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J C Jonas

Publications and source records attributed to J C Jonas.

30 records · Page 2Linked to original sources
PubMed1996

Stable and diffusible pools of nucleotides in pancreatic islet cells.

Adenine nucleotides are thought to serve as second messengers in the control of beta-cell function by glucose, e.g. by regulating the activity of ATP-dependent K+ channels. However, their localization in different intracellular pools may mask the biologically relevant changes and complicate the interpretation of measurements in whole cells. The plasma membrane of mouse islet cells was selectively permeabilized by the alpha-toxin from Staphylococcus aureus to allow diffusion of cytoplasmic nucleotides. After permeabilization of cells from freshly isolated islets, approximately 68% of ATP, 45% of ADP, and 52% of AMP rapidly diffused out of the cells, whereas the insulin content hardly varied. The nondiffusible pool of nucleotides was stable for at least 90 min at 4 C, which suggests that it is contained in cellular organelles. The size of this nondiffusible pool decreased proportionally to insulin stores when these were lowered by stimulating secretion to different degrees during culture before permeabilization. From these results, it can be calculated that nondiffusible nucleotides are mainly contained in insulin secretory granules, with a small proportion in another, probably mitochondrial, compartment. Approximately 80% GTP and 30% GDP were present in the diffusible pool, and their relative proportions in the granular pool were only about 20% that of adenine nucleotides. Incubation of the cells in 20 instead of 2 mM glucose before permeabilization did not affect the nondiffusible pool, which indicates that the increase in the ATP/ADP ratio measured in intact cells occurred in the diffusible pool. Cytoplasmic nucleotide levels could be evaluated by subtracting the nondiffusible pool from the measurements in intact cells. It emerges that glucose induces large changes in the ATP/ADP ratio in the cytoplasmic pool, and that these changes are largely due to a fall in ADP.

Adenosine Diphosphate↗
PubMed1995

Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells: comparison with the effect of Rab3AL peptide.

Most guanine nucleotide binding proteins (G-proteins) possess an S-prenylated C-terminal cysteine whose carboxyl group can be reversibly methylated. The prenylcysteine analog N-acetyl-S-geranylgeranyl-cysteine (AGGC) (50 microM), a competitive inhibitor of prenylcysteine methyl transferases, introduced into streptolysin-O permeabilized HIT-T15 cells doubled the rate of basal (0.1 microM Ca2+) and of stimulated (10 microM Ca2+ or 100 microM GTP gamma S) insulin secretion in a reversible and ATP-dependent manner. N-acetyl-S-farnesylcysteine (AFC) was less potent while N-acetyl-S-geranyl-cysteine was inactive. Prenylcysteine action on exocytosis did not involve inhibition of G-protein methylation, since (1) the methyl ester derivative of AFC, an inefficient inhibitor of methyltransferases in HIT-T15 cell fractions, was as potent as AGGC in stimulating exocytosis; (2) S-adenosyl-homocysteine, a general inhibitor of methylation reactions, did not alter basal or GTP gamma S-triggered secretion while inhibiting Ca(2+)-induced insulin release. The binding of G-proteins to Rab/GDP-dissociation inhibitor, Rab3A/GTPase activating protein or rabphilin-3A was not affected by the prenylcysteine analogs. AGGC or AFC had the same effect on insulin release as a synthetic peptide mimicking the amino acid residues 52-67 of the G-protein Rab3A (Rab3AL). Moreover, the action on secretion of the combination of Rab3AL and prenylcysteines was not additive. We propose that the prenylcysteines and the Rab3AL peptide influence exocytosis by affecting the association of Rab3A with different proteins of the exocytotic machinery of insulin-secreting cells.

Acetylcysteine↗
PubMed1995

Multiple effects and stimulation of insulin secretion by the tyrosine kinase inhibitor genistein in normal mouse islets.

1. Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus-secretion coupling in pancreatic beta-cells. 2. Genistein produced a concentration-dependent (10-100 microM), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non-metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 microM genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3. Although genistein slightly inhibited ATP-sensitive K+ channels, as shown by 86Rb efflux and patch-clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP-sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP-sensitive K+ channels were opened by diazoxide and the beta-cell membrane depolarized by 30 mM K, but ineffective in the presence of diazoxide and normal extracellular K. 4. Genistein paradoxically decreased Ca2+ influx in beta-cells, as shown by the inhibition of glucose-induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura-2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in beta-cells. 5. Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion.6. Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in beta-cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.

Adenine Nucleotides↗
PubMed1995

Possible links between glucose-induced changes in the energy state of pancreatic B cells and insulin release. Unmasking by decreasing a stable pool of adenine nucleotides in mouse islets.

Whether adenine nucleotides in pancreatic B cells serve as second messengers during glucose stimulation of insulin secretion remains disputed. Our hypothesis was that the actual changes in ATP and ADP are obscured by the large pool of adenine nucleotides (ATP/ADP ratio close to 1) in insulin granules. Therefore, mouse islets were degranulated acutely with a cocktail of glucose, KCl, forskolin, and phorbol ester or during overnight culture in RPMI-1640 medium containing 10 mM glucose. When these islets were then incubated in 0 glucose + azide (to minimize cytoplasmic and mitochondrial adenine nucleotides), their content in ATP + ADP + AMP was decreased in proportion to the decrease in insulin stores. After incubation in 10 mM glucose (no azide), the ATP/ADP ratio increased from 2.4 to > 8 in cultured islets, and only from 2 to < 4 in fresh islets. These differences were not explained by changes in glucose oxidation. The glucose dependency (0-30 mM) of the changes in insulin secretion and in the ATP/ADP ratio were then compared in the same islets. In nondegranulated, fresh islets, the ATP/ADP ratio increased between 0 and 10 mM glucose and then stabilized although insulin release kept increasing. In degranulated islets, the ATP/ADP ratio also increased between 0 and 10 mM glucose, but a further increase still occurred between 10 and 20 mM glucose, in parallel with the stimulation of insulin release. In conclusion, decreasing the granular pool of ATP and ADP unmasks large changes in the ATP/ADP ratio and a glucose dependency which persists within the range of stimulatory concentrations. The ATP/ADP ratio might thus serve as a coupling factor between glucose metabolism and insulin release.

Adenosine Diphosphate↗
PubMed1994

The imidazoline SL 84.0418 shows stereoselectivity in blocking alpha 2-adrenoceptors but not ATP-sensitive K+ channels in pancreatic B-cells.

The novel alpha 2-adrenoceptor antagonist SL 84.0418 (2-(4,5-dihydro-1H-imidazol-2-yl)-1,2,4,5-tetrahydro-2-propyl-pyrrolo[3, 2,1- hi]-indole hydrochloride) is a racemic mixture of a (-) enantiomer (SL 86.0714) and a (+) enantiomer (SL 86.0715 or deriglidole). It was recently reported to inhibit alpha 2-adrenoceptors and ATP-sensitive K+ channels in mouse pancreatic B-cells, and to increase insulin release. We have now studied the stereospecificity of these responses with isolated mouse islets. Both enantiomers were equipotent in potentiating insulin release induced by 15 mM glucose alone. SL 86.0714 and deriglidole were also equally effective in inhibiting 86Rb efflux from islets perifused with a low-glucose medium, and in reversing the inhibition of glucose-induced insulin release caused by the opening of ATP-sensitive K+ channels with diazoxide. In contrast, deriglidole was approximately 100-fold more potent than SL 86.0714 in reversing the inhibition of insulin release caused by the activation of alpha 2-adrenoceptors with clonidine. The effects of SL 84.0418 are thus stereoselective on alpha 2-adrenoceptors, but not on ATP-sensitive K+ channels of pancreatic B-cells.

Adenosine Triphosphate↗
PubMed1994

Dynamics of Ca2+ and guanosine 5'-[gamma-thio]triphosphate action on insulin secretion from alpha-toxin-permeabilized HIT-T15 cells.

