oil (SSO) diet were low and probably rate-limiting for tissue lipolytic activities. HEAR feeding of rats did not modify the rate of erucic acid (22∶1) oxidation in heart. Prolonged HEAR and TE feeding led to a decrease in the endogenous TG level, a process in which the increased rate of TG hydrolysis might play an important role. The enhanced breakdown of tissue TG in hearts from TE-and HEAR-fed rats was accompanied by an increased release of fatty acids into the coronary effluent. Erucic acid was a major constituent of the perfusate fatty acids. Evidence is presented that the site of the intracellular TG breakdown is associated with lysosomes, since a subcellular fraction enriched in acid lipase, N-acetyl-β-glucosaminidase and TG could be isolated from heart homogenates of TE-fed rats. Fatty acids seemed to be an important regulator of tissue lipase activity: palmitate inhibited glucagon-stimulated lipolysis, which suggests the tissue lipase is subject to product inhibition by fatty acids.

Effect of fasting and streptozotocin-diabetes on the coronary flow in isolated rat hearts: A possible role of endogenous catecholamines and prostaglandins

SummaryThe coronary flow rate of retrogradely perfused hearts from fasted (group I) and streptozotocin-diabetic rats (group II) is increased when compared with the flow rate of control, fed animals (group III). The enhanced coronary flow is absent when hearts of groups I and II are perfused in the presence of indomethacin (1 μg/ml) in the perfusion fluid and the lowest flow rates are observed after depletion of the endogenous catecholamines by reserpin. Hearts from groups I and II showed a marked increase in prostaglandin-release which was counteracted both in the presence of indomethacin (1 ⧎g/ml) and by reserpinpretreatment. The results suggest that the increased coronary flow rates in hearts from fasted and streptozotocin- diabetic rats are mediated by an effect of released endogenous catecholamines on coronary vascular smooth muscle and by a catecholamine-induced release of vasodilatory, prostaglandin-like substances.ZusammenfassungDie koronare Durchströmung der retrograd perfundierten Herzen der gehungerten (Gruppe I) und Streptozotocin-diabetischen Ratten (Gruppe II) ist erhöht im Vergleich mit der koronaren Durchströmung von Kontrolltieren (Gruppe III). Zunahme der Durchströmung ist nicht vorhanden, wenn mit Indomethacin (1 μg/ml) Herzen aus Gruppen I und II perfundiert werden Die niedrigste Durchströmungsgeschwindigkeit wird gemessen, wenn die Tiere mit Reserpin vorbehandelt werden. Herzen aus Gruppen I und II zeigten eine bedeutende Zunahme der Freisetzung von Prostaglandinen, welche gehemmt wurde, wenn Indomethacin hinzugefügt wurde (1 μg/ml) oder nach Vorbehandlung mit Reserpin. Die Ergebnisse suggerieren daß die erhöhte koronare Durchströmung des Herzens gehungerter oder Streptozotocin-diabetischer Ratten veranstaltet wird von dem Effekt freigesetzter, endogener Katecholamine auf glatten, lonoraren Gefäßmuskeln und von einer katecholamin-induzierten Freisetzung gefäßerweiternder, prostaglandinartiger Substanzen.

Pitfalls in histochemical localization studies of NADPH generating enzymes or enzyme systems in rat small intestine

SummaryIn the presence of excess β-glycerophosphate or p-nitrophenylphosphate NADPH diaphorase of the epithelial cell in rat small intestine has its highest activity in the villi at the basis of the microvilli, where smooth endoplasmic reticulum of the cells is present. In the absence of β-glycerophosphate or p-nitrophenylphosphate the diaphorase activity is much higher and broader localized in crypts and villi. The increased activity is due to the conversion of NADPH to NADH by non-specific (mostly alkaline-) phosphatase activity, so that both NADPH and NADH diaphorase activities are measured.When NADPH is generated by a specific NADP+-linked dehydrogenase, such as glucose-6-phosphate dehydrogenase, and nitroblue tetrazolium is present to trap the reducing equivalents formed, the histochemical localization of the dehydrogenase is identical to that of NADPH diaphorase, although the dehydrogenase may be present in another cell compartment (glucose-6-phosphate dehydrogenase for instance has a cytosolic distribution). Therefore the localization of a dehydrogenase may be falsely interpreted histochemically, according to the diaphorase reaction involved. A different localization may be obtained when the diaphorase reaction is circumvented by the addition of an alternative hydrogen carrier, such as phenazine methosulphate. Also in coupled dehydrogenase assays this may be observed. The hexokinase reaction, coupled to the glucose-6-phosphate dehydrogenase reaction in the presence of nitroblue tetrazolium and the absence of phenazine methosulphate, has a distribution identical to NADPH diaphorase. In the presence of phenazine methosulphate the enzyme has an almost ubiquitous distribution in the small intestinal epithelial cell. When a substrate may react both with NADP+- and NAD+-linked dehydrogenases, such as L-malate: NADP oxido-reductase (decarboxylating) and L-malate: NAD oxidoreductase respectively, the high activity of intestinal alkaline phosphatase may influence the histochemical distribution by converting part of NADP+ to NAD+. The addition of another phosphate ester, such a β-glycerophosphate or p-nitrophenylphosphate, may therefore influence the observed distribution.