High K+ elevates cytosolic free Ca concentration due to mobilization from internal storage sites in rat parotid cells

1. Effects of high K+ on cytosolic free Ca concentration ([Ca2+]i) in rat parotid cells were studied using quin2. 2. High K+ elevated [Ca2+]i in a dose-dependent manner in normal and Ca-free media. The elevation of [Ca2+]i induced by high K+ was less in the latter medium. 3. High K+ depolarized the membrane in a dose-dependent manner in normal and Ca-free media. 4. Although monensin increased [Ca2+]i, high K+ did not affect 22Na uptake into cells. 5. After treatment with oligomycin, high K+ but not carbachol raised [Ca2+]i. 6. We suggest that high K+ increases [Ca2+]i due to mobilizing Ca2+ from the intracellular storage site which does not need energy.     

Calcium requirement of uterine contraction induced by PGE1: importance of intracellular calcium stores

The contraction of the rat uterus in response to PGE1 in high K+ medium and in Ca-free solution which contained EDTA has been investigated in order to examine whether excitation-contraction coupling involves the release of Ca from an intracellular store. In uterus maximally contracted by K+, cumulative concentrations of PGE1 (1.25 - 20 ng/ml) caused maintained concentration-dependent contraction. PGE1 induced sustained contraction of rat uterus in Ca-free medium after incubation with 3mM EDTA for 50 min. In these conditions the involvement of extracellular Ca is highly unlikely. The PGE1-induced contraction could be repeated without exposure to external Ca ions and with only slight reduction in magnitude. The PGE1 concentrations required to elicit uterine contraction in Ca-free solution were about 1000 times higher than the effective doses in KCl-depolarized uterus. In conclusion, the present investigation shows that Ca influx is not essential for PGE1-induced contraction of rat uterus, although extracellular Ca enhances it presumably by increasing the free Ca levels in the cytosol.     

Cytoplasmic Ca2+ is a primary determinant for myosin phosphorylation in smooth muscle cells

Initiation of smooth muscle contraction is associated with Ca2+/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca2+ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca2+ ionophore, ionomycin, increases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca2+ concentrations below 1 microM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca2+ concentration is apparently independent of the source of free intracellular Ca2+ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca2+. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca2+ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.     

Magnesium ion-dependent contraction of skinned frog muscle fibers in calcium-free solution

Skinned frog fibers were reversibly activated in Ca-free solutions containing 0 mM KCl, 23 microM free Mg, and having an ionic strength of approximately 50 mM. Contractile force was nearly maximal at 22 degrees - 25 degrees C and decreased at lower temperatures. Maximal force in Ca-free solution at 50 mM ionic strength was close to twice the calcium-activated force with pCa 5 and 190 mM ionic strength. The force in Ca-free solution could be reduced to zero by raising the concentration of free Mg from 23 microM to 1.0 mM at the same ionic strength (50 mM). On stretching the fiber from 2.0 to 3.2 micron the force decreased; this effect was similar to that seen with Ca-activated fiber and the data support the idea that Ca-free tension is made at the cross-bridge level. Isotonic contraction during Ca-free activation showed a velocity transient as in Ca-activated fiber at 190 mM ionic strength, but the transient in the present case was very much prolonged. This finding suggests that contraction mechanisms for force generation and for shortening are essentially the same in the two conditions, but that certain rate constants of cross-bridge turnover are slower for the Ca-free contraction. Also, the results indicate that, in low ionic strength, Ca binding to thin filaments is not essential for unmasking the cross-bridge attachment sites, which suggests that the steric blocking mechanism is modified under these conditions.     

The vaso-contractile action of zooxanthellatoxin-B from a marine dinoflagellate is mediated via Ca2+ influx in the rabbit aorta

We previously showed that zooxanthellatoxin-B, isolated from dinoflagellate, caused a sustained contraction of the aorta in an external Ca2+-dependent manner. To clarify the role of Ca2+ in this action, we examined the effects of zooxanthellatoxin-B as well as a depolarizing stimulus (60 mM KCl), using the simultaneous recording for cytosolic Ca2+ level (fura-2) and developed tension in the rabbit aorta. KCl (60 mM) elicited a rapid cytosolic Ca2+ elevation followed by a pronounced contraction, and time required for half-maximum contraction was 2 min. Zooxanthellatoxin-B caused an increase in cytosolic Ca2+ followed by a gradual contraction, with a time for half-maximum contraction of 5-10 min in a concentration-dependent manner. We found a strong correlation between Ca2+ elevation and the contraction in zooxanthellatoxin-B action. In a Ca2+-free solution, zooxanthellatoxin-B caused neither the contraction nor the increase in cytosolic Ca2+. Furthermore, both pre- and post-treatment with verapamil, a voltage-operated Ca2+-channel blocker, partially suppressed both an increase in cytosolic Ca2+ and the contraction by zooxanthellatoxin-B. Zooxanthellatoxin-B-induced contraction was also inhibited by other voltage-operated Ca2+-channel blockers: nifedipine or diltiazem. These results suggest that zooxanthellatoxin-B-elicited contraction is caused by a Ca2+ influx into the smooth muscle cells, partially via voltage-operated Ca2+ channels.     

