Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca²⁺ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca²⁺ stimulation of ATP hydrolysis was not discernible above the high background of Ca²⁺-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca²⁺ concentration, suggesting that Ca²⁺ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca²⁺ at half maximal velocity of uptake (S_0.5) was 12.5 micromolar and the Hill coefficient (n_H) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca²⁺, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca²⁺ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca²⁺ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca²⁺ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S_0.5 or n_H, but V_max for ATP-dependent Ca²⁺ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca²⁺ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.     

Calcium uptake into acini from rat pancreas: Evidence for intracellular ATP-dependent calcium sequestration

Summary Intracellular ATP-dependent Ca²⁺ sequestration mechanisms were studied in isolated dispersed rat pancreatic acini following treatment with saponin or digitonin to disrupt their plasma membranes. In the presence of⁴⁵Ca²⁺ concentrations <10^–6 mol/liter, addition of 5 mmol/liter ATP caused a rapid increase in⁴⁵Ca²⁺ uptake exceeding the control by fivefold. ADP mimicked the ATP effect by 50 to 60%, whereas other nucleotides such as AMP-PNP, AMP-PCP, CTP, UTP, ITP, GTP, cAMP and cGMP did not. Maximal ATP-promoted Ca²⁺ uptake was obtained at 10^–5 mol/liter Ca²⁺ uptake by mitochondrial inhibitors was dependent on the Ca²⁺ concentration, indicating the presence of different Ca²⁺ storage systems. Whereas the apparent half-saturation constant found for mitochondrial Ca²⁺ uptake was 4.5×10^–7 mol/liter, in the presence of antimycin and oligomycin (nonmitochondrial uptake) it was 1.4×10^–8 mol/liter. In the absence of Mg²⁺ both ATP- and ADP-promoted Ca²⁺ uptake was nearly abolished. The Ca²⁺ ionophore and mersalyl blocked Ca²⁺ uptake. Electron microscopy showed electrondense precipitates in the rough endoplasmic reticulum of saponintreated cells in the presence of Ca²⁺, oxalate and ATP, which were absent in intact cells and in saponin-cells without ATP or pretreated with A23187. The data suggest the presence of mitochondrial and nonmitochondrial ATP-dependent Ca²⁺ storage systems in pancreatic acini. The latter is likely to be located in the rough endoplasmic reticulum.     

Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca2+ ion transport studies

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy.Voltage-dependent Ca²⁺ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive ⁴⁵Ca²⁺ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K⁺] medium. Time course studies indicated that voltage-dependent Ca²⁺ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase.ATP-dependent Ca²⁺ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported ⁴⁵Ca²⁺ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated ⁴⁵Ca²⁺ from the vesicles. The maximal rate of ATP-dependent Ca²⁺ transport occurred at a [Ca²⁺ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca²⁺ ions.     

Potentiation by supernatant of Ca2+ uptake in rabbit aortic microsomes: effect of pretreatment of animals with reserpine

The effect of 105,000 g supernatant on Ca²⁺ uptake by rabbit aortic microsomes was studied to determine if a membrane-cytosol interaction exists, and to determine whether it is modified by pretreatment of animals with reserpine, which induces postjunctional supersensitivity in this tissue. Aliquots of supernatant from aortae of control and pretreated animals potentiated ATP-dependent microsomal Ca²⁺ uptake nearly equally but had no effect on total ATPase. Uptake-potentiation in control microsomes was significant only in the presence of 5 mM oxalate, whereas uptake-potentiation in reserpine-pretreated microsomes was significant both in the absence or presence of oxalate. A cytosolic factor in smooth muscle cells may be involved in Ca²⁺ transport in a manner reported for erythrocytes. Its action appears to be unmodified by reserpine-pretreatment. Instead, an alteration in the microsomes affects the interaction of the Ca²⁺ transport mechanism with the cytosolic factor.     

Active Ca2+ transport by vesicles reconstituted from triton X-100-solubilized pigeon erythrocyte membrane

Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidylethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of ⁴⁵Ca²⁺. ATP-dependent ⁴⁵Ca²⁺ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca²⁺ uptake rate depended on the square of the Ca²⁺ concentration and had similar $\text{[Ca}{}^{\mathrm{2+}}\text{]}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values, 0.16 μM and 0.18 μM, respectively.     

