Agonist induced release of intracellular Ca2+ in the rabbit aorta.

The effects of hormonal agonists (norepinephrine, angiotensin, and histamine) on 45Ca efflux from the rabbit aorta were studied using a Ca-EGTA buffered efflux medium. Each caused a transient stimulation of efflux rate which probably reflected the release of an intracellular 45Ca store. The size of the stimulation of efflux correlated with the size of the initial rapid phase of contraction. The norepinephrine-sensitive intracellular Ca fraction was estimated to be greater than 21 mumoles/Kg wet tissue weight. This fraction is separate from intracellular Ca which is accumulated during relaxation. Evidence is presented for the lack of cyclic nucleotide involvement in the release of Ca2+, and possible alternative modes of coupling are discussed.     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

The influence of high potassium depolarization and acetylcholine on calcium exchange in the rat uterus

Net and radioactive calcium movements were studied in the rat uterus during stimulation with acetylcholine and high potassium solutions. High potassium did not affect the efflux of intracellular Ca⁴⁵, but was able to release Ca⁴⁵ from a small parallel Ca fraction which was believed to be located in the cell membranes. High potassium did markedly slow the influx of Ca⁴⁵ and caused a net calcium efflux. Acetylcholine had no effect on calcium movements in polarized myometrium, but it increased the Ca⁴⁵ influx in depolarized uteri. Ca⁴⁵ taken up during contraction exchanged more slowly during subsequent efflux than Ca⁴⁵ taken up at rest. The results were interpreted as supporting the hypothesis that myometrial contraction is induced by a release of calcium from the inside of the cell membrane and the endoplasmic reticulum, and relaxation follows the removal of ionic cytoplasmic calcium by these same structures.     

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.     

Ca++ fluxes in isolated cells of rat pancreas. Effect of secretagogues and different Ca++ concentrations

Summary Secretagogues of pancreatic enzyme secretion, the hormones pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, and caerulein as well as the Ca⁺⁺-ionophore A 23187 stimulate⁴⁵Ca efflux from isolated pancreatic cells. The nonsecretagogic hormones adrenaline, isoproterenol, secretin, as well as dibutyryl cyclic adenosine 3,5-monophosphate and dibutyryl cyclic guanosine 3,5-monophosphate have no effect on⁴⁵Ca efflux. Atropine blocks the stimulatory effect of carbamylcholine on⁴⁵Ca efflux completely, but not that of pancreozymin. A graphical analysis of the Ca⁺⁺ efflux curves reveals at least three phases: a first phase, probably derived from Ca⁺⁺ bound to the plasma membrane; a second phase, possibly representing Ca⁺⁺ efflux from cytosol of the cells; and a third phase, probably from mitochondria or other cellular particles. The Ca⁺⁺ efflux of all phases is stimulated by pancreozymin and carbamylcholine. Ca⁺⁺ efflux is not significantly effected by the presence or absence of Ca⁺⁺ in the incubation medium. Metabolic inhibitors of ATP production, Antimycin A and dinitrophenol, which inhibit Ca⁺⁺ uptake into mitochondria, stimulate Ca⁺⁺ efflux from the isolated cells remarkably, but inhibit the slow phase of Ca⁺⁺ influx, indicating the role of mitochondria as an intracellular Ca⁺⁺ compartment. Measurements of the⁴⁵Ca⁺⁺ influx at different Ca⁺⁺ concentrations in the medium reveal saturation type kinetics, which are compatible with a carrier or channel model. The hormones mentioned above stimulate the rate of Ca⁺⁺ translocation.The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca⁺⁺ transport most likely at the level of the cell membrane and that Ca⁺⁺ exchange diffusion does not contribute to the⁴⁵Ca⁺⁺ fluxes.With the technical assistance of C. Hornung.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

Stimulation of 45Ca efflux from rat lacrimal gland slices by carbachol and epinephrine

The effects of carbachol (10^?5M) and epinephrine (10^?5M) on efflux of ⁴⁵Ca from rat exorbital lacrimal gland slices were examined. Both carbachol and epinephrine stimulated a transient release of ⁴⁵Ca from the tissue. The quantity of Ca released was estimated to be of the order of 0.5 μmol/g. Release of ⁴⁵Ca by one agonist prevented subsequent release of ⁴⁵Ca by a different agonist. These data support the hypothesis put forth previously that in the lacrimal gland muscarinic or α-adrenergic receptor activation causes a transient increase in membrane permeability to K by triggering the release of a sizable intracellular pool of Ca common to both receptors.     