Nicorandil is a potent dilator of endothelin-induced vascular contraction

Nicorandil, an antianginal drug, is known to open K+ channel and to increase cGMP production. The effects of nicorandil on vascular contraction induced by endothelin (ET), a potent newly discovered vasoconstrictor peptide, were investigated using helical strips from rat thoracic aorta. ET at a concentration of 5 x 10(-9) M induced strong and persistent contraction in the presence of extracellular Ca2+ and similar persistent but smaller contraction in the absence of extracellular Ca2+. Nicorandil at concentrations greater than 10(-7) M, strongly and dose-dependently inhibited ET-induced contraction in the presence of extracellular Ca2+. Nicorandil also suppressed ET-induced contraction in the presence of 10(-4) M methylene blue, an inhibitor of cGMP production, in the presence of extracellular Ca2+ but not in the absence of extracellular Ca2+. ET-induced contraction was also inhibited to lesser extents by the Ca2+ channel blockers nicardipine and verapamil. Nicorandil also strongly suppressed ET-induced increase in cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells. These results suggest that nicorandil is a potent dilator of ET-induced vasoconstriction.     

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.     

Visualization of biphasic Ca2+ diffusion from cytosol to nucleus in contracting adult rat cardiac myocytes with an ultra-fast confocal imaging system.

In contracting cardiac myocytes, the rapid changes in cytosolic and nuclear Ca2+ make it difficult to determine whether the nuclear Ca2+ transient is caused by diffusion from the cytosol or by Ca2+ release channels on the inner nuclear membrane, or both. The propagation mechanism in the nucleoplasm also remains unknown. We have developed an ultra-fast Nipkow confocal imaging system able to acquire two-dimensional images at approximately 4 ms/full frame speed and employed it to analyze Ca2+ waves and the dynamics of the cytosolic and nuclear Ca2+ transients after electrical stimulation of cardiac myocytes. The pattern of nuclear Ca2+ upon stimulation was well described by a mathematical model of Ca2+ diffusion across the nuclear envelope. No evidence of Ca2+ release from perinuclear Ca2+ stores was obtained. The Ca2+ diffusion constant appeared to change during contraction, with essentially free diffusion of Ca2+ through nuclear pore complexes at low cytosolic Ca2+ and partially restricted diffusion at high cytosolic Ca2+. The Ca2+ in the nucleoplasm propagated by diffusion and no Ca2+ release phenomena were seen in the nucleus.     

Calcium requirement of uterine contraction induced by PGE1: Importance of intracellular calcium stores

The contraction of the rat uterus in response to PGE₁ in high K⁺ medium and in Ca-free solution which contained EDTA has been investigated in order to examine whether excitation-contraction coupling involves the release of Ca from an intracellular store.In uterus maximally contracted by K⁺, cumulative concentrations of PGE₁ (1.25 – 20 ng/ml) caused maintained concentration-dependent contraction. PGE₁ induced sustained contraction of rat uterus in Ca-free medium after incubation with 3mM EDTA for 50 min. In these conditions the involvement of extracellular Ca is highly unlikely. The PGE₁-induced contraction could be repeated without exposure to external Ca ions and with only slight reduction in magnitude. The PGE₁ concentrations required to elicit uterine contraction in Ca-free solution were about 1000 times higher than the effective doses in KCl-depolarized uterus.In conclusion, the present investigation shows that Ca influx is not essential for PGE₁-induced contraction of rat uterus, although extracellular Ca enhances it presumably by increasing the free Ca levels in the cytosol.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.     

Ca2+ homeostasis in unstimulated platelets

Unstimulated platelets maintain a low cytosolic free Ca2+ concentration and a steep plasma membrane Ca2+ gradient. The mechanisms that are required have not been completely defined. In the present studies, 45Ca2+ was used to examine the kinetics of Ca2+ exchange in intact unstimulated platelets. Quin2 was used to measure the cytosolic free Ca2+ concentration. Under steady-state conditions, the maximum rate of Ca2+ exchange across the platelet plasma membrane, 2 pmol/10(8) platelets/min, was observed at extracellular free Ca2+ concentrations 20-fold less than in plasma. Two intracellular exchangeable Ca2+ pools were identified. The size of the more rapidly exchanging pool (t 1/2, 17 min) and the cytosolic free Ca2+ concentration were relatively unaffected by large changes in the extracellular Ca2+ concentration. In contrast, the size of the more slowly exchanging Ca2+ pool (t 1/2, 300 min) varied with the extracellular Ca2+ concentration, which suggests that it is physically as well as kinetically distinct from the rapidly exchangeable Ca2+ pool. The locations of the Ca2+ pools were determined by differential permeabilization of 45Ca2+-loaded platelets with digitonin. 45Ca2+ in the rapidly exchanging pool was released with lactate dehydrogenase, which suggests that it is located in the cytosol. 45Ca2+ in the slowly exchanging pool was released with markers for both the dense tubular system and mitochondria, but inhibition of mitochondrial Ca2+ uptake with carbonyl cyanide m-chlorophenylhydrazone had no effect on the size of the slowly exchangeable Ca2+ pool or the cytosolic free Ca2+ concentration. In contrast, addition of metabolic inhibitors (KCN plus carbonyl cyanide m-chlorophenylhydrazone plus deoxyglucose) or trifluoperazine caused a decrease in the size of the slowly exchangeable Ca2+ pool and an increase in the cytosolic free Ca2+ concentration. These observations suggest that Ca2+ homeostasis in unstimulated platelets is maintained by limiting Ca2+ influx from plasma, actively promoting Ca2+ efflux, and sequestering Ca2+ within an internal site, which is most likely the dense tubular system and not mitochondria.     

