Accumulation In Microsomes Research Articles

Effect of Ryanodine on Sarcoplasmic Reticulum Ca 2+ Accumulation in Nonfailing and Failing Human Myocardium

The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.

Transfer of liposomes containing dolichol into isolated hepatocytes.

Isolated rat hepatocytes were preincubated with egg lecithin liposomes containing [3H]dolichol and [3H]dolichyl ester, and the intracellular levels and distributions of these lipids were subsequently determined after incubation in a liposome-free medium. [3H]Dolichol was recovered initially mainly in microsomes, and no increase with time in the low level of this compound in the mitochondrial/lysosomal fraction could be observed. A small portion of the labeled dolichol was esterified in the endoplasmic reticulum and transferred to the lysosome-containing fraction. [3H]Dolichyl linoleate was initially localized in microsomes and supernatant, but later accumulated in the mitochondria/lysosomes. Dolichyl linoleate was found in the membrane of microsomes, in the membrane and lumen of lysosomes, and in the soluble cytoplasm. Exogenous dolichol recovered in microsomes was not phosphorylated to any significant extent. Liposomal phosphatidylcholine also showed preferential accumulation in microsomes after incubation with hepatocytes. These results indicate that exogenous or endogenously formed dolichyl esters are transferred from the endoplasmic reticulum to lysosomes, probably through the cytoplasm. It appears that fatty acids play a role in targeting these lipids to their intracellular locations.

Rapid kinetics of liver microsomal glucose-6-phosphatase. Evidence for tight-coupling between glucose-6-phosphate transport and phosphohydrolase activity.

Rapid kinetics of both glucose-6-P uptake and hydrolysis in fasted rat liver microsomes were investigated with a recently developed fast-sampling, rapid-filtration apparatus. Experiments were confronted with both the substrate transport and conformational models currently proposed for the glucose-6-phosphatase system. Accumulation in microsomes of 14C products from [U-14C]glucose-6-P followed biexponential kinetics. From the inside to outside product concentrations, it could be inferred that mostly glucose should accumulate inside the vesicles. While biexponential kinetics are compatible with the mathematical predictions of a simplified substrate transport model, the latter fails in explaining the "burst" in total glucose production over a similar time scale to that used for the uptake measurements. Since the initial rate of the burst phase in untreated microsomes exactly matched the steady-state rate of glucose production in detergent-treated vesicles, it can be definitely concluded that the substrate transport model does not describe adequately our results. While the conformational model accounts for both the burst of glucose production and the kinetics of glucose accumulation into the vesicles, it cannot explain the burst in 32Pi production from [32P]glucose-6-P measured under the same conditions. Since the amplitude of the observed bursts is not compatible with a presteady state in enzyme activity, we propose that a hysteretic transition best explains our results in both untreated and permeabilized microsomes, thus providing a new rationale to understand the molecular mechanism of the glucose-6-phosphatase system.

Formation of reactivity of the mesenteric microcirculatory system in pubertal rats

Calmodulin and cAMP were demonstrated to have no stimulating effect on Ca2+ transport in the sarcoplasmic reticulum of the dog heart in experimental myocardial infarction as compared to that in the uninvolved myocardium (control). Introduction to the incubation medium of exogenous protein kinase in addition to calmodulin and cAMP provoked an approximately 35% increase in 45Ca accumulation in microsomes of the impaired myocardium as compared with the system containing no exogenous protein kinase. Under the same conditions, the control showed a 75% increase in 45Ca accumulation. A reduction in the activity of Ca2+-activated ATPase of the reticulum and translocation of calmodulin activity from the membrane fraction of cardiomyocytes to cytosol were recorded in myocardial infarction.

Effect of colchicine on internalization of prolactin in female rat liver: an in vivo radioautographic study.

Binding and internalization of 125I-ovine prolactin into hepatocytes of female rats was visualized by the in vivo radioautographic method (Bergeron, J. J. M., G. Levine, R. Sikstrom, D. O'Shaughnessey, B. Kopriwa, N. J. Nadler, and B. I. Posner, 1977, Proc. Natl. Acad. Sci. USA, 745:051-5055). Receptor-mediated internalization of label was observed into lipoprotein-filled vesicles in the Golgi/bile canalicular region of the hepatocyte. Colchicine treatment had no effect on the internalization of label into the lipoprotein-filled vesicles. However, the location of the radio-labeled lipoprotein-filled vesicles was altered from the Golgi/bile canalicular region to subsinusoidal. Radioactive content of hepatocytes decreased as a function of time after injection of 125I-prolactin; however, colchicine treatment markedly retarded this loss of label. Subcellular fractionation experiments indicated that colchicine treatment led to decreased levels of 125I-prolactin accumulation in microsomes but augmented the accumulation of label in the L fraction. It is concluded that in normal female rats prolactin is internalized into lipoprotein-filled vesicles in the Golgi region before degradation of the hormone. Colchicine treatment accumulates labeled lipoprotein-containing vesicles in a subsinusoidal region and retards hormone catabolism. The labeled vesicles observed after colchicine treatment may correspond to the unique vesicles previously observed in the L fraction and found to be enriched in prolactin receptors (Khan, M. N., B. I. Posner, A. K. Verma, R. J. Khan, and J. J. M. Bergeron, 1981, Proc. Natl. Acad. Sci. USA, 78:4980-4981).

