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PubMed1995

Reversible histochemical modifications of endoplasmic reticulum following arginine vasopressin stimulation of granular cells of toad bladder.

The endoplasmic reticulum is generally absent from schematic representations of transport phenomena, although it shows a well-organized network in most transport epithelial cells. In order to examine the correlation between this organelle and cellular activity, bladders of Bufo marinus were studied under different experimental conditions and fixed by immersion in glutaraldehyde, followed by OsO4 impregnation for 3 days. Normal granular and mitochondria-rich cells showed a rich cytoplasmic network of canaliculi, well-impregnated by osmium deposits. Following a 2 to 15-min stimulation (serosal bath) with arginine vasopressin, the V2 receptor agonist dD-arginine-vasopressin or cyclic AMP (cAMP), the staining of endoplasmic reticulum in granular cells disappeared. After washing out of the hormone or the agonist, impregnation of the endoplasmic reticulum could be observed once again. Arginine vasopressin did not modify the impregnation of endoplasmic reticulum of either mitochondria-rich or basal cells. Our data indicate a correlation between the reactivity of endoplasmic reticulum to osmium, and a cAMP-dependent effect of arginine vasopressin through its V2 receptors. Incubation of arginine vasopressin through its V2 receptors. Incubation of toad bladders carried out with agents interfering with cellular calcium (calcium ionophores, high or low bath calcium) or with calcium release from the endoplasmic reticulum (TMB-8, thapsigargin) suggested that an early step in the cAMP-dependent effect of arginine vasopressin must involve the release of intracellular calcium from the endoplasmic reticulum. However, calcium ATPases in this organelle do not seem to participate in the hormonal effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene.

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K+ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K+ channel blocker barium (10 mmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mumol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mumol/l), by cobalt (100 mumol/l), zinc (100 mumol/l), gadolinium (100 mumol/l), verapamil (10 mumol/l) and diltiazem (10 mumol/l), but are abolished in the presence of 100 mumol/l lanthanum, 1 mmol/l cadmium, 10 mumol/l nifedipine, 25 mumol/l SK & F 96365 and 200 mumol/l TMB-8. Stimulation of calcium entry by 10 nmol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.

Animals↗
PubMed1991

Calcium- and cyclic-AMP-mediated secretory responses in isolated colonic crypts.

Whole-cell recordings were performed at isolated crypts from the distal colon of the rat. Enterocytes in intact crypts, patched from the basolateral side, exhibited a gradient in the resting zero-current potential. Along the axis of the crypt, the highest potentials were measured in the ground region, the lowest in the surface region. The cholinergic agonist, carbachol, induced a hyperpolarization and an increase of the outward current in both the middle and the ground cells of intact crypts. This effect could be prevented by Ba2+ or by the intracellular Ca2+ antagonist, 8-(N, N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8). Its action, however, was not dependent on the presence of external Ca2+. Both ground cells and the cells in the middle part of the crypt responded to forskolin, an activator of the adenylate cyclase, with a depolarization. In the middle part of the crypt, the depolarization induced by forskolin was associated with an increase of the outward current. It could be blocked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, indicating an increase of Cl- conductance. In contrast, the forskolin-induced depolarization in the ground part of the crypt was associated with a decrease of the outward current. This effect could be prevented by Ba2+, indicating a decrease of a potassium conductance. The changes in outward current could be prevented by the presence of an inhibitor of protein kinase A in the pipette solution. In conclusion, these results suggest that carbachol, an agonist acting on the Ca2+ pathway, indirectly causes Cl- secretion by an increase of the driving force, i.e. the membrane potential. Only the activation of cyclic AMP synthesis by forskolin is able to increase Cl- conductance in the rat colon. The latter response seems to be dependent on the state of differentiation of the enterocytes.

Animals↗
PubMed1984

Ca2+ as messenger of 5HT2-receptor stimulation in human blood platelets.

In blood platelets of man, both 5-hydroxytryptamine (5HT) and 80 nM of the Ca2+-ionophore A23187 led to rapid shape change reactions which were inhibited by prostaglandin E1 (PGE1), forskolin, 2-methyl-6-methoxy-8-nitroquinoline ( quin2 ) and chlortetracycline. The IC50-values of the inhibitors were similar in the 5HT- and the A23187-experiments. Higher amounts of A23187 abolished the inhibitory actions of PGE1 and forskolin. Furthermore, 5HT and A23187 enhanced adrenaline-induced platelet aggregation their effects showing similar time dependence. Ketanserin, an antagonist of 5HT2 -receptors, and 8-(N,N-diethyl-amino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca2+-antagonist, counteracted the effects of 5HT much more than those of A23187, whereas acetylsalicylate and indomethacin did not influence the actions of either 5HT or A23187. In addition, 5HT caused a concentration-dependent rise of intracellular free Ca2+ in platelets which was counteracted by ketanserin. PGE1 and forskolin reduced the resting Ca2+-levels. 5HT did not affect either the basal or the PGE1-stimulated activity of adenylate cyclase, whereas the Ca2+-ionophore A23187 slightly raised the basal activity of the enzyme. In conclusion, the functional effects of 5HT2 -receptor stimulation in human blood platelets (shape change reaction and enhancement of adrenaline aggregation) seem to be mediated by a rise of intracellular free Ca2+.

