Alloxan diabetes blocks noradrenergic tachyphylaxis in the isolated rabbit kidney--effects of insulin.

Normal and alloxan treated diabetic rabbit kidneys were perfused with Krebs-Henseleit solution in a non-recirculating system and the effects of norepinephrine (NOR) 10(-6)M were tested by infusion of this drug for three subsequent periods of 20 min each, with an interval of 10 min for drug wash-out. In the control kidneys the infusion of NOR promoted an intense vasoconstriction, which was less intense during the second and the third periods. This was known as tachyphylaxis. In contrast to the controls, kidneys from diabetic animals did not show tachyphylaxis to NOR, but when insulin was added to the perfusate, tachyphylaxis appeared. Normal kidneys perfused with hyperosmolar solutions show, as in controls, the same phenomenon. The data presented here demonstrate a defect of adrenergic vascular receptors in alloxan treated kidneys, which can be acutely reversed by insulin. These facts are of importance for the understanding of the vascular disease in diabetes.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Effect of exogenous ATP upon inositol phosphate production, cationic fluxes and insulin release in pancreatic islet cells

Endogenous ATP is thought to play a key regulatory role in nutrient-stimulated insulin release. The present study deals with the effect of exogenous ATP and its stable analog alpha, beta-methylene ATP upon pancreatic islet function. Both alpha, beta-methylene ATP (5.0 microM to 0.2 mM) and ATP (0.3-3.0 mM) caused a rapid and concentration-related increase in insulin output by rat islets incubated or perfused at an intermediate concentration of D-glucose (8.3 mM). The effect of the ATP analog faded out at both lower and higher D-glucose concentrations. In the presence of 8.3 mM D-glucose, ATP also increased both 86Rb and 45Ca outflow from prelabelled islets. The cationic response to ATP persisted in the absence of extracellular Ca2+ and, hence, was reminiscent of that evoked by cholinergic agents. Like carbamylcholine, ATP caused a dose-related increase in the production of [3H]inositol phosphates from prelabelled islets or tumoral islet cells (RINm5F line). The latter effect was duplicated by alpha, beta-methylene ATP and unaffected by atropine. It is speculated that ATP, liberated together with insulin at the exocytotic site, might participate in a positive feedback control of insulin release.     

Calcium and renin release: inhibition of low sodium-induced renin secretion by high calcium concentration in rat kidney perfusion

The effects of changes in calcium on renin secretion have been studied in the isolated perfused rat kidney. Perfusion with free calcium buffer significantly decreases renin secretion as compared with control experiments (Ca++: 2.5 mM/l). Other calcium concentrations (1.25 mM/l) and 5 mM/l) do not affect basal renin secretion. When the renin release is previously increased by low sodium concentration (Na+: 110 mM/l) however, perfusion with high calcium buffer (Ca++: 5 mM/l) significantly inhibits this stimulation.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

The effects of alpha- and beta-adrenergic agents, Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 microM) and 1 microM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 microM), 50 microM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 microM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 microM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 microM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 microM) and epinephrine (1 microM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 microM but maximal concentrations of epinephrine (e.g., 1 microM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 microM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 microM) was added. Similarly, removal of 0.1 microM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either alpha or beta receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.     

Carbon monoxide effects on calcium levels in vascular smooth muscle

Previously we showed that carbon monoxide (CO) relaxes vascular smooth muscle in the working heart and thoracic aorta preparations perfused with hemoglobin-free, Krebs-Henseleit (KH) solution. The CO-induced relaxation was not caused by hypoxia, nor was it mediated by adrenergic influences, adenosine, or prostaglandins. In these studies the effect of CO on calcium (Ca++) concentrations in vascular smooth muscle was determined using 45Ca as a tracer. Isolated rat thoracic aorta segments were incubated with 45Ca and gassed with O2, N2, or CO for 60 min. Verapamil was used to verify the effectiveness of the test system. Ca++ concentrations were 488 +/- 35 and 515 +/- 26 mM/g tissue (X +/- SE) in aortic rings gassed with O2 and N2, respectively. CO reduced Ca++ concentrations significantly (P less than 0.01) by 29% to 369 +/- 18 mM/g tissue. Verapamil treatment reduced Ca++ concentrations by 40% to 314 +/- 23 mM/g tissue. These results suggest that CO relaxes vascular smooth muscle and dilates blood vessels by decreasing Ca++ concentrations in vascular smooth muscle.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

Ca2+, K+ redistributions and alpha-adrenergic activation of glycogenolysis in perfused rat livers

