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E F Nemeth

Publications and source records attributed to E F Nemeth.

51 records · Page 3Linked to original sources
PubMed1988

Regulation of hormone secretion and cytosolic Ca2+ by extracellular Ca2+ in parathyroid cells and C-cells: role of voltage-sensitive Ca2+ channels.

The two dihydropyridine enantiomers, (+)202-791 and (-)202-791, that act as voltage-sensitive Ca2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca2+ concentrations ([Ca2+]i) and on hormones secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca2+ evoked transient followed by sustained increases in [Ca2+]i, as measured with fura-2. Increases in [Ca2+]i obtained by raised extracellular Ca2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca2+ channel agonist (+)202-791 stimulated whereas the antagonist (-)202-791 inhibited both transient and sustained increases in [Ca2+]i induced by extracellular Ca2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (-)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca2+]i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K+ increased [Ca2+]i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca2+ influx in the regulation of cytosolic Ca2+ and hormones secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca2+ influx pathways and regulate PTH secretion by some alternative mechanism.

Animals↗
PubMed1987

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.

Aminoquinolines↗
PubMed1987

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques.

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [14C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.

Aminoacridines↗
PubMed1986

Cytosolic Ca2+ and the regulation of secretion in parathyroid cells.

The concentration of intracellular Ca2+ [( Ca2+]i) was measured in dissociated bovine parathyroid cells loaded with quin-2 or fura-2. In quin-2-loaded cells, increases in the concentration of extracellular Ca2+ elicited slow, monophasic increases in [Ca2+]i, whereas in fura-2-loaded cells, extracellular Ca2+ evoked rapid, transient increases which were followed by lower, yet sustained increases in [Ca2+]i. Cytosolic Ca2+ transients arose from the mobilization of cellular Ca2+ and could be evoked by a variety of divalent cations. Transient, but not sustained increases in [Ca2+]i were associated with an inhibition of hormone secretion. Secretion was still inhibited, however, when cytosolic Ca2+ transients were blocked by buffering with quin-2, suggesting that changes in [Ca2+]i might not be the essential factor regulating secretion in parathyroid cells.

Aminoquinolines↗
PubMed1986

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+.

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.

Aminoquinolines↗
PubMed1985

Evidence for the involvement of kainate receptors in synaptic transmission in the avian cochlear nucleus.

Previous studies using various excitatory amino acid antagonists have shown that synaptic transmission between the auditory nerve and the cochlear nucleus of chickens (nuc. magnocellularis; NM) is mediated by non-N-methyl-D-aspartate (non-NMDA) receptors. In the present study we have attempted to define the subclass of non-NMDA receptor in the NM by examining the effects of various excitatory amino acid agonists on synaptically evoked field potentials in an in vitro preparation of the chicken brain stem. Both quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), whose actions operationally define the quisqualate receptor class, caused variable and weak depression of evoked responses in the NM, as did L-glutamate. Kainic acid, on the other hand, completely blocked postsynaptic responses at micromolar concentrations. We conclude that kainate-preferring non-NMDA receptors play a predominant role in mediating transmission in the NM.

Animals↗
PubMed1985

Non-N-methyl-D-aspartate receptors mediating synaptic transmission in the avian cochlear nucleus: effects of kynurenic acid, dipicolinic acid and streptomycin.

We have examined the effects of a number of excitatory amino acid antagonists on transmission at the cochlear nerve-nucleus magnocellularis synapse in the chicken. Using an in vitro preparation and bath application of drugs, we studied the effects of kynurenic acid and several related substances, streptomycin and a selective N-methyl-D-aspartate receptor antagonist, DL-alpha-aminosuberate. The last compound had no effect on evoked transmission. Of the various kynurenic acid-related compounds tested, only kynurenic and dipicolinic acid selectively altered responses in nucleus magnocellularis. Quinolinic acid, a kynurenic acid analogue that is structurally akin to dipicolinic acid but which acts selectively at N-methyl-D-aspartate receptors, was without effect. The effect of kynurenic acid was solely inhibitory, completely blocking postsynaptic responses with a potency dependent on the frequency of nerve stimulation. No such frequency dependence was seen with dipicolinic acid although this compound also completely suppressed evoked responses. In addition dipicolinic acid potentiated postsynaptic responses at concentrations only slightly lower than those causing inhibition. Streptomycin inhibited responses in nucleus magnocellularis but this effect seems to result partially from the ability of the drug to inhibit presynaptic calcium influx. Our finding that selective antagonists of N-methyl-D-aspartate receptors were ineffective while antagonists of both receptor types, such as kynurenic and dipicolinic acids, inhibited evoked responses reinforces the conclusion that postsynaptic receptors mediating transmission at this synapse are of the non-N-methyl-D-aspartate type [Nemeth et al. (1983) Neurosci. Lett. 40, 39-44].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Pharmacologic evidence for synaptic transmission mediated by non-N-methyl-D-aspartate receptors in the avian cochlear nucleus.