Adenylyl Cyclases↗
PubMed1984

The role of prostaglandins in the endothelium-mediated vasodilatory response to hypoxia.

The effect of intraluminal hypoxia on vascular tone and the release of prostaglandins (PG) I2 and E2 were investigated in intact isolated segments of canine femoral and coronary arteries as well as in the rat tail artery. Perfusion with hypoxic Tyrode's solution (pO2: 20-40 mm Hg) evoked a marked vasodilation of the segments, precontracted with norepinephrine or serotonin. Simultaneously, a 2-3-fold increase in the release of 6-keto-PGF1 alpha (the stable hydrolysis product of PGI2) could be observed. In parallel to 6-keto-PGF1 alpha, smaller quantities of PGE2 were released. Removal of the endothelium as well as pretreatment with indomethacin abolished both, the dilatory response and the PG-release. After administration of verapamil as well as 3,4,5-trimethoxybenzoic acid 8-diethylaminooctylester (TMB-8) (which binds intracellular calcium) the PG-increase was abolished and hypoxic dilatation could no longer be elicited, although the vessel had still a capacity to dilate. Exogenous administration of PGI2 and PGE2 showed that in canine femoral and coronary arteries PGI2 was the most effective vasodilating prostaglandin, while in the rat tail artery PGE2 had a 10-fold higher dilating potency compared to PGI2. At very high concentrations both PGI2 and PGE2 caused vasoconstriction. Our experiments suggest that the hypoxic endothelium-dependent dilatation may be mediated by an increased PG-release. Hypoxia-induced transmembrane calcium influx into the endothelial cells seems to be the trigger reaction.

6-Ketoprostaglandin F1 alpha↗
PubMed1980

Characteristics of N-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils.

N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated a time- and concentration-dependent release of granule associated beta-glucuronidase and lysozyme but not cytoplasmic lactate dehydrogenase from human neutrophils. Maximum discharge of lysome activity released is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to FMLP (10(-10)-10(-7) M); although 11.2 +/- 1.3 and 12.4 +/- 1.1% of total activity for beta-glucuronidase and lysozyme, respectively, is secreteer, had no effect on FMLP-elicited lysosomal enzyme extrusion. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a purported antagonist of intracidating the role of calcium in the mechanism of lysosomal enzyme release.

Calcium↗
PubMed1990

Inflammatory agonists that increase microvascular permeability in vivo stimulate cultured pulmonary microvessel endothelial cell contraction.

Bovine pulmonary microvessel endothelial cells grown on a flexible substrate contract upon the addition of angiotensin II, thrombin, bradykinin, and U44069, a stable analogue of thromboxane A2. All these agents promote inflammation and increase paracellular permeability in vivo or in vitro. The contractile response is mediated by intracellular and extracellular free calcium: the response is inhibited by TMB-8, an intracellular Ca2+ chelator, and EGTA. Contraction is inhibited by trifluoroperazine, a Ca2(+)-calmodulin antagonist, and by ML-7, an inhibitor of myosin light-chain kinase. Preincubation with PMA, a protein kinase C activator, prevents contraction by angiotensin II. The inactive analogue 4-alpha-phorbol 12,13-didecanoate does not inhibit contraction. In contrast cAMP, carbacyclin (a stable PGI2 analogue), and isoproterenol, agonists known to stabilize the microvascular barrier against inflammatory agents, relax pulmonary microvessel EC. This direct evidence of the contractile potential of microvessel endothelial cells lends support to the theory that endothelial contraction leads to increased junctional permeability.

Angiotensin II↗
PubMed1990

Bacterial lipopolysaccharide enhances polymorphonuclear leukocyte function independent of changes in intracellular calcium.