1. The alpha-adrenergic activation of glycogenolysis was investigated in isolated rat livers perfused in a non-recirculating system. Net uptake and/or release of Ca2+, K+ and H+ by the liver (measured by ion-selective electrodes) were correlated with the glycogenolytic effects of phenylephrine. Uptake and retention of 45Ca by the mitochondria of perfused livers were studied to obtain information on the role played by exchangeable mitochondrial calcium in alpha-adrenergic activation of glycogenolysis. 2. Between 1 and 5 min after starting the addition of phenylephrine a net release of Ca2+ was observed, this was paralleled by an uptake of K+. Production rates of glucose and lactate from endogenous glycogen started to increase at the same time. During the following minutes K+ was released. 2 mM EGTA and a high concentration of Mg2+ strongly diminished the ionic and metabolic responses to phenylephrine, 0.2 mM EGTA was less effective. 3. High concentrations of K+ prevented the metabolic response to phenylephrine but had no effect on the release of Ca2+ into the extracellular medium. Tetracaine activated glycogenolysis and suppressed all the effects of the alpha-adrenergic agonist. 4. Experiments with 45Ca provided no evidence for an alpha-adrenergic release of Ca2+ from the exchangeable mitochondrial pool. Incorporation of 45Ca into the mitochondria of perfused livers was enhanced by phenylephrine. 5. We propose that the alpha-adrenergic release of Ca2+ from a pool located close to the surface of the cell is capable of triggering the glycogenolytic response.     

Tiron exerts effects unrelated to its role as a scavenger of superoxide anion: effects on calcium binding and vascular responses

The effect of tiron (4,5-dihydroxy-1,3-benzene disulfonic acid) on the binding of Ca2+ and its effect on vascular responses of the rat perfused mesenteric bed was studied at concentrations of tiron that are used widely to scavenge superoxide anion. In competition assays in buffered solutions with no tissue present, tiron decreased the fluorescence ratio of fura-FF, a measure of [Ca2+]: the inhibition constant (Ki) of tiron with Ca2+ was 0.692 +/- 0.036 mM. In the mesenteric bed perfused at constant flow and preconstricted with 90 mM KCl, tiron evoked decreases in perfusion pressure of the mesenteric bed in a concentration-dependent manner (Rmax = 43.58 +/- 2.6 mmHg; EC50 = 1.46 +/- 0.33 mM). This vasodilator effect of tiron was similar in the presence of the superoxide anion scavenger, tempol (Rmax = 46.12 +/- 1.87 mmHg; EC50 = 1.34 +/- 0.27 mM). In the presence of 90 mM KCl, increasing concentrations of Ca2+ increased perfusion pressure and tiron shifted the concentration-response curve to Ca2+ to the right. In freshly drawn blood from rats, tiron increased clotting time. The data indicate that tiron binds Ca2+ at concentrations at or below those commonly used to scavenge superoxide anion, an action that may be responsible for a variety of biological responses. The interpretation of effects of tiron in previous work on the role of superoxide anion may need to be re-evaluated.     

Effects of diabetes and insulin on ketone bodies metabolism in heart

This work investigates the effect of alloxan-induced short-term diabetes (24 h) on D-3-hydroxybutyrate metabolism at physiological and non-physiological concentrations of the ketone body in the isolated non-working perfused rat heart. Also the effect of insulin (2 mU.ml⁻¹) on D-3-hydroxybutyrate metabolism was investigated in hearts from normal and diabetic rats. The rates of D-3-hydroxybutyrate utilization and oxidation and of acetoacetate production were proportional to D-3-hydroxybutyrate concentration. The utilization of D-3-hydroxybutyrate showed saturation kinetics in hearts from normal and diabetic rats, in the presence and absence of insulin. Acute short-term diabetes augmented D-3-hydroxybutyrate utilization and oxidation at 1.25 and 2.5 mM DL-3-HB, with no significant effect at higher concentrations, but increased acetoacetate production at all investigated concentrations. In hearts from normal rats, insulin enhanced D-3-hydroxybutyrate utilization and oxidation at 2.5, 5, and 10 mM DL-3-HB, but no effect was observed at the lowest (1.25 mM) and highest (16 mM) DL-3-HB concentrations. Insulin had no effect on D-3-hydroxybutyrate metabolism in hearts from diabetic rats. No significant effect of insulin on the rate of acetoacetate production in normal and diabetic states was observed.     

Effect of beta-hydroxybutyrate and acetoacetate on insulin and glucagon secretion from perfused rat pancreas

To elucidate the physiological significance of ketone bodies on insulin and glucagon secretion, the direct effects of beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc) infusion on insulin and glucagon release from perfused rat pancreas were investigated. The BOHB or AcAc was administered at concentrations of 10, 1, or 0.1 mM for 30 min at 4.0 ml/min. High-concentration infusions of BOHB and AcAc (10 mM) produced significant increases in insulin release in the presence of 4.4 mM glucose, but low-concentration infusions of BOHB and AcAc (1 and 0.1 mM) caused no significant changes in insulin secretion from perfused rat pancreas. BOHB (10, 1, and 0.1 mM) and AcAc (10 and 1 mM) infusion significantly inhibited glucagon secretion from perfused rat pancreas. These results suggest that physiological concentrations of ketone bodies have no direct effect on insulin release but have a direct inhibitory effect on glucagon secretion from perfused rat pancreas.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

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.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

