Stimulation Of Ca Uptake Research Articles

Relationship between cyclic AMP-dependent protein kinase activation and Ca uptake increase of sarcoplasmic reticulum fraction of hog biliary muscles relaxed by cholecystokinin-C-terminal peptides

In hog terminal bile duct cholecystokinin peptides caused an activation of cyclic AMP-dependent protein kinase (A-PK) with cyclic AMP, followed by increase in Ca uptake of sarcoplasmic reticulum fraction (SR-F). By contrast, papaverine showed no activation of A-PK-induced Ca uptake by SR-F with cyclic AMP. The Ca uptake by SR-F was dependent on ATP and Mg 2, but the component phosphorylated was not the phosphoenzyme intermediate in Ca 2-ATPase. The effect of Ca uptake was blocked by the inclusion of a protein inhibitor of A-PK. The correlation coefficient between cyclic AMP-dependent SR-F phosphorylation and stimulated Ca uptake by the phosphorylated SR-F was 0.731 (P < 0.01). These results suggest that one of the mechanisms by which CCK-4, CCK-8, and CCK-33 peptides relax isolated Oddi's sphincters of terminal bile ducts is activation of A-PK-induced Ca uptake by sarcoplasmic reticulum fraction and possibly also by plasma membrane.

Effects of neurotransmitter candidates on 45Ca uptake by cortical slices of rat brain: Stimulatory effect of l-glutamic acid

The effects of neurotransmitter candidates and the characteristics of the stimulatory effect of l-glutamic acid ( l-Glu) on 45Ca uptake by rat brain slices were investigated. 45Ca uptake was significantly stimulated by acetylcholine, serotonin and especially l-Glu, but not by other neurotransmitter candidates. L-Glu caused dose-dependent stimulation of 45Ca uptake ( L-Glu-stimulated 45Ca uptake), its effect being half-maximal at 1 μM. The related compounds D-glutamic acid, D, L-α-aminoadipic acid and N-methyl- D, L-glutamic acid (final conc. of 10 μM) also stimulated 45Ca uptake, but less than 10 μM L-Glu. D, L-α-Methylglutamic acid and L-glutamic acid diethylether (final conc. of 10 μM), which are specific inhibitors of L-Glu, inhibited L-Glu-stimulated 45Ca uptake. Mg, Ca-ATPase activity was hardly affected by a concentration of 10 μM L-Glu that caused maximal stimulation of 45Ca uptake. These findings suggest that L-Glu-stimulated 45Ca uptake by brain cortical slices is linked to L-Glu receptor.

Taurine activation of a bicarbonate-dependent, ATP-supported calcium uptake in frog rod outer segments.

Isolated frog rod outer segments (ROS) with a leaky plasma membrane showed a bicarbonate-dependent, ATP-activated 45Ca accumulation. This calcium uptake requires magnesium and is specific for ATP; other nucleotides, ITP, GTP, UTP and the non-hydrolysable analogue of ATP beta-gamma-methylene ATP did not substitute for ATP. 45Ca accumulation was inhibited by mersalyl, ethylmaleimide, ruthenium red, oligomycin and dicyclohexylcarbodiimide and was unaffected by ouabain. Addiction of taurine to the incubation medium enhanced 45Ca uptake in a concentration-dependent manner; increases of more than 100% being produced by 25 mM taurine. The taurine-induced stimulation of 45Ca uptake was also sensitive to the tested inhibitors. The effect of taurine was only exerted on the bicarbonate-dependent, ATP-activated 45Ca uptake. Calcium accumulation observed in the absence of ATP or in a tris-buffered medium was unaffected by taurine. Other amino acids, glycine, GABA, beta-alanine, glutamic acid and the taurine analogue guanidinoethyl-sulfonate did not stimulate 45Ca uptake. These results suggest that taurine is affecting a Mg-ATPase activity responsible for calcium accumulation in frog ROS.

Inhibitory effect of microtubule-disrupting agents on potassium stimulated 45Ca uptake by rat brain slices.

Inhibitory effect of microtubule-disrupting agents on potassium stimulated 45Ca uptake by rat brain slices.

Does endogenous cyclic GMP inhibit potassium stimulated 45Ca uptake by P 2 fraction from rat brain?

The effects of sodium azide (NaN 3), hydroxylamine (NH 2OH) and sodium nitroprusside on potassium-stimulated 45Ca uptake (K-stimulated 45Ca uptake) by P 2 fraction of Gray and Whittaker from rat brain were investigated. During preincubation with these reagents, the contents of cyclic GMP in synaptosomes increased, reaching maximum levels within 2 min. On preincubation for 2 min, NaN 3, an activator of membrane bound guanylate cyclase, inhibited K-stimulated 45Ca uptake, but NH 2OH and sodium nitroprusside did not affect it. Sodium cyanide, another metabolic inhibitor, had no effect on K-stimulated 45Ca uptake. There was a correlation between inhibition of K-stimulated 45Ca uptake and increase in the cGMP level on preincubation with NaN 3 for various periods. Based on these results role of cGMP in or around the membrane was discussed in relation to the K-stimulated 45Ca uptake by P 2 fraction.