Bacterial lipopolysaccharide (LPS) enhanced expression of C3bi receptors (CR3), phagocytosis of opsonized bacteria, and subsequent hydrogen peroxide (H2O2) production by human polymorphonuclear leukocytes (PMNs). The role of changes in intracellular calcium concentration ([Ca2+]i) in LPS-induced priming was examined by determining the effect of modulators of intracellular calcium on enhanced PMN function, determining the ability of calcium ionophores to reproduce the effects of LPS, and measuring PMN [Ca2+]i following addition of LPS. Inhibition of intracellular calcium-dependent processes with TMB-8 or quin-2 blocked all three measures of LPS-induced priming. LPS did not stimulate an increase in [Ca2+]i, and calcium ionophores failed to reproduce the effect of LPS. Maintenance of [Ca2+]i is necessary for LPS priming, but an increase in [Ca2+]i is not a component of the signal transduction pathway leading to PMN priming by LPS.

Aminoquinolines↗
PubMed1986

Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils.

Aggregated immunoglobulin G (AggIgG) induced a time- and concentration-dependent phagocytic release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Degranulation was significantly enhanced in the presence of calcium or magnesium, and maximum granule exocytosis was observed when both divalent cations were present. AggIgG-stimulated enzyme release was inhibited with the intracellular calcium antagonist, TMB-8[8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate] in the absence of extracellular calcium. DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene), a permeant anion channel blocker, also suppressed AggIgG-induced degranulation. Cycloheximide, an inhibitor of protein synthesis, enhanced granule exocytosis from AggIgG-treated neutrophils. Two inhibitors of transmethylation reactions, 3-deazaadenosine (3-DZA) and homocysteine thiolactone (HCTL) in combination, suppressed AggIgG-elicited granule enzyme release. These data indicate that AggIgG is a useful probe for investigating the requirements for phagocytic enzyme release from human neutrophils.

Calcium↗
PubMed1985

NADPH and "cocktails" containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents.

Human blood leukocytes generated large amounts of superoxide (O2-) following stimulation by certain "cocktails" of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2- generation by the soluble cocktails, markedly depressed luminol-dependent chemiluminescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2- by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2- was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin (LL) and which lost their viability, and their O2- and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2- generation. Since PARG acted both as a cytolytic agent and as a inducer of O2- generation, we postulate that lytic agents might also act as "primers" of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in "multiple hits," PARG could be totally replaced either by LL or by digitonin in the generation of O2- provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble "cocktails" may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.

Cell Membrane↗
PubMed1984

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid.

Exposure of human neutrophils to 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (leukotriene B4, LTB4) resulted in a time- and concentration- (10(-9)-10(-6) M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB4. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB4 prior to contact with cytochalasin B. LTB4-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB4-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) and N-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB4-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive. Neutrophils pretreated with LTB4 or 5(S),12(R),20-trihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB4, an omega-oxidation metabolite of LTB4) were desensitized to the subsequent exposure to LTB4. Cross-desensitization was also demonstrated between LTB4 and 20-OH-LTB4. The stimulus specific nature of LTB4-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB4-pretreated neutrophils. Enzyme release from LTB4-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB4-induced degranulation.

Arachidonic Acid↗
PubMed1989

Possible roles of cAMP and Ca2+ in the regulation of miracidial transformation in Schistosoma mansoni.

The triggering action of physiological saline in the miracidial transformation of Schistosoma mansoni was analyzed using various agents affecting cAMP- and Ca2+-dependent pathways. Potent activators of adenylate cyclase, such as forskolin and serotonin, strongly inhibited the transformation provoked by saline in RPMI-1640. These inhibitory actions were diminished by the combined administration of phosphodiesterase activators such as ammonium salts or imidazole. Furthermore, the exposure of miracidia to ammonium salts or imidazole in dechlorinated tap water "mimicked" the transformation, i.e., the cessation, of swimming and then shedding of epithelial plates. This mimic transformation was also inhibited by serotonin or forskolin. In contrast, treatment of miracidia with Ca2+ antagonists such as TMB-8 (an inhibitor of Ca2+ release), nicardipine (a Ca2+ channel blocker), or W-7 (a calmodulin inhibitor) in tap water produced severe vesiculation on their body surfaces and resulted in death. However, these toxic effects were abolished by a combined administration of these Ca2+ antagonists with saline or NH4Cl, and the transformation was reestablished except with W-7 treatment. W-7 strongly inhibited the triggering action of saline and NH4Cl and the worms swam slowly, whereas W-5, an inactive analogue of W-7, had no inhibitory effect on the transformation. These results suggest that the initiation of micracidial transformation to young sporocysts may be synergistically regulated by cAMP and Ca2+ and that a decrease in cAMP levels and an increase in Ca2+ mobilization may be provoked in worms transformed by saline, ammonium salts, or imidazole.

Animals↗
PubMed1983

Further studies on the role of calcium in the regulation of glutamate decarboxylase activity in brain slices.

