In vitro effects of thyroxine on the mechanical properties of erythrocytes

In vitro effects of thyroxine on erythrocyte deformability and mechanical fragility were observed. Deformability of erythrocytes was improved in a dose dependent manner by thyroxine. Mechanical hemolysis was found to be lower if thyroxine was included in erythrocyte suspensions at concentrations close to the physiological levels (10(-9)M). These changes might be related to the alterations of intracellular calcium concentration, as in the erythrocyte suspensions containing 10(-9)M thyroxine, intracellular calcium concentration was found to be 30 times lower than the control suspensions which did not contain thyroxine. Thyroxine also reduced the mechanical hemolysis ratio in calcium loaded cells. These observations suggest that thyroxine might play some role in the regulation of the mechanical properties of erythrocytes which might be mediated via the effects on calcium metabolism.     

Effects of caffeine, tetracaine, and ryanodine on calcium-dependent oscillations in sheep cardiac Purkinje fibers

Membrane current and tension were measured in voltage-clamped sheep cardiac Purkinje fibers. Elevating the intracellular calcium concentration ([Ca2+]i) results in oscillations of membrane current and tension both at rest and during stimulation. During stimulation, an oscillatory transient inward current and an after contraction follow repolarization. We have examined the effects on the oscillations of changing the extracellular calcium concentration ([Ca2+]o) and of adding various drugs. In agreement with previous work, high concentrations of drugs that affect the sarcoplasmic reticulum, namely caffeine (10-20 mM), tetracaine (1 mM), and ryanodine (10 microM), abolish the oscillations. However, at lower concentrations, these three drugs have different effects on the oscillations. Caffeine (1-2 mM) decreases the oscillation amplitude but increases the frequency. Tetracaine (100-500 microM) has little effect on the magnitude of the oscillations but decreases their frequency. Ryanodine, at all concentrations used (0.1-10 microM), eventually abolishes the oscillations but, in doing so, decreases the magnitude, leaving the frequency unaffected. When [Ca2+]o was changed in order to vary [Ca2+]i, both the frequency and the magnitude of the oscillations always changed in the same direction. This suggests that these three drugs have effects in addition to just changing [Ca2+]i.     

Polyamines Stimulate Mitochondrial Calcium Transport in Rat Brain

The effects of the polyamines spermine and spermidine on rat brain mitochondrial calcium transport were examined using a variety of techniques for measuring the kinetics of calcium uptake and the buffering capabilities of isolated mitochondria. Spermine both increased the rate of calcium accumulation and decreased the set-point to which isolated mitochondria buffer free calcium concentration. In the presence of physiological concentrations of sodium and magnesium, spermine lowered the extramitochondrial calcium level to approximately 0.3 microM, a value close to the resting intracellular calcium concentration. The effect of polyamines was concentration dependent, with a half-maximal effect of spermine observed at approximately 0.1-0.4 mM (respiratory substrate dependent), whereas spermidine was approximately 10 times less potent. Calcium transport by hippocampal mitochondria was stimulated markedly more by spermine than was calcium transport by mitochondria isolated from brainstem. The stimulatory effect of spermine was not due to an increase in the transport of respiratory substrates inside the mitochondria nor to an effect on the enzymes using these respiratory substrates. An examination of the effect of spermine on the kinetics of calcium uptake indicated that spermine increased calcium uptake maximally at low calcium concentrations. Beyond that level, the stimulatory effect of spermine decreases, and spermine can even inhibit calcium uptake. These results are in good agreement with previous reports on the effects of polyamines on calcium transport in mitochondria from peripheral tissue. They support the hypothesis that spermine increases the rate of calcium uptake by mitochondria by increasing the affinity of the uniporter for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of anti-Ig on cytosolic Ca2+ in Daudi lymphoblastoid cells

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.     

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

Simultaneous recording of calcium transients and reactive oxygen intermediates of human polymorphonuclear granulocytes in response to formyl-Met-Leu-Phe and the environmental agent sulfite

