Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA.These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells.

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA. These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Effect of various agents on the cytoplasmic calcium concentration in cultured human muscle cells

1. We determined the cytoplasmic Ca2+ concentration ([Ca2+]i) in cultured human muscle cells using the fluorescent indicator Quin-2. 2. The [Ca2+]i was dependent on the external Ca2+ concentration. Acetylcholine in the presence of external Ca2+ caused a transient increase in [Ca2+]i. Inhibition by nifedipine indicated that this response was mediated through activated voltage-operated channels. In nominally Ca2(+)-free buffer acetylcholine did not markedly increase [Ca2+]i. Therefore, the increase in [Ca2+]i as a response to depolarization is mainly due to influx of external Ca2+. 3. Various concentrations of caffeine did not influence the [Ca2+]i. Dantrolene decreased [Ca2+]i, both in the presence and absence of external Ca2+. The reduction probably resulted from an action of dantrolene on the intracellular Ca2+ stores, since dantrolene did not influence 45Ca2+ influx or efflux and caffeine partially counteracted the reduction.     

Effects of various agents on the R bodies of certain bacteria

We have shown that urea and temperature affect the unrolling of the R bodies from the bacteria Caedibacter taeniospiralis, Pseudomonas taeniospiralis and Pseudomonas avenae. The R bodies of P. taeniospiralis are not, however, affected by pH changes nor by EDTA or EGTA unlike the other two types of R body. Antibodies prepared against the R bodies of C. taeniospiralis have been shown, following gold-labelling and electron microscopy, to be specific for the proteins of the homologous R body. Pre-treatment of R bodies with pronase or sodium periodate had no effect on these reactions.     

Effects of calcium on the permeability of isolated adult rat heart cells to sodium

The permeability of rat heart myocytes to Na increases when extracellular Ca (Ca₀) is decreased. This increased permeability is reflected in elevated $\text{Na}\text{K}$ ratios in nonenergized myocytes and in increased ouabain-sensitive lactate production in anaerobic myocytes supplemented with glucose. Myocytes treated with ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid (1 mm) maintain low $\text{Na}\text{K}$ ratios, but expend considerable glycolytic ATP in ouabain-sensitive cation cycling. The data suggest that Ca₀ bound to the sarcolemma can restrict transmembrane movement of Na via pathways that are not yet defined. The lack of significant net accumulation of Ca argues against the explanation that Ca₀ maintains low internal Na levels as a result of NaCa exchange. Both the increased uptake of Na and increased utilization of ATP in the absence of Ca₀ may be relevant to the phenomenon of “Ca-paradox” in situ.     

Characterization of neurokinin receptors in various isolated organs by the use of selective agonists

The three mammalian neurokinins, substance P, neurokinin A and neurokinin B, as well as some agonists selective for their respective receptors, NK-P, NK-A and NK-B, were tested in a variety of pharmacological preparations in order to evaluate if the biological responses of the various tissues were mediated by single or multiple receptor types. Previous observations that the dog carotid artery, the rabbit pulmonary artery and the rat portal vein are selective preparations respectively for SP, NKA and NKB were confirmed in the present study by showing that only the respective selective agonists were active on these tissues. Multiple functional sites were demonstrated in intestinal tissues (guinea pig ileum, rat duodenum), which apparently contain the three neurokinin receptors. A large number of NK-P, together with some NK-A receptor sites were found in the guinea pig and rat urinary bladder. Similarly, the guinea pig trachea and the rabbit mesenteric vein contain NK-A and NK-P functional sites. Rat and rabbit vas deferens stimulated electrically respond as typical NK-A preparations, since they are almost insensitive to SP or NKB selective agonists. A mixture of NK-A and NK-B receptor sites has been shown to be present in the hamster urinary bladder: dog and human urinary bladder definitely contain NK-A receptors and the dog bladder also some NK-P functional sites.