[3H]GABA synthesis in brain slices was used as a model to study the role of Ca2+ in the regulation of GAD activity. Experimental conditions were chosen to increase and decrease the flux of Ca2+ and to promote the increase in free intracellular Ca2+. The blockade of electron transport and the inhibition of oxidative phosphorylation in the slices inhibited [3H]GABA synthesis. High K+ depolarization stimulated [3H]GABA synthesis and this effect was not blocked by lidocaine, trifluoperazine, or verapamil, but the stimulation was blocked by the intracellular Ca2+ antagonist TMB-8. The data do not differentiate between the relative contributions of extra- and intracellular Ca2+ but reflect that GAD activity is modulated by a dynamic balance between these two compartments as well as between stored and free Ca2+ within the cells.

2,4-Dinitrophenol↗
PubMed1981

A simplified triple-test cross analysis of alcohol preference in the rat.

Reanalyses of first-degree biometrical genetic data from previous studies of alcohol preference in the mouse revealed little consistency beyond a basic additive genetic component. A simplified triple-test cross in the rat investigated the genetic architecture of alcohol preference for a 10% (w/v) alcohol solution or water. An initial survey of eight selected and inbred strains identified high- and low-scoring strains, the MNR and the ACI respectively, which were crossed as tester lines to six strains ( the RHA, RLA, TMB, TMD, MNR, and ACI) to produce the required set of largely F1 families. The additive-dominance model proved adequate for males, and directional dominance for low alcohol preference was found on all three measures: alcohol intake, alcohol preference ratio, and alcohol calorie contribution ratio. For females the model was adequate only for alcohol preference ratio, which showed ambidirectional dominance. The relevance of such genetic architecture to an animal model of alcoholism and to the evolution of alcohol drinking in the rat is discussed.

Alcohol Drinking↗
PubMed1984

A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures.

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T3. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intracellular calcium mobilization, TMB-8, was found to inhibit the release of T3 in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T3 secretion by TSH and itself stimulated the release of T3 to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23187 produced a biphasic effect on T3 secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T3 secretion but above a concentration of 1 microM, T3 secretion was increased. The calmodulin antagonist W7 was found to inhibit T3 release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T3 secretion.

Calcimycin↗
PubMed1983

Calcium, calmodulin, and the production of prostacyclin by cultured vascular endothelial cells.

The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell lysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.

Animals↗
PubMed1993

Roles of Ca2+ and protein kinase C in regulation of prostaglandin E2 release by cultured rabbit gastric epithelial cells.

Prostaglandin (PG) has been reported to be one of the important protective factors in the gastric mucosa. However the mechanism of the regulation of endogenous PG production has not been well studied. We investigated the possible roles of Ca2+, cAMP, and protein kinase C (PKC) in the regulation of PGE2 release from cultured rabbit gastric mucosal cells. PGE2 was measured by radioimmunoassay. A23187 (Ca2+ ionophore) at 2 x 10(-6) M significantly increased PGE2 release. Deprivation of Ca2+ from the medium blocked the A23187-induced increase of PGE2. TMB-8 (a putative inhibitor of Ca2+ release from intracellular stores) did not have any significant effects on the increase of PGE2-induced by A23187. Thus, A23187 increased PGE2 through the influx of extracellular Ca2+. W7 or compound 48/80 (calmodulin inhibitors) did not alter the response of PGE2 caused by A23187. Exogenous administration of cAMP, forskolin (an activator of adenylate cyclase), or 2-chloroadenosine (a possible activator of adenylate cyclase through adenosine A2 receptor) had neither significant effects on PGE2 release nor an effect on A23187-induced increase of PGE2 release. 12-O-tetradecanoylphorbol 13-acetate (TPA, an activator of PKC) significantly stimulated PGE2 release in a dose-dependent fashion, whereas another phorbol ester with no biological activity did not. A23187 at 0.8 x 10(-6) M, but not cAMP, potentiated the TPA-induced increase of PGE2. Mepacrine (a phospholipase A2 inhibitor) reduced the A23187- and TPA-induced increase of PGE2. These results suggest that Ca2+ and protein kinase C may play important roles in the regulation of PGE2 release by cultured rabbit gastric cells.

2-Chloroadenosine↗
PubMed1995

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells.

[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca(2+)-free medium + 0.5 mM ethylene glycol-bis(beta-aminoethylether)N,N,N',N'-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 microM modified [3H]purine outflow whereas 0.1 microM omega-conotoxin and 0.03-0.1 microM nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 microM omega-conotoxin + 0.1 microM nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor omega-conotoxin influenced the uptake of 45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the 45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 microM Fura-2/AM. 10-30 microM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 microM thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC, seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