The hypothesis that synaptic transmission between the auditory nerve and the cochlear nucleus is mediated by an excitatory amino acid acting through N-methyl-D-aspartate (NMDA) receptors was examined in an in vitro preparation of the chicken brainstem. The ability of various bath-applied excitatory amino acid receptor antagonists to inhibit synaptically-evoked responses was assessed by recording field potentials from nucleus magnocellularis (NM) following electrical stimulation of the cochlear nerve. Antagonists that selectively block responses mediated by NMDA receptors, such as D-alpha-aminoadipate and 2-amino-5-phosphonovalerate, were without effect on evoked transmission in NM. In contrast, antagonists that additionally act on non-NMDA receptors, such as cis-2,3-piperidine dicarboxylate and gamma-D-glutamylglycine, reversibly suppressed transmission. The results indicate that (1) transmission in the chicken auditory system is mediated by non-NMDA receptors, and (2) a substance(s) chemically akin to aspartate and glutamate may be the transmitter used by the auditory nerve in NM.

Animals↗
PubMed1983

Calmodulin, phospholipase, and exocytosis. p-Bromophenacyl bromide inhibits but mepacrine stimulates secretion in rat mast cells.

p-Bromophenacyl bromide (10-50 microM), a reagent that reacts with and inhibits phospholipase A2, completely suppressed exocytotic responses in rat serosal mast cells elicited by A23187. In contrast, mepacrine (0.01-0.1 mM), an antimalarial drug which has likewise been reported to inhibit phospholipase and additionally calmodulin, did not inhibit exocytosis elicited by concanavalin A (con A) or ionophore A23187. Rather, mepacrine (0.1-0.5 mM) alone increased histamine release up to 70%. Light microscopic observations following ruthenium red staining demonstrated the mepacrine-induced response to be exocytosis. Like the response to compound 48/80, that elicited by mepacrine was maximal within 40 s and was inhibited by phosphatidylserine. In the absence of extracellular calcium, secretion elicited by mepacrine and by con A was inhibited by 87 and 90%, respectively, whereas that elicited by 48/80 was unaffected. Incubation of mast cells in the presence of 2 mM EDTA for 2 h inhibited responses to 48/809 by 76% and nearly abolished those to mepacrine and to con A. 5,8,11,14-Eicosatetraynoic acid (50 and 100 microM) inhibited secretion elicited by con A but not that evoked by mepacrine or polymyxin B. The phenothiazines thioridazine (10-50 microM) and chlorpromazine (25-125 microM) inhibited secretory responses to mepacrine, to 48/80 and to con A. Both phenothiazines inhibited secretion elicited by con A more effectively than that elicited by mepacrine or 48/80. The results indicate that mepacrine, like con A, elicits exocytosis in mast cells by drawing on extracellular sources of calcium, but the early events of secretion initiated by these two secretagogues are pharmacologically distinct.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetophenones↗
PubMed1982

Lipoxygenase inhibitors exert secretagogue-specific effects on mast cell exocytosis.

The lipoxygenase inhibitors HTYA (5,8,11,14-henicosatetraynoic acid, 25-150 microM) and ITYA (4,7,10,13-icosatetraynoic acid, 25-75 microM), inhibited histamine secretion from rat mast cells evoked by concanavalin A but that elicited by compound 48/80 or polymyxin B. Arachidonic acid (100 microM) did not inhibit concanavalin A-induced histamine secretion. The results are consistent with the notion that lipoxygenase inhibitors affect an early stage of stimulus-secretion coupling in the mast cell, peculiar to antibody-directed secretagogues, perhaps that involving calcium influx.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1982

On the calcium receptor activating exocytosis: inhibitory effects of calmodulin-interacting drugs on rat mast cells.