Active chloride secretion by rabbit colon: Calcium-dependent stimulation by ionophore A23187

Summary Addition of Ca ionophore, A23187, to the solution bathing the mucosal surface of descending rabbit colon resulted in a reversal of active Cl absorption to active Cl secretion, a twofold increase in short-circuit current and a 40% increase in tissue conductance without affecting the rate of active Na absorption. These alterations in electrolyte transport are quantitatively similar to those previously observed in response to cyclic 3,5-AMP (cAMP) (R.A. Frizzell, M.J. Koch & S.G. Schultz,J. Membrane Biol. 27:297, 1976). When medium Ca concentration was reduced to 10^–6 m, the secretory response to A23187 was abolished but the response to cAMP was unaffected. The ionophore did not influence the cAMP levels of colonic mucosa. Addition of cyclic AMP to colonic strips preloaded with⁴⁵Ca elicited a reversible increase in Ca efflux from the tissue. These results suggest that an increase in intracellular Ca concentration stimulates colonic electrolyte secretion and that the secretory response to cAMP may be due, at least in part, to a release of Ca from intracellular stores.     

Computer simulation and interpretation of45Ca efflux profile patterns

Summary Stimulations or inhibitions by various agents of⁴⁵Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of⁴⁵Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because⁴⁵Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of⁴⁵Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance (⁴⁰Ca), the tracer (⁴⁵Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries.     

Chronic ethanol inhibits rat hippocampal “stimulus-secretion” coupling mechanism for 5-hydroxytryptamine in vitro

Effects of ethanol on serotonergic neurotransmission were investigated in crude mitochondrial fraction (P₂ fraction) from rat brain hippocampus and hypothalamus. The [¹⁴C]5-HT preloaded P₂ fraction was exposed to 45 mM KCl to induce 5-hydroxytryptamine release in vitro. Ethanol in vitro did not produce any significant inhibition of [¹⁴C]5-HT release until its concentration was greater than 100 mM. The K⁺-evoked⁴⁵Ca uptake of hippocampal P₂ fraction was unaffected b6 100 mM. However, 200 mM ethanol inhibited approximately 63% of K⁺-evoked⁴⁵Ca uptake. Chronic ethanol (10g/kg/day) for 6 days inhibited [¹⁴C]5-HT release from hippocampus whereas it did not affect [¹⁴C]5-HT release from hypothalamus. Results indicate that chronic ethanol treatment may decrease serotonergic neurotransmission in selective brain regions. The reduction in 5-hydroxytryptamine release was the result of inhibition in stimulus-secretion coupling mechanism.The views expressed in this paper are those of the authors and do not necessarily reflect those of the Addiction Research Foundation.     

The Action of Serotonin on Calcium-45 Efflux from the Anterior Byssal Retractor Muscle of Mytilus edulis

⁴⁵Ca efflux was studied in resting anterior byssal retractor muscle. The data are described by a three-compartment system. The most rapidly exchanging compartment, with an average time constant of 7 min, contains about 0.9 mM Ca/liter muscle, and probably represents extracellular space. A second compartment, with a time constant of 83 ± 5 min, contains 1.2 mM Ca/liter, and may represent a membrane calcium store. The presence of a third, or more, compartments, probably representing sarcoplasmic reticulum and contractile proteins, is indicated by the fact that the final time constant is 10 times the 83 min time constant of the second compartment. Serotonin (5HT), on initial application, increases ⁴⁵Ca efflux from this third compartment(s). This effect has a typical dose-response relationship with a maximum response appearing at 10^-7 M5HT. In addition, removal of 5HT causes a secondary increase in ⁴⁵Ca efflux which has a maximum at a 5HT concentration of 10^-7 M and declines at both higher and lower doses.     

