Assessment of the role of glutathione conjugation in the protection afforded by anethol dithiolthione against hexachloro-1,3-butadiene-induced nephrotoxicity

Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca(2+)-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca2+ ionophore ionomycin. The Ca2+ dependence of this effect was established, as removal of Ca2+ eliminated evoked release and the lipid-dependent potentiation. Also, Ca2+ channel blockers attenuated ionomycin- and KCl-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca2+, it may not involve an enhancement of evoked Ca2+ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca2+ accumulation. Also, the facilitation may not depend on inhibition of K+ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCl- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca2+ and involves multiple presynaptic effects, some of which depend on protein kinase C.     

Inhibition of acetylcholine release from presynaptic terminals of skate electric organ by calcium channel antagonists: a detailed pharmacological study

Release of acetylcholine (ACh) from the presynaptic terminals in skate electric organ was tested for its sensitivity to calcium channel antagonists. A pharmacological profile was established by measuring inhibition of K(+)-stimulated release of [3H]ACh from prelabelled tissue slices. Peptide antagonists of N-type (omega-conotoxins GVIA and MVIIA) and P-type (omega-agatoxin-IVA) channels had no effect, whereas both omega-conotoxins MVIIC and SVIB produced concentration-dependent inhibition and could completely block ACh release. omega-Conotoxin GVIA and omega-agatoxin IVA did not attenuate the block by omega-conotoxin MVIIC. The inorganic ions, Cd2+ and Ni2+, also produced a full inhibition of release (Cd2+ > > Ni2+) and Gd3+ a partial one. Drugs targeting L-type channels (diltiazem, nifedipine and verapamil) at low microM concentrations and a synthetic analogue of the polyamine toxin from funnel web spider venom (sFTX) at 1 mM were all non-inhibitory. Inhibition by omega-conotoxins MVIIC (IC50 25 nM) and SVIB (IC50 500 nM) was reversible and modulated by external concentrations of Ca2+. Inhibitory potency was increased by lowering and decreased by elevating external Ca2+. This "antagonistic" effect of Ca2+ was also seen with Cd2+ inhibition. The inhibitory potency of omega-conotoxin MVIIC was unaffected by predepolarisation. End plate potentials generated by release of endogenous ACh in electrically-stimulated slices were also reversibly blocked by Cd2+ and omega-conotoxins MVIIC and SVIB but were unaffected by omega-conotoxin GVIA and omega-agatoxin IVA. It is concluded that ACh release in skate electric organ depends on presynaptic calcium channels which have different pharmacological properties from established sub-types.     

Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus

The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent Ki against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca(2+)-activated K+ current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa.     

Modulation of transmitter release by action potential duration at the hippocampal CA3-CA1 synapse

Presynaptic Ca2+ influx through voltage-dependent Ca2+ channels triggers neurotransmitter release. Action potential duration plays a determinant role in the dynamics of presynaptic Ca2+ influx. In this study, the presynaptic Ca2+ influx was optically measured with a low-affinity Ca2+ indicator (Furaptra). The effect of action potential duration on Ca2+ influx and transmitter release was investigated. The K+ channel blocker 4-aminopyridine (4-AP) was applied to broaden the action potential and thereby increase presynaptic Ca2+ influx. This increase of Ca2+ influx appeared to be much less effective in enhancing transmitter release than raising the extracellular Ca2+ concentration. 4-AP did not change the Ca2+ dependence of transmitter release but instead shifted the synaptic transmission curve toward larger total Ca2+ influx. These results suggest that changing the duration of Ca2+ influx is not equivalent to changing its amplitude in locally building up an effective Ca2+ concentration near the Ca2+ sensor of the release machinery. Furthermore, in the presence of 4-AP, the N-type Ca2+ channel blocker omegaCgTx GVIA was much less effective in blocking transmitter release. This phenomenon was not simply due to a saturation of the release machinery by the increased overall Ca2+ influx because a similar reduction of Ca2+ influx by application of the nonspecific Ca2+ channel blocker Cd2+ resulted in much more inhibition of transmitter release. Rather, the different potencies of omega-CgTx GVIA and Cd2+ in inhibiting transmitter release suggest that the Ca2+ sensor is possibly located at a distance from a cluster of Ca2+ channels such that it is sensitive to the location of Ca2+ channels within the cluster.     

Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors

The effects of external pH (pHout) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pHout 6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 microM) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Na(i)] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Ca(i)] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pHout 6. On the contrary, the external alkalinization (pHout 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pHout was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pHout changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pHout variations on [3H]GABA release were independent on the presence of HCO3-. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels.     

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.     

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.     

Design and synthesis of small molecule interleukin-1 receptor antagonists based on a benzene template

Effects of the antiepileptic drug carbamazepine on nerve action potential and transmitter release in mouse neuroblastoma-glioma hybrid cells (NG108-15) and the frog neuromuscular junction were studied. Carbamazepine within a concentration range of 0.1-0.5 mmol/L reduced the peak height of the action potential of the NG108-15 cells, whereas the membrane potential and membrane resistance were unaffected. Voltage clamp revealed that the decrease in the action was due to the blockage of the Na+, delayed K+ and transient Ca2+ currents. Carbamazepine did not affect Ca(2+)-activated and A type K+ currents and long-lasting Ca2+ current. In the frog neuromuscular junction, carbamazepine decreased the mean quantal content by a parallel shift in the frequency augmentation-potentiation (FAP) relation. It is concluded that carbamazepine blocks the voltage-dependent Na+, delayed K+, and transient Ca2+ currents and quantal transmitter release through a decrease of nerve excitation.     

Sulphonylureas do not increase insulin secretion by a mechanism other than a rise in cytoplasmic Ca2+ in pancreatic B-cells

The following sequence of events is thought to underlie the stimulation of insulin release by hypoglycaemic sulphonylureas. Interaction of the drugs with a high-affinity binding site (sulphonylurea receptor) in the B-cell membrane leads to closure of ATP-sensitive K+ channels, depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx and rise in cytoplasmic [Ca2+]i. Recent experiments using permeabilized islet cells or measuring changes in B-cell membrane capacitance have suggested that sulphonylureas can increase insulin release by a mechanism independent of a change in [Ca2+]i. This provocative hypothesis was tested here with intact mouse islets. When B-cells were strongly depolarized by 60 mM K+, [Ca2+]i was increased and insulin secretion stimulated. Under these conditions, tolbutamide did not further increase [Ca2+]i or insulin release, whether it was applied before or after high K+, and whether the concentration of glucose was 3 or 15 mM. This contrasts with the ability of forskolin and phorbol 12-myristate 13-acetate (PMA) to increase release in the presence of high K+. Tolbutamide also failed to increase insulin release from islets depolarized with barium (substituted for extracellular Ca2+) or with arginine in the presence of high glucose. Glibenclamide and its non-sulphonylurea moiety meglitinide were also without effect on insulin release from already depolarized B-cells. In the absence of extracellular Ca2+, acetylcholine induced monophasic peaks of [Ca2+]i and insulin secretion which were both unaffected by tolbutamide. Insulin release from permeabilized islet cells was stimulated by raising free Ca2+ (between 0.1 and 23 microM). This effect was not affected by tolbutamide and inconsistently increased by glibenclamide. In conclusion, the present study does not support the proposal that hypoglycaemic sulphonylureas can increase insulin release even when they do not also raise [Ca2+]i in B-cells.     

The effect of a reduction in temperature on the quantal release of transmitter at the mouse neuromuscular junction

1. The effects of a reduction in temperature were examined on evoked and spontaneous release of transmitter quanta and on presynaptic negative signals, blocked by Cd2+, measured externally at neuromuscular junctions in mouse diaphragm muscles in low-Ca2+, high-Mg2+ Krebs-Ringer solutions. 2. The evoked release was enhanced with lowering of the temperature, whereas the extent of spontaneous release was reduced. Cooperativity of Ca2+ in the evoked release was slightly reduced by lowering the temperature. 3. The presynaptic negative signals increased in duration with lowering of the temperature. 4. These results support the hypothesis that the effect of a reduction in temperature reflects the improved efficacy of the calcium-mediated mechanism of transmitter release, manifested as a prolongation of the inflow of Ca2+. The process involved in the evoked release is probably attributable to an almost passive mechanism.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process