1. A series of neuroleptic drugs (five phenothiazines, imipramine, and pimozide) and the smooth muscle relaxant W-7, which all inhibit calcium-calmodulin-activated processes inhibited rat mast cell secretion elicited by antigen, by 48/80, and by the calcium ionophore A23187. 2. Neither the phenothiazines nor W-7 reduced 45Ca uptake in response to A23187. The drugs thus exert an inhibitory action distal to the rise in intracellular Ca ions that activates exocytosis. 3. Chlorpromazine sulphoxide, which shares several membrane-perturbing actions of the phenothiazines but is a weak inhibitor of calmodulin, did not inhibit secretion. Moreover, the inhibitory effects of the phenothiazines were not overcome by a 5- or 10-fold increase in the concentration of calcium, which should counter unspecific membrane effects. 4. The inhibitory effects of the various neuroleptic drugs appeared to be related to their ability to inhibit calmodulin because the individual potencies of these compounds on secretion evoked by 48/80 or A23187 correlated significantly with their reported potencies in inhibiting calmodulin-activated processes. (The greater potency and different rank order of these compounds on secretion evoked by antigen suggests an additional inhibitory action, perhaps involving Ca entry.) 5. These results, which parallel those obtained with drugs of this sort in smooth muscle where calmodulin seemingly functions as the Ca receptor activating contraction, strengthen the view that calmodulin, or some calmodulin-like protein, is the Ca receptor activating exocytosis.

Animals↗
PubMed1980

Differential inhibitory effects of the arachidonic acid analog ETYA on rat mast cell exocytosis evoked by secretagogues utilizing cellular or extracellular calcium.

ETYA (5,8,11,14-eicosatetraynoic acid; 50-100 microM), which inhibits both cyclo-oxygenase and lipoxidase, inhibited histamine release evoked by secretagogues dependent on extracellular calcium (antigen, dextran, and concanavalin A) but failed to inhibit secretion elicited by secretagogues capable of mobilizing calcium from intracellular sites (48/80, polymyxin B, protamine sulfate and poly-L-lysine). Responses to these latter secretagogues were inhibited only by higher concentrations of ETYA (100-200 microM) that were cytotoxic. Secretion evoked by the calcium ionophore A23187 (0.1 microgram/ml) was inhibited at much lower concentrations of ETYA (1-10 microM) but this inhibition could not be overcome by increasing the concentration of calcium. Responses to higher concentrations of ionophore were not inhibited by ETYA except in amounts affecting cell viability. Like ETYA, each of several fatty acids, including arachidonic acid, were inhibitory towards histamine release evoked by A23187 or 48/80. The results indicate that EYTA acts at some early stage of stimulus-secretion coupling rather than on the final, common, calcium-activated steps of exocytosis. Moreover, this action may be unrelated to inhibition of lipoxidase or cyclooxygenase.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1978

Effects of microfilament-active drugs, phalloidin and the cytochalasins A and B, on exocytosis in mast cells evoked by 48/80 or A23187.

Rat peritoneal mast cells were used as a model system to study the effect, on exocytosis, of three agents known to interact with microfilaments. Mast cell secretion was evaluated by fluorimetric assay of histamine and by ruthenium red staining, the latter method allowing a direct visualization and quantitation of exocytosis at the light microscopic level. Phalloidin, in concentrations up to 300 microgram/ml, was without effect on either spontaneous or 48/80-evoked secretion, even after cells were exposed to the drug for 28 h. The failure of even high doses of phalloidin to influence cellular morphology and exocytosis in the mast cell may reflect the absence of a specific membrane receptor. Cytochalasin B was likewise without effect on the response to 48/80 in normally respiring cells; but inhibited this response in the presence of Antimycin A. This inhibitory effect probably reflects the ability of cytochalasin B to block glucose transport. In normally respiring cells, neither phalloidin nor cytochalasin B affected the active expulsion of granules from exocytotic pits. Cytochalasin A, without concomitant treatment with Antimycin A, completely inhibited secretion in response to both 48/80 and A23187, and did so in low concentration. Whether this striking inhibitory effect results from an interaction with microfilaments is uncertain for the inhibition could be mimicked by nonpenetrating thiol-oxidizing agents and prevented by impermeant thiol-protecting agents suggesting that cytochalasin A may inhibit histamine release by thiol-oxidation at the cell surface. Possible surface sulfhydryls are important for membrane rearrangements accompanying exocytosis.

Animals↗