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Biomedical subjects

J B Hoek

Publications and source records attributed to J B Hoek.

At least 91 records · Page 5Linked to original sources
PubMed1988

Ethanol stimulates shape change in human platelets by activation of phosphoinositide-specific phospholipase C.

Administration of ethanol to human platelets resulted in a rapid shape change which was maximal within 30 s. Ethanol did not cause aggregation or secretion of ATP at any time and inhibited aggregation induced by collagen. In platelets that were loaded with the intracellular calcium indicator fura2, ethanol induced a rapid mobilization of calcium from internal, thrombin-sensitive pools. Cytosolic calcium increased to a maximum within 5 s and decreased slowly over the ensuing 5 min to near basal levels. The mobilization of calcium by ethanol coincided with the rapid formation of phosphatidic acid and a decrease in the level of phosphatidylinositol 4,5-bisphosphate, as measured in 32P-labeled platelets. In platelets labeled with myo-[2-3H]inositol, ethanol caused a 20-30% increase in the levels of inositol (1,4,5)-trisphosphate and inositol bisphosphate within 10 s. Ethanol also induced the transient phosphorylation of myosin light chain (20 kDa) and a 40 kDa protein, a known substrate for protein kinase C. The results indicate that ethanol activates phosphoinositide-specific phospholipase C in human platelets. The subsequent mobilization of intracellular calcium and activation of protein kinase C can account for the shape change induced by ethanol.

Adult↗
PubMed1987

Ethanol-induced mobilization of calcium by activation of phosphoinositide-specific phospholipase C in intact hepatocytes.

The short-term effects of ethanol on calcium homeostasis were studied in isolated hepatocytes. Ethanol caused a rapid transient activation of phosphorylase not associated with changes in cAMP levels which peaked after 20-30 s and declined slowly over a period of 5-10 min. Maximal activation was found with 200 mM ethanol, and a significant effect was observed at 25 mM ethanol. Similar effects were induced by other organic solvents and by halothane, with more hydrophobic agents being effective at lower concentrations. In hepatocytes loaded with the intracellular calcium indicator quin2, the addition of ethanol caused a transient increase in cytosolic free calcium, with a kinetic pattern compatible with its involvement in the activation of phosphorylase. Pretreatment of the hepatocytes with phenylephrine or vasopressin to deplete the hormone-sensitive calcium pools in the cells prevented the ethanol-induced calcium mobilization. In 32P-labeled hepatocytes addition of ethanol caused a small (5-7%) decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate and a 10-15% increase in [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidic acid. In hepatocytes labeled with myo-[3H]inositol, ethanol induced a 50-100% increase in the levels of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol bisphosphate. The changes in the inositol 1,4,5-trisphosphate level due to ethanol paralleled the time course of the elevation of cytosolic free calcium levels and activation of phosphorylase a. The effects of ethanol were comparable to those of a physiologic (1 nM) dose of vasopressin; however, unlike with vasopressin, the inositol phosphates and cytosolic calcium levels declined to basal levels 2 min after the addition of ethanol. These results indicate that ethanol, in common with calcium-mobilizing hormones, activates hormone-sensitive phosphoinositide-specific phospholipase C. The resulting changes in inositol 1,4,5-trisphosphate can account for the mobilization of intracellular calcium and the consequent activation of phosphorylase by ethanol.

Animals↗
PubMed1987

Effect of ethanol on amylase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol-fed rats.