Doc2alpha and Munc13-1 proteins are highly concentrated on synaptic vesicles and the presynaptic plasma membrane, respectively, and have been implicated in Ca2+-dependent neurotransmitter release. Doc2alpha interacts with Munc13-1 through the N-terminal region of Doc2alpha (the Mid domain; amino acid residues 13-37). Here we examine whether the interaction between Doc2alpha and Munc13-1 is required for Ca2+-dependent neurotransmitter release from intact neuron. A synthetic Mid peptide (the Mid peptide), but not a control mutated Mid peptide or a scrambled Mid peptide, inhibited the interaction between Doc2alpha and Munc13-1 in vitro. Introduction of the Mid peptide into presynaptic neurons of cholinergic synapses, formed between rat superior cervical ganglion neurons, reversibly inhibited synaptic transmission evoked by action potentials. In contrast, the control peptides did not inhibit synaptic transmission. This inhibitory effect depended on the presynaptic activity and was affected by extracellular Ca2+ concentrations. The onset of the Mid peptide effect was shortened when the neuron was stimulated at a higher frequency, and the inhibition was more potent at 1 mM Ca2+ than at 5.1 mM Ca2+. These results suggest that the Doc2alpha-Munc13-1 interaction plays a role in a step before the final fusion step of synaptic vesicles with the presynaptic plasma membrane in the evoked neurotransmitter release process.     

Role of the S3 stalk segment in the thapsigargin concentration dependence of sarco-endoplasmic reticulum Ca2+ ATPase inhibition

The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is specifically inhibited by thapsigargin (TG), whereas the Na+,K+-ATPase is not. Large chimeric exchanges between Ca2+ and Na+,K+-ATPases (Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem. 269, 26598-26601), as well as photolabeling with a TG azido derivative (Hua, S., and Inesi, G. (1997) Biochemistry 36, 11865-11872), suggest that the S3-M3 (stalk and membrane-bound) region of the Ca2+ ATPase is involved in TG binding. We produced small site-directed changes in the S3 stalk segment of the Ca2+ ATPase and found that mutation of five amino acids to the corresponding Na+,K+-ATPase residues increases by 3 orders of magnitude the TG concentration required for inhibition of Ca2+ ATPase and coupled Ca2+ transport. A single mutation in the S3 stalk segment (Gly257 --> Ile) is sufficient to increase by 1 order of magnitude the TG concentration required to produce 50% inhibition. By comparison, mutations yielding a nine-amino acid homology in the M3 transmembrane segment, or a 25-amino acid homology in the S4 stalk segment, do not affect the ATPase sensitivity to TG. We suggest that specific binding of TG to the S3 stalk segment, in addition to stacking of the TG ring structure at the membrane interface, determines the high affinity of the ATPase for the inhibitor.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: cytoplasmic cation binding and release

In the preceding publication (. Biophys. J. 76:000-000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2-0.3 s-1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

Possible involvement of intracellular Ca2+ in hyposmosis-evoked catecholamine release from adrenal chromaffin cells

The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.     

GABAB receptor-mediated inhibition of tetrodotoxin-resistant GABA release in rodent hippocampal CA1 pyramidal cells

Tight-seal whole-cell recordings from CA1 pyramidal cells of rodent hippocampus were performed to study GABAB receptor-mediated inhibition of tetrodotoxin (TTX)-resistant IP-SCs. IPSCs were recorded in the presence of TTX and glutamate receptor antagonists. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs by a presynaptic action. The inhibition by (R)-(-)-baclofen was concentration-dependent, was not mimicked by the less effective enantiomer (S)-(+)-baclofen, and was blocked by the GABAB receptor antagonist CGP 55845A, suggesting a specific effect on GABAB receptors. The inhibition persisted in the presence of the Ca2+ channel blocker Cd2+. There was no requirement for an activation of K+ conductances by (R)-(-)-baclofen, because the inhibition of TTX-resistant IPSCs persisted in Ba2+ and Cd2+. Because the time courses of TTX-resistant IPSCs were not changed by (R)-(-)-baclofen, there was no evidence for a selective inhibition of quantal release from a subgroup of GABAergic terminals. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs in guinea pigs and Wistar rats, whereas the inhibition was much smaller in Sprague Dawley rats. In Cd2+ and Ba2+, beta-phorbol-12,13-dibutyrate and forskolin enhanced the frequency of TTX-resistant IPSCs. Only beta-phorbol-12, 13-dibutyrate reduced the inhibition by (R)-(-)-baclofen. We conclude that GABAB receptors inhibit TTX-resistant GABA release through a mechanism independent from the well known effects on Ca2+ or K+ channels. The inhibition of quantal GABA release can be reduced by an activator of protein kinase C.     

