Cytoplasmic Calcium Levels Research Articles

Effects of hydralazine and verapamil on phosphorylase activity and guanosine cyclic 3',5'-monophosphate levels in guinea-pig taenia coli.

1 The roles of guanosine cyclic 3',5'-monophosphate (cyclic GMP) and calcium in the relaxation produced by hydralazine and verapamil in potassium-depolarized guinea-pig taenia coli have been investigated.2 Depolarization of isolated strips of guinea-pig taenia coli by 124 mM KCl caused sustained contractures and increases in tissue levels of cyclic GMP.3 The KCl-induced increases in cyclic GMP levels appeared to be calcium dependent. No increases in cyclic GMP levels were seen in strips of taenia coli depolarized in the absence of calcium. Readdition of calcium to the depolarizing solution contracted the muscles and increased cyclic GMP levels. When calcium was removed from the depolarizing solution during the sustained, tonic phase of a KCl-induced contracture, both tension and cyclic GMP levels returned to control values.4 Administration of 50 muM verapamil to KCl-contracted muscles completely relaxed the muscles and caused cyclic GMP levels to return to control values within 14 min. Hydralazine, 2 mM, on the other hand, relaxed the depolarized muscles without lowering cyclic GMP levels. No significant changes in cyclic AMP levels were seen in any of these experiments.5 Similar results were obtained in an analogous series of experiments in which glycogen phosphorylase activity was measured instead of cyclic GMP levels. Activation of phosphorylase during contractions of guinea-pig taenia coli had previously been reported to be a calcium-dependent phenomenon.6 It was concluded that increases in tissue levels of cyclic GMP (or of cyclic AMP) were not responsible for the relaxant effects of hydralazine or verapamil in these experiments. It was also suggested, based on our results, that verapamil exerted its relaxant effects in the depolarized taenia coli by lowering cytoplasmic calcium levels, whereas hydralazine relaxed the depolarized muscles without lowering intracellular calcium activity.

X-ray microanalysis of calcium around the membrane of platelets

Since the function of platelets is linked to an increase in the cytoplasmic calcium level, it would be of great interest to establish its subcellular distribution. Recently we found the existence of the surface connected membrane system (SCS) in the chicken thrombocyte(1,2) and succeeded in detecting calcium on the membrane of the SCS using ultrahistochemical methods in combination with an electron probe X-ray microanalyzer(1). This study is undertaken to demonstrate calcium accumulation around the membrane of the SCS of mammalian platelets.Ultrahistochemistry: Human platelet rich buffy coat was fixed in the potassium antimonate-OsO4. method or in the potassium oxalate-glutaraldehyde method without post-fixation with OsO4. After incubation, specimens were dehydrated with ascending concentrations of ethyl alcohol and embedded in Epon 812.

Intracellular calcium and the regulation of sodium transport in the frog skin.

Exposure of frog skins to Na-free solutions on the basal border causes a reduction in transepithelial Na transport. Since Na-free solutions produce an increase in cell Ca concentration in some cell types, we have explored whether such increase plays a rôle in their inhibitory effects on transepithelial transport. Measurements of 45 Ca uptake in isolated epithelia showed that Na-free solutions caused a threefold stimulation of Ca uptake; nearly all the increased uptake occurred through the basal border. The reduction in transepithelial Na transport caused by Na-free solutions was absent when Ca was also eliminated from the basal solution. These findings suggest that the reduction in transepithelial Na-free solutions is caused by increased cytoplasmic calcium levels. We also found that during the action of metabolic inhibitors, Ca efflux from the epithelium was increased markedly. Since such as increase in efflux is probably due to increased (Ca) in the cytoplasm, we suggest that the reduced epithelial Na permeability observed during metabolic inhibition may be due to increased cytoplasmic calcium.

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3′,5′-cyclic monophosphate and protein kinase

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca 2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such as hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 × g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 × g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl 3. These data indicate that calcium was being actively concentration within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca 2 is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 · 10 −6 M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca 2+ from the cytoplasm.

Calcium distribution in islets of langerhans: A study of calcium concentrations and of calcium accumulation in b cell organelles

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.

Energy-dependent calcium sequestration activity in rat liver microsomes.

MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires MgATP. Substitution of CTP, UTP, GTP, or ADP will not support calcium uptake. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle microsomes, is required to maintain net accumulation of calcium. The reaction is temperature-dependent and has an apparent Vmax of 11.2 nmol/mg of protein/min. The apparent Km for calcium is 23.2 muM calculated from total calcium concentration, and approximately 4.6 muM based on free calcium concentration, and apparent Km for ATP is 1.8 mM. Calcium uptake activity normally measured in presence of 100 mM KCl is only slightly depressed if 100 mM NaCl is substituted and is considerably depressed when 200 mM sucrose replaces KCl. An appropriate hydrolysis of ATP is associated with the calcium uptake. Separation of smooth and rough endoplasmic reticulum on sucrose gradients indicates a considerably lower specific activity per mg of protein in the fraction enriched with rough endoplasmic reticulum. Azide, at a level which completely inhibits liver mitochondrial calcium sequestration, has no effect on the liver microsomal system. Oligomycin, which inhibits ATP-dependent calcium uptake of liver mitochondria, has a considerably lesser effect on calcium uptake of liver microsomes. p-Chloromercuribenzoate and mersalyl inhibit the liver microsomal calcium pump at levels as low as 10- minus 7 M. Calcium uptake activity is considerably reduced in adult female rats. Weanling rats, both male and female, have calcium uptake activities like that of the adult males. Because of the higher activity in the male rat, the fatty acid composition of the liver microsomal phospholipids was analyzed. The male rat had a higher percentage of linoleic and palmitic acids in the microsomal phospholipids. Endoplasmic reticulum and plasma membrane are postulated to play a role in regulation of the levels of free cytoplasmic calcium in the mammalian liver.

Effects of hypoxia on the distribution of calcium in arterial smooth muscle cells of rats and swine.

Exposure to hypoxia caused an increase in the hematocrit and right heart weight of experimental rats, but did not affect calcium-45 uptake by pulmonary arterial smooth muscle cells. However, autoradiographic studies showed that hypoxia apparently caused a shift of 45-Ca from primarily extracellular sites in arteries of control rats to intracellular sites in tissues of hypertensive rats. Cytochemical studies of calcium distributions in pulmonary arterial smooth muscle cells support the autoradiographic data and show that in both rats and swine the majority of pyroantimonate granules occur extracellulary in control tissues. In contrast, hypoxic tissues displayed a greatly reduced number of granules in extracellular sites and an increase in the amount of precipitate in intracellular sites. In pulmonary arterial smooth muscle cells from hypoxic rats most of the precipitate was associated with the caveolae intracellulares, while in corresponding cells from hypoxic swine in the majority of the pyroantimonate granules were localized to the sarcoplasmic reticulum. Hypoxia may produce pulmonary hypertension by interfering with the ability of the arterial smooth muscle cells to maintain transmembrane ionic gradients, thus producing an effective increase in cytoplasmic calcium levels. The increased calcium may then activate the contractile apparatus to produce a sustained vasoconstriction

Electron Probe Analysis and Cryoultramicrotomy of Cardiac Muscle: Mitochondrial Granules

The contribution of mitochondria to the regulation of cytoplasmic calcium levels in the heart is an unsettled question. In certain pathological conditions, mitochondrial granules thought to contain calcium have been found in fixed myocardium.Mouse ventricular myocardium, papillary muscle and trabeculae, unfixed and without cryoprotectants, were quick-frozen in Freon 22 cooled in liquid N2 or in liquid N2 slush. Sections were cut with glass knives at -100-120° C, placed on Cu or carbon coated nylon grids, and dried on a cold block at l0-2-10-5 Torr. Some sections were vapor stained with crystalline Os under vacuum, but the analyses reported are of unstained sections. Even 5 minute “pre-fixation” with glutaraldehyde, before cryoultramicrotomy, or staining of unfixed frozen thin sections with NH4 molybdate removed elements of interest (e.g.: K).The morphological detail detectable by inherent contrast or Os vapor staining varied in different preparations and even in different regions of the same sections. In fortuitous examples (Fig. 1) the filament lattice could be clearly demonstrated.

The regulatory role of divalent cations in human granulocyte chemotaxis. Evidence for an association between calcium exchanges and microtubule assembly.

Optimal human granulocyte chemotaxis has been shown to require both calcium and magnesium. Exposure of granulocytes to three different chemotactic factors (C5a, kallikrein, and dialyzable transfer factor) yielded a rapid calcium release, depressed calcium uptake, and was associated with a shift of calcium out of the cytoplasm and into a granule fraction. Colchicine, sodium azide, and cytochalasin B, in concentrations that inhibited chemotaxis, also inhibited calcium release while low concentrations of cytochalasin B, which enhanced chemotaxis, also enhanced calcium release. Microtubule assembly was visualized both in cells suspended in C5a without a chemotactic gradient and in cells actively migrating through a Micropore filter. The data suggest microtubule assembly is regulated, at least, in part, by the level of cytoplasmic calcium. It is proposed that asymmetric assembly of microtubules may be instrumental in imparting the net vector of motion during chemotaxis.