BACKGROUND: Human polymorphonuclear granulocytes (PMN) are an essential component in the immunological defense network against a variety of harmful pathogens. We have studied the effects of the airborne pollutant sulfite on the calcium metabolism and respiratory burst of these cells simultaneously. METHODS: A flow cytometric method was developed using the fluochromes Indo-1 and DHR-123. This method allowed us to investigate the real-time kinetics of intracellular free calcium and reactive oxygen intermediates in viable cells with a temporal resolution of 1 s over a time course of 17 min. An additional feature was the possibility to discriminate between reacting and nonreacting cells after treatment with defined stimuli, thus gaining additional insight into the behavior of cell subpopulations. RESULTS: We analyzed the effects of sulfite on PMN before and after stimulation with formyl-Met-Leu-Phe (FMLP). Treatment with sulfite alone (0.001-1 mM) caused a small, nontransient increase in intracellular calcium. Preincubation with sulfite reduced the maximal calcium response elicited by FMLP. A significant increase in steady-state calcium levels after stimulation with FMLP was observed after treatment with sulfite in concentrations of 10 and 100 mM. Regarding the respiratory burst, treatment with sulfite alone in concentrations of 0.001-1 mM induced a significant increase in DHR-123-derived fluorescence, whereas concentrations of 5 and 10 mM caused a significant depression of this fluorescence below baseline values. Sulfite caused a maximal twofold increase of DHR-123-derived fluorescence compared with the FMLP response. Similar results were obtained after preincubation with sulfite before treatment with FMLP, showing that the effect of sulfite on the respiratory burst was additive to the FMLP response. Regarding the fractions of responding cells, treatment with sulfite up to 1 mM induced a concentration-dependent increase of burst-reactive PMN, whereas preincubation before stimulation with FMLP showed no correlation between sulfite concentration and fraction of burst-reacting cells. CONCLUSIONS: By simultaneous registration of [Ca(2+)](i) and [H(2)O(2)](i) of PMN after treatment with FMLP and sulfite, the essential responses were already observed within a short time interval (15 min). Striking differences were found in the response of calcium as second messenger and respiratory burst in PMN treated with sulfite. Until a critical concentration (0. 5-1 mM), sulfite caused a concentration-dependent increase of [H(2)O(2)](i), in addition to the FMLP-induced response. The [Ca(2+)](i) changes induced by sulfite alone, however, were found to be small and showed no correlation with the respiratory burst response.     

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.     

Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes

TRPM2 (transient receptor potential melastatin 2) is a Ca2+-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO4 cell extracts. The reversed-phase ion-pair HPLC method with an Mg2+-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 muM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD+ and fluorimetric determination of NAD+. Intracellular cADPR concentrations were approx. 0.2 microM and not altered by fMLP. In patch-clamp experiments, ADPR (0.1-100 microM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (<10 nM) or a high (1 microM) intracellular Ca2+ concentration. TRPM2 currents were significantly larger at high than at low [Ca2+] (e.g. -225+/-27.1 versus -7+/-2.0 pA/pF at 5 muM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentration < or =0.3 microM). cADPR (0.1, 0.3 and 10 microM) was without effect even in the presence of subthreshold ADPR (0.1 microM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca2+. Thus ADPR and Ca2+ in concert behave as a messenger system for agonist-induced influx of Ca2+ through TRPM2 in granulocytes.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i.     

Mechanism of activation of liver glycogen synthase by swelling.

The mechanism linking the stimulation of liver glycogen synthesis to swelling induced either by amino acids or hypotonicity was studied in hepatocytes, in gel-filtered liver extracts, and in purified preparations of particulate glycogen to which glycogen-metabolizing enzymes are bound. High concentrations of KCl, but not of potassium glutamate, were found to inhibit glycogen synthesis in permeabilized hepatocytes. Similarly, physiological concentrations (30-50 mM) of Cl- ions were also found to inhibit synthase phosphatase in vitro, whereas 10-20 mM Cl- ions, a concentration found in swollen hepatocytes, did not inhibit synthase phosphatase. Synthase phosphatase activity was more sensitive to inhibition by Cl- ions at low (0.1%) than at high (1%) concentrations of glycogen. By contrast, 10 mM glutamate and aspartate, a concentration observed in hepatocytes incubated with glutamine or proline, stimulated synthase phosphatase in vitro. Therefore, it is proposed that the fall in intracellular Cl- concentration as well as the increase in intracellular glutamate and aspartate concentrations, that are observed in swollen hepatocytes in the presence of amino acids, are responsible, at least in part, for the stimulation of synthase phosphatase and, hence, of glycogen synthesis.     

Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism

Current knowledge suggests that normal parathyroid glands and parathyroid adenomas have different sensitivities to environmental calcium. In search for morphological equivalents, 5 normal human and 10 porcine parathyroid glands, as well as 10 human parathyroid adenomas were investigated with regard to intracellular and extracellular calcium distribution. The glands were incubated for 2, 4, 6 and 20 h in tissue cultures using HAM's F10 medium with various calcium concentrations. For visualization of the calcium distribution in the tissue the method of pyroantimonate precipitation was applied. Specificity of the reaction was controlled by X-ray microanalysis. Shifts of the calcium pyroantimonate precipitates were quantitated by morphometry using an area-counting system. The results demonstrate that in normal parathyroid glands calcium precipitates are distributed randomly. Incubation of normal glands in medium with low calcium concentration (0.6 mM) provoked reduced amounts of intracellular and extracellular calcium complexes. When the incubations were performed in medium with high calcium content (2.6 mM), calcium accumulated inside parathyroid and stroma cells. In contrast to normal parathyroid glands, parathyroid adenomas fixed immediately after surgery showed an atypical calcium distribution with low amounts of intracellular and high amounts of extracellular calcium grains. The data suggest that in normal parathyroid glands the intracellular calcium concentration follows the extracellular environmental calcium concentration. Thus, calcium modulates parathyroid hormone (PTH) secretion via intracellular regulatory mechanisms. In parathyroid adenomas the calcium transport via the tumor cell membrane appears to be disturbed resulting in lowered intracellular calcium levels. This is remarkable since the environmental calcium concentration is elevated due to the hypercalcemia of primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.     