Far-red light irradiation of intact corn seedlings affects mitochondrial and calmodulin-dependent microsomal Ca2+ transport

Microsomal fractions isolated from coleoptiles of dark or far-red light grown corn show ATP-dependent Ca²⁺ accumulation. The microsomal transport from dark but not from far-red light grown tissue could be stimulated by calmodulin. Ca²⁺ accumulation into mitochondria from coleoptiles of far-red light grown corn in also inhibited as compared to the dark controls. Light irradiation of isolated microsomes and mitochondria had no effect on either Ca²⁺ uptake nor efflux.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

ATP-Stimulated Ca2+ Transport into Cholinergic Torpedo Synaptic Vesicles

Abstract: Activation of the Ca²⁺/Mg²⁺ ATPase associated with highly purified Torpedo synaptic vesicles results in ⁴⁵Ca²⁺ uptake. The accumulated ⁴⁵Ca²⁺ is released by hypoosmotic buffer and by the Ca²⁺ ionophore A23187. Density-gradient centrifugation and permeation chromatography reveal that vesicular acetylcholine and the membrane-bound ⁴⁵Ca²⁺ co-migrate, thus implying that ⁴⁵Ca²⁺ is transported into cholinergic vesicles. ATP-dependent ⁴⁵Ca²⁺ uptake follows saturation kinetics, with K_mCa²⁺= 50 μM, K_mATP= 5 μM, and V_max= 3 ± 0.3 nmol Ca²⁺/mg protein/min. Treatment of the vesicles with mersalyl, dicyclohexyl-carbodiimide, and quercetin leads to inactivation of the Ca²⁺/Mg²⁺ ATPase and to comparable inhibition of ⁴⁵Ca²⁺ transport. Ruthenium red and ouabain have no effect on either of these activities. Nigericin in the presence of external K⁺ is a potent inhibitor of ⁴⁵Ca²⁺ translocation, whereas gramicidin activates transport. The proton translocator carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and FCCP + the ionophore valinomycin partially inhibit ⁴⁵Ca²⁺ transport. By contrast, the above ionophores do not affect Ca²⁺/Mg²⁺ ATPase activity. Tentative mechanisms for ATP-dependent Ca²⁺ transport into cholinergic synaptic vesicles and the physiological significance of this process are discussed.     

The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca²⁺-binding glycoprotein and its Ca²⁺-transporting activity may be as low as 10–20% of the starting value. Both the rate of Ca²⁺ uptake and the capacity to maintain a high Ca²⁺ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca²⁺-transporting ability. The active component in the supernatant is the Ca²⁺-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 μM Mg²⁺, the glycoprotein alone may restore fully the Ca²⁺-transporting ability of the particles. The maximal velocity is already reached at 0.1 μg glycoprotein/mg mitochondrial protein.     

Effect of calmodulin antagonists on Ca2+ uptake by boar spermatozoa

Calcium uptake by washed boar sperm suspensions is markedly stimulated by the calmodulin antagonists trifluoperazine and calmidazolium. Both ⁴⁵Ca²⁺ uptake and net Ca²⁺ uptake are increased by these drugs. Drug stimulated Ca²⁺ uptake is blocked by verapamil (1 mM), by ruthenium red (25 μM) and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Calmodulin antagonists do not slow ATP-dependent Ca²⁺ extrusion from plasma membrane vesicles, and they do not inhibit plasma membrane Ca²⁺-ATPase. It is proposed that calmodulin is involved in the control of Ca²⁺ entry in boar spermatozoa. Most entering Ca²⁺ in uncapacitated spermatozoa is sequestered by mitochondria or rapidly extruded by plasma membrane pumps. In contrast to the uptake mechanism, ATP-dependent Ca²⁺ extrusion does not appear to be regulated by calmodulin.     

Phosphodiesterase protein activator stimulates calcium transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum.

