Calcium metabolism and enzyme secretion in guinea pig pancreas. Uptake, storage and release of calcium in whole cells and mitochondrial and microsomal fractions

In isolated pancreatic acinar cells from the guinea pig stimulation of enzyme secretion by carbamoylcholine is slightly diminished in the absence of extracellular Ca. LaCl3 in a concentration, which does not influence the secretory response to carbamoylcholine, nearly completely abolishes 45Ca uptake by cells, indicating that Ca uptake is not necessary for secretion. In cells preloaded with 45CaCl2, addition of carbamoylcholine leads to an immediate release of 45Ca, which can be blocked by atropine or 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate and is not influences by LaCl3 in concentrations, which do not inhibit secretion. A similar release of 45CaCl2 from preloaded cells is obtained by addition of the mitochondrial inhibitors antimycin A, carbonylcyanide p trifluoromethoxyphenylhydrazone (FCCP), and oligomycin. Possibly due to markedly diminished ATP levels, neither antimycin A nor FCCP act as secretagogues, both compounds being inhibitors of secretion. Oligomycin, which decreases ATP levels only to 20%, stimulates secretion. Mitochondria and microsomes from pancreatic tissue are able to accumulate 45Ca. Mitochondrial 45Ca uptake can be driven by ATP or active respiration and is inhibited by NaN3, oligomycin, antimycin A or FCCP. Microsomal 45Ca uptake is ATP-dependent. NaN3 and mitochondrial inhibitors have no influence on microsomal 45Ca uptake, which is stimulated several-fold by oxalate. The results support the assumption, that in the guinea pig pancreas Ca mobilization from intracellular stores is necessary to initiate secretion. Due to their ability for an active accumulation of45Ca both mitochondria and microsomes could serve as intracellular calcium stores.     

Kinetic characteristics of the two glucose transport systems in Neurospora crassa

Glucose is transported across the cell membrane of Neurospora crassa by two physiologically and kinetically distinct transport systems. System II is repressed by growth of the cells in 0.1 m glucose. System I is synthesized constitutively. The apparent K(m) for glucose uptake by system I and system II are 25 and 0.04 mm, respectively. Both uptake systems are temperature dependent, and are inhibited by NaN(3) and 2,4-dinitrophenol. Glucose uptake by system II was not inhibited by fructose, galactose, or lactose. However, glucose was shown to be a noncompetitive inhibitor of fructose and galactose uptake. The transport rate of [(14)C]3-0-methyl-d-glucose (3-0-MG) was higher in cells preloaded with unlabeled 3-0-MG than in control cells. The rate of entry of labeled 3-0-MG was only slightly inhibited by the presence of NaN(3) in the medium. Further, NaN(3) caused a rapid efflux of accumulated [(14)C]3-0-MG. These data imply that the energetic step in the transport process prevents efflux.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Differences between substrate specificities of l-glutamate uptake by neurons and glia,studied in cell lines and primary cultures

High affinity uptake of [³H]l-glutamate was studied in cultures of continuous cell lines, originating either from mouse neuroblastoma or rat glioma, and in two types of primary cultures containing cerebellar granule cells and astrocytes from cerebral cortex, respectively. In the continuous lines, d- and l-aspartate-4-hydroxamate were found to interact preferentially with the uptake of [³H]l-glutamate in glioma cells while l-glutamate-5-hydroxamate and 2-aminoadipate interacted more strongly with [³H]l-glutamate uptake in neuroblastoma cells, d-Aspartate-4-hydroxyamate, l-glutamate-5-hydroxamate and 2-aminoadipate were inactive as inhibitors of [³H]l-glutamate uptake by either granule cells or astrocytes, grown in primary culture, but several other glutamate analogues, which did not differentiate between neuroblastomal and gliomal uptake of [³H]l-glutamate, were somewhat stronger inhibitors of [³H]l-glutamate uptake in astrocytes as compared to that in granule cells. However, all of these compounds (N-acetyl-l-glutamate, formimino-l-aspartate, d-homocysteate, l-homocysteate and dl-2-methylglutamate) were only very weak inhibitors and, consequently, it is unlikely that any of them could be useful in experiments with central nervous tissue in vivo or, at least, in brain slices in vitro, attempting to resolve the uptake of l-glutamate into glia- and neuron-localized components.     

Transport and metabolism of folates by bacteria.