Potassium stimulated 45Ca uptake by cortical slices of rat brain: effects of cyclic nucleotide derivatives

The effects of dibutyryl cyclic GMP (db-cGMP) and dibutyryl cyclic AMP (db-cAMP) on potassium-stimulated 45Ca uptake by cortical slices of rat brain were investigated. db-cAMP specifically inhibited the initial rate of potassium-stimulated 45Ca uptake in a dose-dependent manner. Our findings supported the suggestion that cyclic GMP may play a regulatory role in depolarization-elicited Ca 2+ influx in nerve endings in situ.

Diltiazem and verapamil inhibit norepinephrine-stimulated 45Ca uptake in rabbit aorta.

The effects of two Ca antagonists, diltiazem (DZ) and verapamil (VP), on norepinephrine (NE)-stimulated 45Ca uptake in vascular smooth muscle from New Zealand White rabbit aortas were studied. Data were collected before, at 10, 30, and 60 min after drug addition, and during a simultaneous control period without drug addition. NE alone (6 X 10(-6) M) significantly increased 45Ca uptake from the extracellular space presumably by activating the receptor-operated Ca channel during excitation-contraction coupling. This effect was maximal by 10 min after NE addition and stable through the 60-min time point. Both VP (5 X 10(-5) M) and DZ (2.2 X 10(-7) M) inhibited the NE-stimulated 45Ca uptake at the 10- and 60-min time points, respectively. These data demonstrate that both DZ and VP inhibit 45Ca uptake from the extracellular space during activation of the receptor-operated Ca channel with NE. The effects of DZ and VP to inhibit NE-stimulated 45Ca uptake are demonstrated at concentrations which have been previously shown to cause dilation of vascular smooth muscle.

Binding of scorpion neurotoxins to chick embryonic heart cells in culture and relationship to calcium uptake and membrane potential

Stimulation of 45Ca uptake by scorpion neurotoxins in cultured chick embryonic heart cells has been shown to be directly linked to their effect on sodium channels. This property was used to compare the activity of 15 neurotoxins from five different species to their lethal effect in the mouse and immunological properties. As scorpion neurotoxins, the alkaloid neurotoxin veratridine enhanced 45Ca uptake, and an apparent positive cooperativity between the two drugs was observed. 125I-Labeled toxin II from the scorpion Androctonus australis Hector was shown to bind to chick heart cells specifically, saturably, and reversibly with high affinity (KD = 1--3 nM in sodium-free medium) and low capacity (10--20 fmol/mg cell protein). As shown by 45Ca uptake and radioactive toxin binding experiments, the affinity of scorpion neurotoxin to heart cell receptors was dependent on external K+ concentration. Toxin binding was lowered by increasing Na+ concentration in the medium and was abolished by veratridine in a sodium (140 mM) containing medium. As previously reported for neuroblastoma cells, all these results are in agreement with the membrane potential dependence of scorpion neurotoxin affinity for its membrane receptor.

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.

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase b kinase (EC 2.7.1.38). (b) The absence of Ca 2+ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca 2+ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of 45Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate 45Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced 45Ca uptake and the phosphorylase activation. We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca 2+ is most probably phosphorylase b kinase.

Inhibition of calcium uptake by somatostatin in isolated rat islets of Langerhans.

Somatostatin (1 mug/ml) inhibited glucose (16.7 mM)-stimulated 45Ca uptake by isolated rat islets incubated in media containing no added calcium or calcium at a low concentration (0.2mM). Epinephrine (50 mug/ml) and mannoheptulose (20mM) also inhibited 45Ca uptake by islets incubated in the presence of calcium (0.2mM). Addition of glucose (16.7 mM) caused a small but significant increase in insulin release from islets incubated in media containing no added calcium. In the presence of a low concentration of calcium (0.2 mM), glucose caused a much greater increase in insulin secretion which was inhibited by addition of somatostatin, epinephrine or mannoheptulose.

Calcium ion uptake in isolated pancreas cells induced by secretagogues

1. 1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein the Ca 2+ inophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3′,5′-monophospate and dibutyrylic cylic guanosine 3′,5′-monophospate have no effect on 45Ca uptake. 2. 2. A graphical analysis of the Ca 2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca 2+ to the membrane and a slow phase representing Ca 2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. 3. The 45Ca-exchangeable pool size is increased by both carbamylchloline and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca 2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca 2+ tranfer into the cell most probably through an increase of the cell membrane permeability for Ca 2+.

Effects of potassium, veratridine, and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro.