Effects of diuretics on calcium uptake and release in renal microsomes

A variety of diuretics were tested for their effects on Ca 2+ uptake and release by microsomes isolated from rat kidneys. Mersalyl acid, ethacrynic acid, furosemide and bumetanide inhibited Ca 2+ uptake by microsomes. Cysteine abolished the inhibitory effects of mersalyl acid and ethacrynic acid on Ca 2+ uptake. No appreciable differences in the inhibition were seen between microsomes from kidney cortex and those from medulla. Acetazolamide had no significant effect on Ca 2+ uptake; however. hydrochlorothiazide stimulated Ca 2+ uptake activity. The effects of the diuretics on Ca 2+ uptake by microsomes were entirely on the process of Ca 2+ accumulation in microsomes, since release of Ca 2+ that had been taken up previously was not influenced by any of the diuretics tested. These results provide information concerning the biochemical mechanisms of action of diuretics on Ca 2+ reabsorption in the kidney.

Inhibitory Action of Calcium Antagonists on ATP-Dependent Calcium Uptake by the Renal Cortical Microsomes

Effects of Ca2+ antagonists, verapamil and diltiazem, on uptake and release of Ca2+ by microsomes of the renal cortex were studied. Verapamil inhibited the ATP-dependent Ca2+ uptake by renal microsomes. Addition of 0.8 mM verapamil was required to produce 50% inhibition of the ATP-dependent Ca2+ uptake. Diltiazem also depressed the Ca2+ uptake of the microsomes. The inhibitory effect of both drugs on the Ca2+ uptake was not due to an increased permeability for Ca2+, since release of Ca2+ from the microsomes was not significantly affected by either drug. It is proposed that verapamil and diltiazem inhibit Ca2+ transport by interfering with an active process of Ca2+ accumulation in microsomes of the renal cortex.

Inhibitory action of calcium antagonists on ATP-dependent calcium uptake by the renal cortical microsomes.

Effects of Ca2+ antagonists, verapamil and diltiazem, on uptake and release of Ca2+ by microsomes of the renal cortex were studied. Verapamil inhibited the ATP-dependent Ca2+ uptake by renal microsomes. Addition of 0.8 mM verapamil was required to produce 50% inhibition of the ATP-dependent CA2+ uptake. Diltiazem also depressed the Ca2+ uptake was not due to an increased permeability for Ca2+, since release of Ca2+ from the microsomes was not significantly affected by either drug. It is proposed that verapamil and diltiazem inhibit Ca2+ transport by interfering with an active process of Ca2+ accumulation in microsomes of the renal cortex.

Subcellular distribution, a factor in risk evaluation of pentachlorophenol

Pentachlorophenol (PCP) is a potent uncoupler of mitochondrial phosphorylation in vitro and also interferes with microsomal detoxication functions in vitro. This favours flavin mediated oxygenation compared with flavin cytochrome P-450 dependent reactions. Gas chromatographic analysis of subcellular fractions, obtained by zonal centrifugation showed markedly lower PCP concentration in mitochondria and a high accumulation in microsomes compared with cytosol. This increases the likelihood that PCP in vivo causes a malfunction in microsomal detoxication.

Effects of temperature and inorganic ions on calcium accumulation in microsomes from intestinal smooth muscle.

Energy dependent calcium binding in microsomal vesicles from the longitudinal smooth muscle of the guinea pig intestine was investigated at two different temperatures (30 degrees C and 10 degrees C) and in the absence and presence of CdCl2, BaCl2 and MnCl2. The investigation was carried out to determine whether the effects of temperature and the effects of the divalent ions on microsomal calcium binding could be correlated with the effects of these interventions on the mechanical activity of the intact longitudinal fibers. A reduction in temperature from 30 degrees C to 10 degrees C inhibited both the uptake of calcium into the microsomes and the rate of release of calcium ions from the microsomes to the external medium. This exchange in temperature also slowed the rate of relaxation of the intact longitudinal muscle after it had been induced to contract with acetylcholine and subsequently allowed to relax by removing calcium ions from the bathing medium and adding 1 X 10(-3) M EGTA. The presence of CdCl2, like the reduction in temperature, decreased the uptake of calcium into the microsomal vesicles. However, the release of calcium from the microsomes was accelerated. BaCl2, produced the same effects as did CdCl2 on the uptake of calcium into microsomes but to a lesser extent. It had very little effect on the release of calcium ions from the microsomes. MnCl2 had no significant effects on either the uptake or release of calcium ions in the microsomal preparation. Both CdCl2 and MnCl2 exerted an inhibitory action on acetylcholine-induced contractile responses of the intact longitudinal fibers; whereas BaCl2 served to initiate a contractile response in the smooth muscle fibers. Thus, it would appear that the effects of a temperature change on microsomal calcium binding and on mechanical activity in intact fibers can be correlated; but the effects of CdCl2, BaCl2 and MnCl2 on these two cellular processes do not follow any consistent pattern.