The effects of ethanol on stimulus-secretion coupling were assessed by studying amylase release, Ca2+-homeostasis, and changes in physical properties of membranes in isolated rat pancreatic acini. In acini from normal rats, ethanol (50 mM and above) in vitro caused a dose-dependent stimulation of amylase release and an increase in cytosolic free Ca2+ concentration. Ethanol did not affect amylase secretion stimulated by cholecystokinin-octapeptide (CCK8), a secretagogue that acts by increasing cytosolic free Ca2+ levels, but did potentiate the secretion of amylase induced by vasoactive intestinal peptide (VIP) which raises intracellular cAMP. Ethanol also increased the rate of 45Ca2+ exchange. In acini labeled with the spin-probe 12-doxyl stearic acid, ethanol disordered the pancreatic plasma membranes. By contrast, in acini from animals that had chronically (6-7 weeks) ingested ethanol, the membranes were resistant to this disordering effect of ethanol. Chronic ethanol feeding lowered the total cellular calcium content and ionophore (A23187)-releasable pools of acinar calcium (11 and 24% respectively), and led to a 15-30% decrease in the rate of 45Ca2+ exchange. Chronic ethanol ingestion also lowered the basal rate of amylase secretion, but ethanol in vitro stimulated amylase secretion more than in control preparations. However, these differences in basal and ethanol-induced amylase secretion were not accompanied by corresponding changes in intracellular free Ca2+. The data suggest that ethanol perturbs cell membranes and also disturbs cellular Ca2+ homeostasis. These effects may explain its actions as a weak Ca2+-mediated secretagogue. However, the membrane alterations induced by chronic ethanol feeding do not prevent the ethanol-induced interference with cellular calcium homeostasis.

Amylases↗
PubMed1987

Ethanol does not stimulate guanine nucleotide-induced activation of phospholipase C in permeabilized hepatocytes.

Guanine nucleotides are thought to mediate the interaction of the receptors for calcium-mobilizing hormones and phosphoinositide-specific phospholipase C. In the present study the characteristics of guanine nucleotide-dependent phospholipase C activation were studied in [3H]inositol-labeled permeabilized hepatocytes. The nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanyl-5'-yl imidodiphosphate stimulated the production of inositol phosphates by phospholipase C. The effect was concentration-dependent with half-maximal and maximal stimulation occurring with 0.6 and 10 microM GTP gamma S, respectively. The guanine nucleotide-induced stimulation of phosphoinositide breakdown was selective for phosphatidylinositol (4,5)-bisphosphate over phosphatidylinositol (4)-phosphate. The individual inositol phosphates formed after maximal GTP gamma S exposure were analyzed by high-performance liquid chromatography. Inositol 1,4,5-trisphosphate was rapidly produced, followed by the formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate. Ethanol is known to activate hormone-sensitive phospholipase C in intact rat hepatocytes. Ethanol (0.3 M) was ineffective in altering the characteristics of GTP gamma S-stimulated phospholipase C activation, in both digitonin-treated and sonicated hepatocytes. The metabolism of the various inositol phosphate isomers was unaffected by ethanol. The findings demonstrate the potential for the use of permeabilized hepatocytes in the analysis of phospholipase C activation by guanine nucleotides. Ethanol does not activate phospholipase C by altering this process.

Animals↗
PubMed1986

Calcium-dependent and calcium-independent mechanisms of irreversible cell injury in cultured hepatocytes.

It has been proposed that alterations in intracellular calcium homeostasis mediate the genesis of lethal cell injury with an acute oxidative stress. It is shown here, however, that such changes can be dissociated by two different means from the cell death occurring with the exposure of cultured hepatocytes to hydrogen peroxide generated either in the medium by glucose oxidase or intracellularly by the mechanism of menadione. The chelation of intracellular ferric iron with deferoxamine inhibits the formation of hydroxyl radicals from hydrogen peroxide and prevents cell killing. Deferoxamine did not prevent, however, an elevation of the cytosolic Ca2+ ion concentration detected as an activation of phosphorylase alpha. Sulfhydryl reagents inhibited the rise in phosphorylase alpha activity in deferoxamine-pretreated hepatocytes. Conversely, cultured hepatocytes were depleted of Ca2+ ions by treatment with EGTA in a calcium-free medium. Calcium-depleted cells were not resistant to the toxicity of hydrogen peroxide despite the virtual elimination of the activation of phosphorylase alpha. In contrast, it was possible to kill cultured hepatocytes by a mechanism dependent upon a disordered intracellular calcium homeostasis using hepatocytes pretreated in calcium-free medium with the ionophore A23187. These cells were killed in a dose-dependent manner by the addition of calcium ions to the culture medium in concentrations ranging from 0.1 to 2.0 mM. There was a similar dose-dependent activation of phosphorylase alpha, but phosphorylase alpha activities were higher than with H2O2 at comparable cell killing. Deferoxamine pretreatment and sulfhydryl reagents had no effect on the loss of viability with this calcium-dependent cell killing.