Protein activator of cyclic nucleotide phosphodiesterase from bovine brain (9 μg/0.5 ml) was found to stimulate ATP-dependent Ca²⁺ - transport in dog cardiac microsomal preparations enriched in sarcoplasmic reticulum. Both oxalate-dependent calcium uptake and ATP-dependent calcium binding were increased. Cyclic AMP-dependent protein kinase (Sigma, type 1, 25 μg/0.5 ml) and cyclic AMP (1 μM) also stimulated calcium uptake and the presence of a maximal stimulatory concentration of the phosphodiesterase activator produced an additive elevation of calcium uptake indicating separate mechanisms of action and potentially different modulatory roles for these two systems in the control of calcium transport.     

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca2+ uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of ⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

Pharmacological characterization of the effects of taurine on calcium uptake in the rat retina

Summary Taurine is known to increase ATP-dependent calcium ion (Ca²⁺) uptake in retinal membrane preparations and in isolated rod outer segments (ROS) under low calcium conditions (10M) (Pasantes-Morales and Ordóñez, 1982; Lombardini, 1991). In this report, ATP-dependent Ca²⁺ uptake in retinal membrane preparations was found to be inhibited by 5M cadmium (Cd²⁺), suggesting the involvement of cation channel activation. The activation of cGMP-gated cation channels, which are found in the ROS, is a crucial step in the phototransduction process. An inhibitor of cGMP-gated channels, LY83583, was found to inhibit taurine-stimulated ATP-dependent Ca²⁺ uptake but had no effect on ATP-dependent Ca²⁺ uptake in the absence of taurine, indicating that taurine may be increasing ATP-dependent Ca²⁺ uptake through a mechanism of action involving the opening of cGMP-gated channels. The activation of cGMP-gated channels with dibutyryl-cGMP and with phosphodiesterase inhibition using zaprinast caused an increase in ATP-dependent Ca²⁺ uptake in isolated ROS, but not in taurine-stimulated ATP-dependent Ca²⁺ uptake. LY83583 had the same effects in isolated ROS as in retinal membrane preparations. Another inhibitor of cGMP-gated channels, Rp-8-Br-PET-cGMPS, produced the same pattern of inhibition in isolated ROS as LY83583. Thus, there appears to be a causal link between taurine and the activation of the cGMP-gated channels in the ROS under conditions of low calcium concentration, a connection that suggests an important role for taurine in the visual signalling function of the retina.     

Catharanthine: A novel stimulator of pancreatic enzyme release

Summary The plant alkaloid, catharanthine, was shown to stimulate release of amylase from pancreatic fragments and to cause extensive degranulation of pancreatic acinar cells with accumulation of membrane material in the Golgi region. The extent and time course of maximal catharanthine stimulation was comparable to that induced by the cholinergic analog bethanechol. Antimycin inhibited the action of catharanthine while atropine did not. Removal of Ca²⁺ from the incubation medium inhibited amylase release induced by catharanthine but did not affect release induced by bethanechol. Catharanthine induced a delayed release of ⁴⁵Ca²⁺ from prelabeled pancreatic fragments as compared to bethanechol. It is suggested therefore that catharanthine activates the physiological pathway controlling amylase release by causing a rise in cytoplasmic Ca²⁺ but the mechanism by which this occurs is different from that caused by physiological secretagogues.Supported by a grant from the NIH (GM-19998)I am indebted for technical assistance to E. Roach and S. Bennett     

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Summary ATP-dependent Ca²⁺ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca²⁺-specific electrode and⁴⁵Ca²⁺. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na⁺,K⁺(Mg²⁺)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,⁴⁵Ca²⁺ uptake correlated with the RNA content and not with the Na⁺,K⁺(Mg²⁺)ATPase activity. The characteristics of nonmitochondrial Ca²⁺ uptake into leaky isolated cells and⁴⁵Ca²⁺ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg²⁺, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca²⁺ concentration of 2 and 2 mol/liter, respectively. Calcium uptake decreased at higher free Ca²⁺ concentration.⁴⁵Ca²⁺ uptake was dependent on monovalent cations (Rb⁺>K⁺>Na⁺>Li⁺>choline⁺) and different anions (Cl^–>Br^–>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–>SCN^–) in both preparations. Twenty mmol/liter oxalate enhanced⁴⁵Ca²⁺ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca²⁺ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca²⁺ concentration to 0.4 mol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca²⁺ concentration in pancreatic acinar cells.