Calcium-induced membrane metabolic alterations modify the sex steroids binding into dog brain synaptosomal plasma membranes

The binding of 45Ca2+ into synaptosomal plasma membranes (SPM) of dog brain follows a sigmoid path. In graphical analysis of this binding the mean Hill coefficient (h) was 1.64 +/- 0.09 (r2 = 0.96 +/- 0.02). Binding of Ca2+ into SPM was saturable, with an apparent binding constant of 1.2 +/- 0.1 microM. At saturation, such calcium specific binding sites corresponded to 11.2 +/- 0.9 nmol/mg SPM protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of calcium into SPM of at least one class of high affinity specific binding sites. [14C]estradiol, [14C]estrone and [14C]progesterone, when incubated with SPM up to a concentration of 10 microM for 2 hr at 37 degrees C, bind into SPM at nmolar concentrations. Ca2+ ions up to 5 mM considerably increase steroids binding into SPM. This effect of calcium was concentration-dependent, reached saturation at approx 4-5 mM. Once calcium has promoted steroids binding, the subsequent addition of 25 mM EGTA failed to displace bound steroids. Molecular interactions between calcium and SPM was assessed by measuring the steady-state fluorescence polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the production of malondialdehyde (MDA) during 2 hr incubation of Ca2+ (5 mM) with SPM at 37 degrees C. The effect of Ca2+ on the SPM structure was to increase both the rigidity of the membrane and the MDA production. Chelation of Ca2+ (5 mM) with EGTA (25 mM) did not reverse the increase in the rigidity owing to metabolic alterations of SPM lipids (e.g. production of MDA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic effect of local anesthetics on the adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes

Previous studies have shown that an adenosine triphosphate-dependent calcium uptake activity in lysed brain synaptosomes is attributable to the neuronal endoplasmic reticulum elements. The present study has examined the effects of tetracaine, lidocaine, and dibucaine on this calcium uptake process. The adenosine triphosphate-dependent uptake of 45Ca2+ was measured (in the absence and in the presence of drug) by Millipore filtration and liquid scintillation spectrometry. The local anesthetics studied exhibited a biphasic effect on 45Ca2+ uptake by lysed synaptosomes from rat brain cortex. High concentrations (5 mM tetracaine, 50 mM lidocaine, 0.6 mM dibucaine) inhibited the uptake of 45Ca2+; the order of potency for this effect was dibucaine greater than tetracaine greater than lidocaine. Lower concentrations of these local anesthetics produced either no effect on 45Ca2+ uptake (2 mM tetracaine or 30 mM lidocaine) or a stimulation of 45Ca2+ uptake (1 mM tetracaine, 10 mM lidocaine, and 0.3 mM or 0.1 mM dibucaine); the order of potency for stimulation of 45Ca2+ uptake was dibucaine greater than tetracaine greater than lidocaine.     

The Ca2+-dependent actions of the alpha-adrenergic agonist phenylephrine on hepatic glycogenolysis differ from those of vasopressin and angiotensin

The stimulation of hepatic glycogenolysis by the Ca2+-dependent hormones phenylephrine, vasopressin and angiotensin II was studied as a function of intracellular and extracellular Ca2+. In the isolated perfused rat liver the decline in glucose formation was monophasic ('half-life' approximately equal to 3 min) with vasopressin (1 nM) or angiotensin II (0.05 microM), but biphasic (half-life of 4.8 min and 17.6 min) in the presence of the alpha-agonist phenylephrine (0.01 mM), indicating either a different mode of mobilization or the mobilization of additional intracellular calcium stores. Under comparable conditions an elevated [Ca2+] level was maintained in the cytosol of hepatocytes for at least 10 min in the presence of phenylephrine, but not vasopressin. Titration experiments performed in the isolated perfused liver to restore cellular calcium revealed differences in the hormone-mediated uptake of Ca2+. The onset in glucose formation above that seen in the absence of exogenous calcium occurred at approximately 30 microM or 70-80 microM Ca2+ in the presence of phenylephrine or vasopressin respectively. The shape of the response curve was sigmoidal for vasopressin and angiotensin II, but showed a distinct plateau between 0.09 mM and 0.18 mM in the presence of phenylephrine. The plateau was also observed at phenylephrine concentrations as low as 0.5 microM. The formation of plateaus observed after treatment of the liver with A 23187, but not after EGTA, is taken as an indication that intracellular calcium stores are replenished. A participation of the mitochondrial compartment could be excluded by pretreatment of the liver with the uncoupler 2,4-dinitrophenol. Differences in the Ca2+ dependence of the glycogenolytic effects of these hormones were also revealed by kinetic analysis. It is concluded that phenylephrine differs from vasopressin and angiotensin II in that, in addition to a more common, non-mitochondrial pool, which is also responsive to the vasoactive peptides, the agonist mobilizes Ca2+ from a second, non-mitochondrial pool. The results are consistent with the proposal that Ca2+ transport across subcellular membranes may be subject to different hormonal control.