Effects of Na readmission on cellular45Ca fluxes in Na-Depleted guinea pig taenia coli

Summary The removal of Na from the medium causes a cellular Ca uptake in the smooth muscle of the guinea pig taenia coli which is rapidly reversed if medium Na is readmitted. This net extrusion was characterized in tissues which were first Na-depleted in a zero-Na (sucrose) solution. Li was able to substitute for Na in mediating this effect. K was also able to mimic Na in this respect if the depolarization-mediated Ca influx caused by the isotonic K solution was blocked with 10^–5 m D-600. The net Ca extrusion upon Na readmission was due to a small decrease in Ca influx, as well as a marked increase in the transmembrane Ca efflux rate, as revealed by⁴⁵Ca washout experiments. The increased⁴⁵Ca efflux upon Na readmission could be mimicked by Li, K, choline and tris. We conclude that the Na/Ca-exchange hypothesis is insufficient to explain these data, in that both Ca extrusion and⁴⁵Ca efflux can be stimulated in the absence of a Na gradient, or in the absence of any monovalent cationic gradient. These observations are discussed in terms of a possible intracellular competition of Ca and monovalent cations for anionic binding sites, as well as with regard to a possible direct stimulation of a plasmalemmal CaATPase by monovalent cations.     

Calcium and pancreatic β-cell function: 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools

β-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of ⁴⁵Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37°C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable ⁴⁵Ca. The stimulatory effect of phosphate was additive to that produced by glucose. ⁴⁵Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of ⁴⁵Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated ⁴⁵Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic β-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Calcium and pancreatic β-cell function 7. Evidence for cyclic AMP-induced translocation of intracellular calcium

The effect of cyclic AMP on calcium movements in the pancreatic β-cell was evaluated using an experimental approach based on in situ labelling of intracellular organelles of ob/ob-mouse islets with ⁴⁵Ca. Whereas the glucose-stimulated ⁴⁵Ca incorporation by mitochondria and secretory granules was increased under a condition known to reduce cyclic AMP (starvation), raised levels of this nucleotide (addition of 3-isobutyl-1-methylxanthine or N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic monophosphate) reduced the mitochondrial accumulation of ⁴⁵Ca. Conditions with increased cyclic AMP were associated with a stimulated efflux of ⁴⁵Ca from the secretory granules but not from the mitochondria. The microsomal fraction differed from both the mitochondrial and secretory granule fractions by accumulating more ⁴⁵Ca after the addition of 3-isobutyl-1-methylxanthine. The results suggest that cyclic AMP potentiates glucose-stimulated insulin release by increasing cytoplasmic Ca²⁺ at the expense of the calcium taken up by the organelles of the pancreatic β-cells.     

Hormone-evoked calcium release from intracellular stores is a quantal process

Ca2+ mobilization by hormones, ionomycin, and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) were studied to determine whether Ca2+ release is a continuous or a quantal process. Hormone-mediated Ca2+ release occurs only during the first 2-4 s of stimulation. Stimulation of acini with a maximal hormone concentration following stimulation with a submaximal concentration resulted in free cytosolic Ca2+ concentration ([Ca2+]i) increase and 45Ca efflux. The peak [Ca2+]i increase induced by a maximal concentration of agonist was nearly constant when cells were prestimulated with a submaximal dose for 1-15 min. Submaximal hormone concentrations release only a fraction of intracellular 45Ca2+, after which intracellular Ca2+ content remains constant. The partially released stores remain depleted until cell stimulation is terminated, at which time the stores reload with Ca2+. For comparison, increasing concentrations of ionomycin resulted in increasing rates of Ca2+ release. Each ionomycin concentration released all the Ca2+ from intracellular stores. We therefore conclude that hormone-evoked Ca2+ release is a quantal rather than a continuous process. In permeabilized cells, increasing concentrations of Ins-1,4,5-P3 resulted in an increased fraction of Ca2+ release. No submaximal Ins-1,4,5-P3 concentration was capable of releasing all the Ins-1,4,5-P3-mobilizable Ca2+. Therefore, it appears that the quantal properties of hormone-evoked Ca2+ release reflect the quantal properties of Ins-1,4,5-P3-mediated Ca2+ release from intracellular stores.     

Quinine and caffeine effects on 45Ca movements in frog sartorius muscle

1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of ⁴⁵Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of ⁴⁵Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on ⁴⁵Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of ⁴⁵Ca by normally polarized muscle. However, there are variable effects of quinine upon ⁴⁵Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release ⁴⁵Ca from muscles that have been washed in Ringer''s solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca²⁺ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K⁺ or an action potential. In respect to the myoplasmic Ca²⁺ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.     

Extrusion of calcium from a single isolated neuron of the snailHelix pomatia

Summary Simultaneous optical measurements of extra- and intracellular Ca²⁺ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca²⁺. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the microchamber containing the cell. The velocity of Ca²⁺ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2–0.5 m, the velocity of Ca²⁺ extrusion from the neuron was approximately 0.3–4.6 m/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca²⁺ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 m/sec per cell volume.