The time course of Ca2+ and GTP-analogue effects on insulin secretion was investigated in HIT-T15 cells permeabilized with Staphylococcus alpha-toxin. These cells responded to Ca2+ in the range 0.1-10 microM and could be used in a dynamic perifusion system because of the minimal run-down of the secretory response. High Ca2+ (10 microM) elicited a monophasic ATP-dependent stimulation of insulin secretion that reached a peak within 5 min (approximately 20-fold increase) and rapidly decreased during the subsequent 15 min to a plateau remaining above basal rates (0.1 microM Ca2+). The decrease in Ca(2+)-induced insulin secretion with time could not be attributed to decreased capacity to respond to Ca2+ after prolonged perfusion at low Ca2+ (run-down), nor to depletion of a particular secretory-granule pool. It was rather due to desensitization of the secretory machinery to Ca2+ that was not reversed by selective inhibition of the Ca2+/calmodulin-dependent kinase II with KN-62. However, an intermediate Ca2+ concentration (2 microM) increased insulin secretion to stable level without causing any desensitization. Imposed oscillations of Ca2+ (0.1-10 microM) produced phasic oscillations of insulin secretion, but did not prevent desensitization to Ca2+. Poorly hydrolysable GTP analogues increased insulin secretion at low Ca2+, whereas they strongly inhibited Ca(2+)-induced insulin secretion. By contrast, GTP did not affect basal secretion, and slightly increased Ca(2+)-evoked secretion. These results indicate the following. (1) Oscillations of insulin secretion are tightly coupled to cytosolic Ca2+ oscillations. (2) Oscillations of Ca2+ do not prevent high-Ca(2+)-induced desensitization to Ca2+; this result does not support the idea of a greater efficiency of oscillations compared with sustained Ca2+ rises in triggering exocytosis. (3) Activation of G-proteins modulates exocytosis in a bimodal manner.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

In vitro stimulation of insulin release by SL 84.0418, a new alpha 2-adrenoceptor antagonist.

SL 84.0418 (2-(4,5-dihydro-1H-imidazol-2-yl)-1,2,4,5-tetrahydro-2-propyl-pyrrolo[3, 2,1- hi]-indole hydrocholoride) is a novel alpha 2-adrenoceptor antagonist which possesses anti-hyperglycaemic properties in vitro study, we tested its effects on insulin release from isolated mouse islets. In the presence of 15 mM glucose, SL 84.0418 produced a concentration-dependent (0.1-100 microM) increase of insulin release with a slightly higher potency than tolbutamide. SL 84.0418 antagonized the inhibition of glucose-induced insulin release caused either by the alpha 2-adrenoceptor agonist clonidine or by diazoxide, a selective opener of ATP-sensitive K+ channels in the beta-cell membrane. Its potency was greater on the inhibition by clonidine than on that by diazoxide, complete antagonism of the inhibition being achieved by 0.9 microM and 6 microM SL 84.0418 respectively. When alpha 2-adrenoceptors were blocked by the antagonist idazoxan, the low concentrations of SL 84.0418 (0.1-0.3 microM) no longer increased insulin release, whereas the effect of higher concentrations (> or = 1 microM) was not affected. SL 84.0418 (> or = 1 microM) inhibited 86Rb efflux from islets perifused with a medium containing 3 mM glucose, i.e. under conditions where many ATP-sensitive K+ channels are open. It also reduced the acceleration of 86Rb efflux that diazoxide caused in the presence of 6 mM glucose. Moreover, SL 84.0418 directly inhibited ATP-sensitive K+ currents measured in single beta-cells by the whole-cell mode of the patch-clamp technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-2 Receptor Antagonists↗
PubMed1994

Culture duration and conditions affect the oscillations of cytoplasmic calcium concentration induced by glucose in mouse pancreatic islets.