Acetylcysteine↗
PubMed1986

Dietary carbohydrate level determines the effect of long-term ethanol ingestion on rat pancreatic amylase content.

We studied the effects of long-term ethanol feeding on pancreatic amylase content in the rat. Because the level of pancreatic amylase depends on the carbohydrate content in the diet, the effects of ethanol ingestion were investigated at different levels of dietary carbohydrate, with fat providing the balance of calories. Animals were maintained on these diets for 3 weeks. In control animals, a reduction in carbohydrate from 47% to 11% of total calories lowered pancreatic amylase content by 60% (from 76.1 +/- 7 U/mg protein to 30.6 U/mg protein). However, in rats that consumed 11% of total calories as carbohydrate, long-term ethanol ingestion (36% of total calories) lowered the amylase content by a further 60% (from 30.6 U/mg protein to 11.8 U/mg protein). By contrast, when the dietary carbohydrate level was maintained at a high (40%) level, long-term ethanol ingestion slightly increased pancreatic amylase content. Pancreatic lipase was not affected by long-term ethanol ingestion at either low (6%) or high (35%) levels of fat. The reduction in the level of amylase caused by ethanol feeding with a low dietary carbohydrate intake was not associated with changes in the fasting blood levels of glucose, insulin, or glucagon. Long-term ethanol ingestion did not lead to morphologic changes in the acini or in the size, shape, or number of granules, as observed by light and electron microscopy. These results suggest that long-term ethanol ingestion has differential effects on pancreatic amylase, depending on the level of carbohydrate in the diet.

Amylases↗
PubMed1984

Ethanol treatment selectively decreases neutral amino acid transport in cultured hepatocytes.

Primary cultures of adult rat hepatocytes were used to study the effects of 100 mM ethanol on various neutral amino acid transport systems. Ethanol exposure for 24 h selectively decreased amino acid uptake by the A and N systems by 40-70%, but had no significant effect on the ASC and L systems. The decrease in the A system was significant after 3 h of ethanol exposure, and the activity was not affected by the presence or absence of ethanol during the uptake measurements. Kinetic analysis showed that ethanol treatment affected predominantly the high-affinity component of A system activity by decreasing the apparent Vmax without significantly changing the apparent Km. Ethanol treatment did not prevent the cells from increasing A system activity in response to insulin and glucagon, but the magnitude of hormone-stimulated uptake was reduced.

Amino Acids↗
PubMed1983

Kinetics of glutamate efflux in rat liver mitochondria.

The transport of glutamate was studied in isolated rat liver mitochondria preloaded with glutamate in the presence of respiratory inhibitors. Glutamate efflux was initiated by dilution of the loaded mitochondria into a glutamate-free medium, and the rate of transport was measured by following the disappearance of glutamate from the mitochondrial matrix following rapid centrifugation through silicone oil. Glutamate efflux was inhibited extensively by bromcresol purple and partially by N-ethylmaleimide, compounds which are both known to inhibit mitochondrial glutamate uptake. The efflux process was stereospecific for L-glutamate and exhibited an activation energy of 19.2 kcal/mol. The rate of glutamate efflux was not affected by changes in the mitochondrial membrane potential. However, a good correlation was observed between the rate of glutamate efflux and the matrix pH, the efflux rate being stimulated by a decrease in matrix pH in the range from 8.0 to 7.2. In contrast, acidification of the incubation medium in the pH range 7.4 to 6.5 inhibited the rate of glutamate efflux. A kinetic analysis was made of the efflux reaction by a computer curve-fitting procedure which fits the experimental data to an integrated rate equation (Williamson, J.R., and Viale, R.O. (1979) Methods Enzymol. 56, 252-278). The results indicated that a fall in the matrix pH primarily caused a decrease in the K'm for matrix glutamate, with little change in V'max. In contrast, a low external pH had an effect on the V'max but not on the K'm for intramitochondrial glutamate. The results are in agreement with a symmetrical sequential model of glutamate transport where the glutamate anion binds to the protonated carrier.