Transport of labeled folic acid (PteGlu), pteroylpolyglutamates (PteGlu3-5), 5-methyl-tetrahydrofolate (5-methyl-H4PteGlu), and methotrexate in late-log phase cells of Lactobacillus casei was active, and subject to inhibition by unlabeled pteroylmonoglutamates, pteroylpolyglutamates, and iodoacetate, but not glutamate or glutamate dipeptides. Pteroylpolyglutamates were transported without prior hydrolysis and shared a common uptake system with pteroylmonoglutamates. The affinity and maximum velocity of PteGlun uptake decreased with increasing glutamate chin length (Km:PteGlu1, 0.03 mum; PteGlu3, 0.32 mum; PteGlu4, 1.9 mum; PteGlu5, 3.7 mum) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by L. casei, once transported, than monoglutamate. No concentration of 5-methyl-H4PteGlu3-8 inside the cells was observed. The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 mum) were 10-formyl-H4PteGlu2 and 10-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No 10-formyl-Pte-Glu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 nm) metabolized the vitamin to polyglutamate forms, the major derivatives being H4PteGlun. First order exit rates of labeled folate from preloaded L. casei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 min-1 to greater than 0.002 min-1 as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n larger than or equal to 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released. Pediococcus cerevisiae actively transported 5-methyl-H4PteGlu but did not take up to 5-methyl-H4PTeGlu3-8. No active accumulation of 5-methyl-H4PteGlu was observed in Streptococcus faecalis.     

Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.     

ENTRY AND EXIT OF INDOLEACETIC ACID IN CORN COLEOPTILES

The entry and exit phases of radioactive indoleacetic acid transportwere investigated in corn coleoptile sections. Compounds capableof inhibiting auxin transport, particularly p-chloromercuribenzoicacid and N-1-naphthylphthalamic acid, were found to only slightlyblock auxin entry but severely suppress auxin exit. An oxygendeficiency had little effect on auxin entry but was found tostrongly inhibit auxin transport and auxin exit. While indoleaceticacid uptake was proportional to concentration, the exit phasebecame apparently saturated at concentrations above 10^–5M. Both entry and exit were found to have temperature coefficientsof about 2 or more. The low sensitivity of auxin entry to inhibitors,or to oxygen deficiency, and the linearity of entry over a wideconcentration range suggest a diffusion component in entry.The strong sensitivity of exit to inhibitors and to oxygen deficiencyconfirms the involvement of active processes in exit, as expectedof a secretion process. ¹Present address: Research Division, Ontario Water ResourcesCommission, Toronto, Canada. Indiana, U.S.A     

Allantoin transport in Saccharomyces cerevisiae.

Allantoin uptake in both growing and resting cultures of Saccharomyces cerevisiae occurs by a low-Km (ca. 15 micrometer) transport system that uses energy that is likely generated in the cytoplasm. This conclusion was based on the observation that transport did not occur in the absence of glucose or the presence of dinitrophenol, carbonyl cyanide-m-chloro-phenyl hydrazine, fluoride, or arsenate ions. Normal uptake was observed, however, in the presence of cyanide. The rate of accumulation was maximal at pH 5.2. In contrast to the urea transport system, allantoin uptake appeared to be unidirectional. Preloaded, radioactive allantoin was not lost from cells suspended in allantoin-free buffer and did not exchange with exogenously added, nonradioactive allantoin. Treatment of preloaded cells with nystatin, however, released the accumulated radioactivity. Allantoin accumulated within cells was isolated and shown to be chemically unaltered.     

Characteristics and energy requirements of an alpha-aminoisobutyric acid transport system in Streptococcus lactis.

Galactose-grown cells of Streptococcus lactis ML3 acculated alpha-aminoisobutyric acid (AIB) by using energy derived from glycolysis and arginine catabolism. The transport system displayed low-affinity Michaelis-Menten saturation kinetics. Using galactose or arginine as energy sources, similar V max and K m values for AIB entry were obtained, but on prolonged incubation the intracellular steady-state concentration of AIB in cells metabolizing arginine was only 65 to 70% that attained by glycolyzing cells. Efflux of AIB FROM PRELOADED CElls was temperature dependent and exhibited the characteristics of a first-order reaction. The rate of AIB exit was accelerated two- to threefold in the presence of metabolizable energy sources. Metabolic inhibitors including p-chloromercuribenzoate, dinitrophenol, azide, arsentate, and N, N'-dicyclohexylcarbodiimide either prevented or greatly reduced AIB uptake. Fluoride, iodoacetate and N-ethylmaleimide abolished galactose-dependent, but not arginine-energized, AIB uptake. K+ and Rb+ reduced the steady-state intracellular AIB concentration by approximately 40%, and these cations also induced rapid efflux of solute from actively transporting cells. Equivalent concentrations (10 mM) of Na+, Li+, or NH4+ were much less inhibitory. The proton-conducting ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenlyhydrazone abolished uptake and induced AIB efflux even though glycolysis and arginine catabolism continued at 60 and 140%, respectively, of control rates. A proton motive force is most likely involved in the active transport of AIB, whereas data from efflux studies suggest that energy is coupled to AIB exit in cells of S. lactis ML3.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Regulation of the Na+-Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astrocytes

The regulation of the Na⁺-dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with l-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca²⁺/ diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [³H]-d-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the l-glutamate–dependent [³H]-d-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the l-glutamate effect. In sharp contrast, in cortical astrocytes, both l-glutamate and TPA pre-exposure result in an augmentation of the [³H]-d-aspartate uptake. These findings suggest that the Na⁺-dependent glutamate uptake GLAST undergoes a region-specific regulation.     