The pattern of the increase in cytoplasmic Cai2+ that glucose produces in beta cells has been reported to be highly variable. Here, we evaluated the influence of the culture duration (1-4 days) and conditions (5-10 mmol/l glucose) on Cai2+ in normal mouse islets stimulated by glucose. After 1 day of culture in 10 mmol/l glucose, a rise of the glucose concentration from 3 to 15 mmol/l induced a triphasic change of Cai2+ in the islets. A small initial decrease was followed by a large peak increase and then by regular fast oscillations (approximately 2.5/min). When the culture was prolonged to 2, 3 and 4 days, the initial decrease became inconsistent and the peak occurred earlier, whereas the oscillations decreased in frequency, increased in duration and eventually disappeared; on day 4 the Cai2+ rise was sustained. After culture in 5 mmol/l glucose, the pattern of Cai2+ changes induced by 15 mmol/l glucose was different. The initial decrease was very pronounced, the first peak was delayed and clearly separated from the subsequent oscillations. These were of a mixed type (fast Ca2+ transients on top of slow ones) after 1 day, and of a slow type only after 4 days. These alterations in the Cai2+ oscillations triggered by glucose could not be ascribed to desynchronization of the signal between different regions of the islets. In conclusion, culturing normal mouse islets in 5 or 10 mmol/l glucose for 1-4 days, markedly alters the characteristics of the changes in Cai2+ produced by glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.

Actin Cytoskeleton↗
PubMed1992

Imidazoline antagonists of alpha 2-adrenoceptors increase insulin release in vitro by inhibiting ATP-sensitive K+ channels in pancreatic beta-cells.

1. Islets from normal mice were used to study the mechanisms by which imidazoline antagonists of alpha 2-adrenoceptors increase insulin release in vitro. 2. Alinidine, antazoline, phentolamine and tolazoline inhibited 86Rb efflux from islets perifused with a medium containing 3 mM glucose, i.e. under conditions where many adenosine 5'-triphosphate (ATP)-sensitive K+ channels are open in the beta-cell membrane. They also reduced the acceleration of 86Rb efflux caused by diazoxide, an opener of ATP-sensitive K+ channels. 3. ATP-sensitive and voltage-sensitive K+ currents were measured in single beta-cells by the whole-cell mode of the patch-clamp technique. Antazoline more markedly inhibited the ATP-sensitive than the voltage-sensitive current, an effect previously observed with phentolamine. Alinidine and tolazoline partially decreased the ATP-sensitive K+ current. 4. The four imidazolines reversed the inhibition of insulin release caused by diazoxide (through opening of ATP-sensitive K+ channels) or by clonidine (through activation of alpha 2-adrenoceptors) in a concentration-dependent manner. Only the former effect correlated with the ability of each drug to increase control insulin release stimulated by 15 mM glucose alone. 5. It is concluded that the ability of imidazoline antagonists of alpha 2-adrenoceptors to increase insulin release in vitro can be ascribed to their blockade of ATP-sensitive K+ channels in beta-cells rather than to their interaction with the adrenoceptor.

Adenosine Triphosphate↗
PubMed1991

Clonidine inhibits ATP-sensitive K+ channels in mouse pancreatic beta-cells.

1. The effects of clonidine and adrenaline on adenosine 5'-triphosphate (ATP)-sensitive K+ channels were studied in pancreatic beta-cells from normal mice. 2. When perifused with a medium containing 1 mM glucose, many of the ATP-sensitive K+ channels in the beta-cell membrane are open. Under these conditions, clonidine (5-100 microM) reversibly decreased 86Rb efflux from the islets, whereas adrenaline was ineffective at concentrations up to 100 microM. 3. In 6 mM glucose, most of the ATP-sensitive K+ channels in the beta-cell membrane are closed. Opening these channels by diazoxide (100 microM) caused a marked acceleration of 86Rb efflux from the islets, which was attenuated by 100 microM clonidine. 4. ATP-sensitive K+ currents were measured in single beta-cells by the whole cell mode of the patch-clamp technique. At concentrations above 4 microM, clonidine reversibly inhibited the ATP-sensitive K+ current in a dose-dependent manner. 5. Voltage-sensitive K+ currents were unaffected by 20 microM but decreased slightly by 100 microM clonidine. 6. Calcium currents, measured by the whole cell or perforated patch technique, were unaffected by clonidine at concentrations up to 100 microM. 7. It is concluded that high concentrations of the alpha 2-adrenoceptor agonist clonidine, but not of adrenaline, can inhibit ATP-sensitive K+ channels in pancreatic beta-cells. Other ionic channels are only slightly affected or unaffected.

Adenosine Triphosphate↗