Animals↗
PubMed1982

Chronic ethanol ingestion increases calcium uptake and resistance to molecular disordering by ethanol in liver microsomes.

Calcium uptake and molecular ordering were studied in hepatic microsomes from rats treated chronically with ethanol and were compared with the effects of ethanol in vitro. Calcium uptake was increased by 30% in microsomes from ethanol-fed animals, the Vmax being increased and the Km remaining unchanged. In Arrhenius plots of calcium uptake, the transition temperature in the 25-30 degrees C range was higher by 1.5 degrees C in microsomes from ethanol-fed rats. Chronic ethanol administration resulted in more rigid microsomal membranes, as evidenced by EPR spectra of the spin probe 5-doxylstearic acid. In a plot of the order parameter, S, as a function of temperature, microsomes from ethanol-fed rats showed a discontinuity at a higher temperature than those from controls (29.9 versus 24.2 degrees C). Ethanol, in vitro, inhibited microsomal calcium uptake in both preparations. However, the degree of inhibition was greater in controls. The rate of calcium uptake by microsomes from ethanol-treated animals in the presence of 100 mM ethanol was the same as those from controls in the absence of ethanol. In vitro, increasing concentrations of ethanol progressively decreased molecular ordering of control microsomes, whereas little change was noted in microsomes after chronic ethanol treatment.

Adenosine Triphosphate↗
PubMed1980

Determination of the mitochondrial protonmotive force in isolated hepatocytes.

The intracellular distribution of lipophilic ions and weak acids and bases was studied in intact hepatocytes as indicators of gradients of pH and membrane potential. Mitochondrial and plasma membrane potentials of 161 and 32.8 mV (negative inside), respectively, were calculated from the intracellular accumulation of triphenylmethylphosphonium (TPMP+) and thiocyanate in the intact hepatocyte, assuming a two-compartment model. The predominantly mitochondrial localization of TPMP+ was confirmed using disruption procedures that selectively break the plasma membrane but not the mitochondrial membrane. The intracellular distribution of 5,5-dimethyloxazolidine-2,4-dione and methylamine was studied with similar techniques. A cytosolic pH of 6.95 to 7.00 and a mitochondrial delta pH of 0.97 units was observed. From these results, a total mitochondrial protonmotive force in the intact cell in excess of -200 mV was obtained.

Animals↗
PubMed1980

Origin of the ammonia found in protein-free extracts of rat-liver mitochondria and rat hepatocytes.

1. Protein-free extracts of isolated rat-liver mitochondria contain 5.17 +/- 0.19 nmol ammonia/mg protein [cf. Harris, E. J. and Bassett, D. J. (1971) FEBS Lett. 19, 214-217]. 2. The ammonia found in the protein-free extracts does not originate from lysosomes contaminating the mitochondrial preparation. 3. When isolated mitochondria are incubated with ornithine, 14CO2 and a source of ATP a small amount of citrulline is formed. This amount is stoichiometrically equivalent to the ammonia that disappears from the extramitochondrial space, whereas the amount of ammonia found in the protein-free extracts of the mitochondria remains unchanged. Similar results were obtained when the reductive amination of 2-oxoglutarate was used as an ammonia-consuming reaction. 4. When isolated mitochondria are incubated under conditions such that the glutamate dehydrogenase and 3-hydroxybutyrate dehydrogenase reactions reach equilibrium, the thermodynamically active concentration of ammonia is not equal to the concentration measured in the protein-free extracts. 5. About 80% of the ammonia found in protein-free extracts of rat-liver mitochondria is derived from a component or components with a molecular weight of greater than or equal to 50,000. 6. Protein-free extracts of isolated rat-liver cells contain considerable amounts of ammonia. After digitonin fractionation this ammonia is found in the protein-free extract of the particulate fraction. 7. It is concluded that the ammonia found in protein-free extracts of rat-liver tissue is derived from a component or components in the mitochondria and is released during deproteinization.

Ammonia↗