Increased free calcium in endothelial cells under stimulation with adenine nucleotides

The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 microM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 +/- 3 (SEM) nM. ATP (adenosine-5'-triphosphate) raised Cai++ dose-dependently and reversibly to 458 +/- 60 nM at a concentration of 10 microM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A-5'-PP) and AMP (A-5'-P) had smaller effects with a maximal Cai++ of 287 +/- 72 nM at 30 microM ADP and 176 +/- 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 microM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 microM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium-derived relaxant factor.     

Alpha 1-adrenergic stimulation increases the Vmax of isolated myocardial papillary muscles.

alpha-Adrenergic agonists have been shown to increase the tension developed by myocardial muscle. However, their effects on the maximum velocity of unloaded muscle shortening (Vmax) have not been rigorously examined. In this study, the contractile effects of the alpha-adrenergic agonist phenylephrine were examined in the presence of propranolol in papillary muscles of two species, the dog and the rabbit. In rabbit papillary muscles studied at physiological calcium concentrations (1.25 mM), phenylephrine increased all indices of contractility (Vmax, tension, and maximum rate of tension developed (dT/dt)) starting at 10(-8) M. The percent increase in Vmax (121 +/- 8%) was less than that of tension (188 +/- 20%, p less than 0.05) and dT/dt (262 +/- 35%, p less than 0.01). These findings occurred at both 29 and 35 degrees C and were inhibited by adding 10(-5) M prazosin. Increasing extracellular calcium concentration from 1.25 to 15 mM caused changes in twitch configuration that were significantly different from those of phenylephrine. Calcium increased all indices of contractility more than did phenylephrine. This was particularly true for dT/dt (502 +/- 82 vs. 262 +/- 35% for phenylephrine, p less than 0.01). Nonetheless, the ratio of increase in tension to increase in Vmax under both experimental conditions was similar (the increase in Vmax was 64% of that of tension with phenylephrine and 69% with increased calcium). At 1.25 mM calcium, the increase in contractility caused by phenylephrine was much smaller in dog myocardium as compared with rabbit myocardium. Rather, the effects of phenylephrine on dog myocardium studied at 1.25 mM calcium resembled that of rabbit myocardium studied at 15 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial uncoupling does not influence the stability of the intracellular signal activating plasma membrane calcium channels.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by Ca2+, allowed to estimate the half-time for the process of cell recovery after activation of store-operated calcium channels. By this approach we have found that carbonyl cyanide m-chlorophenylhydrazone, which has been documented to inhibit calcium entry into Jurkat cells, does not influence the stability of the intracellular signal involved in the activation of store-operated calcium channels.     

Ecto-calcium-dependent ATPase activity of mammalian taste bud cells.

Histochemistry was utilized to characterize Ca-ATPases associated with lingual taste buds in the golden hamster. Taste buds showed elevated staining for magnesium- or calcium-dependent ATPase (Ca-ATPase) relative to the surrounding epithelium. At low calcium concentrations (0.1-0.5 mM), intracellular staining predominated. Most of the studies were conducted at calcium concentrations of > or = 10 mM, in which most of the staining was localized to the external face of plasma membranes of taste bud cells (including receptor and basal cells) located in the core of fungiform taste buds, or the entire vallate or foliate taste buds. The peripheral fungiform taste bud cells stained much less intensely, but the peripheral cells adjacent to the core showed intermediate levels. GTP and ITP were just as effective substrates as ATP. Millimolar concentrations of magnesium were as effective as calcium. Inhibitors of intracellular ATPases, including quercetin, sodium azide, and 2,4-dinitrophenol, had no effect on the staining. Therefore, the Ca-ATPase staining of plasma membranes at mM concentrations of calcium is thought to correspond to one or more ecto-Ca-ATPase activities with unknown functions. Roles related to increased energy requirements or to the possible function of ATP as a neurotransmitter or -modulator are proposed.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

Extracellular calcium modulates generation of reactive oxygen species by the contracting diaphragm.

Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 +/- 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18. 90 +/- 2.70 ng/mg) or high-calcium (19.30 +/- 4.50 ng/mg) studies (P < 0.001). Control, noncontracting diaphragms (in 2.5 mM calcium) had little ROS production (3.40 +/- 0.80 ng/mg; P < 0.001). To further investigate this issue, we added nimodipine (20 microM), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 +/- 0.85 ng ethidium/mg tissue). These data indicate that ROS generation by the contracting diaphragm is strongly influenced by extracellular calcium concentrations and may be dependent on calcium transport through L-type calcium channels.