Genistein inhibits Ca2+ influx and glutamate release from hippocampal synaptosomes: putative non-specific effects

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events.Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.     

High affinity transport of taurine by the Drosophila aspartate transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.     

Role of L-glutamate in systemic AVP-induced hypothermia.

It has been reported that systemic injection of arginine vasopressin (AVP) induces a drop in body core temperature (T(c)), but little is known about the mechanisms involved. Because glutamate is an important excitatory neurotransmitter involved in a number of thermoregulatory actions, in the present study, we tested the hypothesis that glutamate plays a role in systemic AVP-induced hypothermia. Wistar rats were pretreated intracerebroventricularly (icv) with kynurenic acid, an antagonist of l-glutamate ionotropic receptors, alpha-methyl-(4-carboxyphenyl)glycine (MCPG), an antagonist of l-glutamate metabotropic receptors, or saline 15 min before intravenous injection of AVP (2 microg/kg) or saline. T(c), brown adipose tissue (BAT) temperature, blood pressure, heart rate, and tail skin temperature were measured continuously. Administration of saline icv followed by intravenous AVP caused a significant drop in T(c) brought about by a reduction in BAT thermogenesis and an increase in heat loss through the tail. MCPG treatment (icv) did not affect the fall in T(c) induced by AVP. Treatment with kynurenic acid (icv) abolished AVP-induced hypothermia but did not affect the AVP-evoked rise in blood pressure or drop in heart rate and BAT temperature. Heat loss through the tail was significantly reduced in animals injected with AVP and pretrated with kynurenic acid. These data indicate that ionotropic receptors of l-glutamate in the central nervous system participate in peripheral AVP-induced hypothermia by affecting heat loss through the tail.     

Transamination and asymmetry in glutamate transport across the basolateral membrane of frog small intestine

Transport of L-glutamate across the basolateral membrane of frog small-intestinal epithelium, unlike that of L-alanine, is highly asymmetric; thus the rate constant (K entry) describing the entry of glutamate into the epithelium from the vascular bed across this membrane is one order of magnitude greater than the rate constant (K exit) describing its exit. This asymmetry, which appears not to depend upon the Na gradient, may be important in maintaining a high intracellular concentration of glutamate relative to alanine thereby favouring the production of alanine from glutamate by transamination.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

A novel type of coupling between proline and galactoside transport in Escherichia coli.

Exit of thiomethylgalactoside (TMG) from preloaded cells induced the accumulation of proline. Likewise, proline exit stimulated TMG accumulation. Since a proton ionophore (carbonylcyanide-m-chlorophenylhydrazone) abolished these effects, a protonmotive force was implicated as the "intermediate" in the coupling reaction. The evidence suggests that the exit of TMG resulted in proton exit, which produced either a membrane potential (inside negative or a pH gradient (outside acid) or both. This inwardly directed protonmotive force provided the energy for proline entry and accumulation. Thus the energy coupling was not via a common transport protein but by proton movements which coupled the two separate H+-dependent transport processes.     

Sodium and Potassium Requirements for Active Transport of Glutamate by Escherichia coli K-12

Active transport of glutamate by Escherichia coli K-12 requires both Na(+) and K(+) ions. Increasing the concentration of Na(+) in the medium results in a decrease in the K(m) of the uptake system for glutamate; the capacity is not affected. Glutamate uptake by untreated cells is not stimulated by K(+). K(+)-depleted cells show a greatly reduced capacity for glutamate uptake. Preincubation of such cells in the presence of K(+) fully restores their capacity for glutamate uptake when Na(+) ions are also present in the uptake medium. Addition of either K(+) or Na(+) alone restores glutamate uptake to only about 20% of its maximum capacity in the presence of both cations. Changes in K(+) concentration affect the capacity for glutamate uptake but have no effect on the K(m) of the glutamate transport system. Ouabain does not inhibit the (Na(+)-K(+))-stimulated glutamate uptake by intact cells or spheroplasts of E. coli K-12.     

The uptake and metabolism of L-glutamate by tissue slices of the cestode Hymenolepis diminuta

The in vitro uptake of L-[3H]glutamate by tissue slices of the cestode Hymenolepis diminuta, denuded of tegument, was investigated. Two sodium concentration-dependent mechanisms, one of high affinity (Kt 1.8 X 10(-5) M; Vmax 4.76 pMoles/min/mg wet weight) and another of low affinity (Kt 2.2 X 10(-4) M; Vmax 50.7 pMoles/min/mg wet weight), were identified, in addition to a sodium insensitive component. Exchange of preloaded [3H]glutamate did not occur in tissue slices incubated in dilute unlabelled glutamate. Acidic amino acids, imipramine and fluoxetine were effective inhibitors of high and low affinity uptake, while glutamate receptor ligands, neurotransmitters and some antihelminthics generally were not. The concentrations present in, and the metabolism of glutamate by, tissue slices was examined by HPLC. The significance of the three modes of glutamate uptake and their possible role in the physiology of H. diminuta are discussed.