1. 45-Ca uptake by pinched-off nerve terminals (synaptosomes) of rat brain incubated in standard physiological saline (including 132 mM-Na + 5mM-K + 1-2 mM-Ca) at 30 degrees C averages about 0-5 mumole Ca per g protein per minute. This may be equivalent to a Ca influx of about 0-03 p-mole/cm-2 sec. 2. The rate of 45-Ca uptake is increased when the concentration of K in the medium is increased above 15-20 mM, K replacing Na isosmotically. Maximum stimulation, a three- to six-fold increase in the rate of Ca uptake, occurs when [K]o is about 60 mM. The effect of increased [K]o is reversible. 3. The K-stimulated Ca uptake is associated primarily with the nerve terminal fraction of brain homogenates. The entering Ca is not accompanied by extracellular markers such as mannitol or inulin. Replacement of external chloride by methylsulphate or sulphate does not prevent the stimulation by K. 4. The effects of external K are quantitatively mimicked by Rb. Caesium also stimulates Ca uptake, but is only about one fifth as effective as K or Rb; Li is ineffective. 5. Two other depolarizing agents also stimulate Ca uptake by synaptosomes: veratridine (7-5 times 10- minus 6 to 7-5 times 10- minus 5 M) and scorpion (Leirus quinquestriatus) venom (6-7 times 10- minus 7 to 6-7 times 10- minus g/ml.). The stimulatory effects of veratridine and scorpion venom, but not of increased [K] are blocked by 2 times 10- minus 7 M tetrodotoxin. 6. Internal K also influences the rate of 45-Ca uptake by synaptosomes: lowering [K]i reduces the stimulatory effect of external K and veratridine. 7. Replacement of external Na by choline markedly inhibits the response to veratridine, but has a much smaller effect on the response to increased [K]o. 8. The Ca uptake mechanism has an apparent dissociation constant for Ca (KCa) of about 0-8 mM. Increasing [K]o increases the maximal rate of Ca uptake, but has no effect on KCa. The K-induced 45-Ca uptake is competitively inhibited by Mg-2+, Mn-2+ and La-3+. 9. The release of acetylcholine and noradrenaline was also studied. Increasing [K]o stimulates external Ca-dependent acetylcholine release. Scorpion venom stimulates noradrenaline release from synaptosomes; this effect could be prevented by adding tetrodotoxin or removing external Ca. 10. These results indicate that synaptosomes may increase their permeability to Ca, accumulate Ca and release neural transmitter substances, when stimulated by depolarizing agents under appropriate physiological conditions.

The effect of veratridine on sodium-sensitive radiocalcium uptake in frog sartorius muscle

Veratridine increases the ability of sodium to displace calcium from muscle binding sites at physiological Ca 0 2+ concentrations of 1.0 mM. Lithium and hydrazine both of which can substitute for sodium with regard to the muscle action potential are much less effective in depressing calcium binding in the presence of veratridine. Lithium or hydrazine substitution for sodium prevents or delays the veratrine response. The uptake of 45Ca into the slow component of muscle as measured by 45Ca desaturation curves is enhanced by veratridine when the TTX treated sartorius muscle is exposed to 20 mM K +. Substitution of sodium by lithium or hydrazine does not interfere with the veratridine stimulated 45Ca uptake in the presence of high potassium. These findings indicate that veratridine affects calcium binding sites in the surface of the muscle fiber so as to increase the effectiveness of sodium in competing with calcium for these sites. Veratridine also appears to increase the transient calcium influx observed upon depolarization of the muscle fibers by high external potassium.

The stimulus-secretion coupling of glucose-induced insulin release. 3. Uptake of 45 calcium by isolated islets of Langerhans.

The role of calcium in glucoseinduced insulin secretion was examined by measuring 45Ca uptake by isolated islets of Langerhans. The islets were incubated for 90 min in the presence of 45Ca, submitted to successive washings, and finally examined for their radioactive content. Glucose, but not galactose, stimulated 45Ca uptake by the islets. A sigmoidal curve characterized the relationship between 45Ca uptake and the glucose concentration of the incubation medium. Glucose-induced calcium uptake was increased in media in which Na+ had been partially replaced by K+, and inhibited by mannoheptulose, 2-deoxyglucose, epinephrine, diazoxide, low temperature, low pH, high Mg++ concentration, low Ca++ concentration, and in media in which Na+ had been partially replaced by Li+. Under these experimental conditions, the, changes in 45Ca uptake paralleled those in insulin secretion by the islets. However, although glucose failed to induce insulin release at low Ca++ concentration, the reduced uptake of calcium found under this experimental condition was still markedly decreased by the addition of mannoheptulose to the incubation medium. Thus, the rate of calcium uptake is not merely a function of the rate of insulin secretion. The data rather suggest that glucose, when metabolized within the beta-cell, stimulates calcium uptake, which in turn triggers the release of insulin. (Endocrinology88: 72, 1971)