Two signaling pathways, from the upper glycolytic flux and from the mitochondria, converge to potentiate insulin release

In the rat pancreatic beta cell, low concentrations of glucose potentiate D-glyceraldehyde (GA)-induced insulin release without any potentiation of the triose-induced elevation of cytosolic free Ca2+ concentration. Namely, 2-3 mM glucose strongly potentiates 5 mM GA-induced insulin release, and the combination of stimulatory concentration of glucose (10 mM) and 5 mM GA elicits far more than additive insulin release: this glucose action is independent of ATP-sensitive K+ channel closure because it can be seen in the presence of diazoxide, an opener of the K+ channel. The triose-induced elevation of cytosolic free Ca2+ concentration was not potentiated by the presence of 3 mM glucose, and oxidation of labeled GA by the islet cells was not enhanced by the presence of glucose. The glucose action can be mimicked by mannose, but not by galactose, and was suppressed by inhibition of glucose phosphorylation with mannoheptulose or 2-deoxyglucose. Glucose also potentiates 2-ketoisocaproate-induced insulin release. In contrast, a combination of GA and 2-ketoisocaproate elicits only additive insulin release. Strikingly, 3 mM glucose does not potentiate insulin release in response to a depolarizing concentration of K+. Therefore, at least two signal pathways, one from upper glycolytic flux and one from mitochondrial metabolism, must converge to provide the potentiation of insulin release. We conclude that the upper glycolytic flux, acting at a site unrelated to the elevation of cytosolic free Ca2+, potentiates insulin release triggered by triose and mitochondrial fuels.     

Magnesium ions but not ATP inhibit cyclic ADP-ribose-induced calcium release

The pharmacology of the cyclic ADP-ribose (cADPR)-dependent Ca2+ release mechanism is very similar to that of the ryanodine receptor (RyR). Here we showed that MgCl2, a known inhibitor of RyR, blocked cADPR-induced Ca+2 release in sea urchin egg homogenates with a half maximal concentration of about 2.5 mM. The effect was specific since up to 10 mM Mg+2 had no effect on the Ca+2 release induced by inositol trisphosphate. K2ATP, another known modulator of RyR, at up to 10 mM did not affect the half-maximal concentration of cADPR, which remained at about 96 nM. These results indicate cADPR is a specific Ca+2 release activator and not merely an adenine nucleotide acting on the ATP-site. The inhibitory effects of Mg+2 further demonstrate the similarity between RyR and the cADPR-dependent Ca+2 release system.     

Maturation of rat renal tubular response to alpha-adrenergic agonists and neuropeptide Y: a study on the regulation of Na+,K+-ATPase

Na+,K+-ATPase in tubular cells plays a pivotal role for the regulation of renal sodium excretion. In adult rats the activity of this enzyme is inhibited by natriuretic hormones and stimulated by antinatriuretic hormones. Here we have examined the tubular response to alpha-adrenergic agonists and neuropeptide Y (NPY) in both infant and adult rats. In the adult kidney, alpha-adrenergic agonists and NPY stimulate Na+,K+-ATPase activity via Ca2+-dependent pathways. Oxymetazoline, a selective alpha-adrenergic agonist, and NPY failed to stimulate proximal tubular (PT) Na+,K+-ATPase activity in 10-d-old rats in doses of 10(-8) to 10(-5) M and 10(-8) to 10(-6) M, respectively, but when tubules were incubated simultaneously with both oxymetazoline 10(-8) M and NPY 5 x 10(-9) M, stimulation was observed in both 10- and 40-d-old rat PT. This effect was abolished by FK 506, an inhibitor of Ca2+ and calmodulin-dependent protein phosphatase 2B in both age groups. A23187, a calcium ionophore, stimulated Na+,K+-ATPase in both infant and adult PT, but 10-fold higher doses were required for the infant tubules. The effect of alpha-adrenergic agonists and NPY on free intracellular Ca2+ was studied in PT cells in primary culture. The Ca2+ response to each agent was less pronounced in infant than in adult cells. Preincubation with NPY, which increases Ca2+ influx into the cells, enhanced the response to the alpha-adrenergic agonist in both infant and adult cells. The results support the concept that the systems regulating renal tubular Na+, K+-ATPase and sodium metabolism undergo postnatal maturation.