Oxidation of Analogs of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Monoamine Oxidases A and B and the Inhibition of Monoamine Oxidases by the Oxidation Products

Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+-channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin-2-loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 microM EGTA or 100 microM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 microM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration-dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+-evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.     

Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and beta-adrenergic agonists

The alteration in the concentration of cytosolic free calcium ([Ca2+]i) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+]i of unstimulated cells was estimated to be 162.7 +/- 3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2+]i induced by CCh and ISP were approximately 100% and 25% of resting level, respectively. In Ca-free medium, CCh produced a small, rapid rise in [Ca2+]i, followed by a slow decay and a return to resting level within 3-4 min, while all doses of ISP tested failed to change [Ca2+]i. These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2+]i, whereas ISP may slightly mobilize only the extracellular Ca pool.     

Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.     

Endothelin-induced Ca-independent contraction of the porcine coronary artery

Front surface fluorometry and porcine coronary arterial strips loaded with fura-2 were used to investigate the effect of endothelin on cytosolic Ca concentrations, [Ca]i, and on contractile force, the objective being to elucidate the mechanism of action. Both in the presence and absence of extracellular Ca, endothelin induced rapid and dose-dependent increases in [Ca]i and in contraction. When caffeine-sensitive and histamine-sensitive intracellular Ca stores were depleted, in Ca-free medium, a transient contraction but no increase in [Ca]i followed the subsequent application of endothelin. This Ca-independent component was largely inhibited by the relative protein kinase C inhibitor, H-7, but not inhibited by W-7, calmodulin antagonist. This component is probably linked to activation of protein kinase C.     

Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.     

Mediator secretion in the nerve-muscle synapse in the frog following long-term effect of calcium-free solutions

The changes of spontaneous and evoked transmitter release in condition of long time (1-4 hours) incubation in Ca-free solution with EGTA adding, were investigated with extracellular recordings in experiments on the nerve-muscular junction of the frog cutaneous-pectoris muscle. Using the method of three extracellular microelectrodes recordings of the monoquantal postsynaptic signals, it was shown that during action of Ca-free solutions the topography of transmitter release changed, the specific spatial organization of points of transmitter release was disrupted. These changes remained after returning to the initial solution. The obtained data suggest that the Ca2+ free solution leads to disruption of active zones of nerve ending. In condition of low initial extracellular Ca2+ concentrations (0.15-0.4 mmol/l), the active zones disorganization led to decreasing of average amplitude of the end-plate currents (EPC) by decreasing their quantal content, increasing their time-course and decreasing the frequency of the miniature end-plate currents (MEPC). The sharp displacement of dependence of quantal contents of EPC in extracellular Ca2+ concentration to a higher Ca2+ concentration without significant changes of slope was revealed. In condition of high (1.8 mmol/l) concentration of Ca2+, the long action of Ca-free solutions leads to decreasing of amplitude of EPC too, but it was less obvious than in condition of initial low Ca2+ concentration. It is supposed that intra- and extracellular Ca concentration provides the support of the typical morpho-functional organization of the mechanisms of transmitter release at the nerve ending of the frog. The disorganization of active zones leads to separation of the elements, which take part at the transmitter release process and reduces the efficiency of secretion.     

Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.     

A comparison of uterine contraction induced by PGE1 and oxytocin in Ca-free solution

The contraction of the rat uterus incubated in Ca-free EDTA-containing solution in response to PGE1, oxytocin and vanadate has been investigated in order to examine the mechanism of the release of Ca from intracellular stores. The results obtained show that PGE1 evoked a sustained contraction the magnitude of which diminishes slightly after successive additions of PGE1 but not after long exposure to Ca-free medium. Oxytocin induced two different contractions: one of them was transient and observed only after incubating for 5 min in Ca-free solution; the other remained constant during prolonged incubation in Ca-free medium. Vanadate, an inhibitor of Ca-ATPase, induced sustained contraction after prolonged exposure to Ca-free medium, and isoprenaline, which stimulates Ca re-uptake by intracellular organelles, counteracted the sustained contractile response induced by the three agonists.