Presence Of Diazoxide Research Articles

Activin A increases intracellular free calcium concentrations in rat pancreatic islets

Activin A stimulated insulin secretion in rat pancreatic islets, an effect that was attenuated by reduction of extracellular Ca 2+ and abolished by either nitrendipine or verapamil. Activin A increased intracellular the free Ca 2+ concentration, [Ca 2+] i in fura-2-loaded islets. Activin A-mediated elevation of [Ca 2+] i was abolished by the reduction of extracellular Ca 2+ or the addition of nifedipine. In addition, activin A did not increase [Ca 2+] i in the presence of diazoxide, an opener of ATP-sensitive K + channels. These results suggest that activin A increases insulin secretion by stimulating Ca 2+ entry.

Dopamine and somatostatin inhibition of prolactin secretion from MMQ pituitary cells: role of adenosine triphosphate-sensitive potassium channels.

The sulfonylurea glibenclamide, which is known to block ATP-sensitive potassium channels, increases, in a dose-dependent manner, the release of PRL from MMQ pituitary cells. Glibenclamide does not reduce the dopaminergic inhibition of forskolin-stimulated PRL secretion; conversely it almost completely abolishes the inhibitory effect of somatostatin (SRIF) on this parameter. The sulfonylurea dose dependently increases basal [Ca++]i, without affecting the increase in [Ca++]i induced by high concentrations of extracellular potassium. Glibenclamide does not modify dopamine-induced [Ca++]i reduction, whereas it abolishes the inhibitory effect of SRIF on basal [Ca++]i. In the presence of diazoxide, an opener of ATP-sensitive potassium channels, which lowers basal [Ca++]i, dopamine still reduces [Ca++]i whereas SRIF does not induce a further decrease. Glibenclamide induces the depolarization of the cell membrane and prevents the SRIF-evoked hyperpolarization. The hyperpolarization of the cell membrane induced by dopamine is not modified by glibenclamide. Diazoxide induces a cell membrane hyperpolarization that is enhanced by dopamine but not by SRIF. Finally, glibenclamide does not affect basal and stimulated adenylate cyclase activity. In conclusion, our findings show that, in MMQ cells, glibenclamide stimulates PRL release, suggesting an involvement of ATP-sensitive potassium channels in the regulation of PRL secretion. The reversal by glibenclamide of the effects of SRIF on calcium homeostasis, membrane potential, and PRL release suggests that this type of potassium channel participates to the somatostatinergic inhibition of PRL secretion. Conversely, we found that glibenclamide does not modify the dopaminergic inhibition of PRL secretion and second messenger systems, suggesting that ATP-sensitive potassium channels may not be involved in the inhibitory effect of dopamine on PRL release.

The relationship between glucose-induced K+ATP channel closure and the rise in [Ca2+]i in single mouse pancreatic beta-cells.

1. Intracellular calcium [Ca2+]i and channel activity were simultaneously recorded in single, dissociated mouse beta-cells kept in culture for 1-3 days. [Ca2+]i was estimated from microfluorometric ratio methods using Indo-1. Channel activity was measured using the cell-attached configuration of the patch-clamp technique. 2. At low glucose concentrations (0.3 mM), resting K+ATP channel activity was prevalent. Increasing glucose up to 16 mM, produced a gradual decrease in K+ATP channel activity over a time course of 90-120 s (temperature = 23 degrees C) and an increase in [Ca2+]i. 3. In the majority of experiments, glucose elicited biphasic action currents (action potentials) which preceded the rise in [Ca2+]i. There was a close correlation between spike frequency and the levels of [Ca2+]i. 4. The sulphonylurea tolbutamide (1 mM) blocked K+ATP channels in 10-20 s. K+ATP channel blockade was associated with a quick rise in [Ca2+]i. 5. When K+ATP channel activity was stimulated in the presence of diazoxide (100 microM), increasing the glucose concentration from 3 to 16 mM produced a decrease in [Ca2+]i. Only when diazoxide was removed did glucose produce an increase in [Ca2+]i. 6. In a small population of cells, glucose (16 mM) produced a small decrease in K+ATP channel activity but not an increase in [Ca2+]i. In such cells, tolbutamide blocked K+ATP channels and produced an increase in [Ca2+]i. 7. These results demonstrate a close correlation between K+ATP channel activity and [Ca2+]i in beta-cells. The findings are consistent with the model in which glucose metabolism produces a rise in [Ca2+]i through the blockade of K+ATP channels, membrane depolarization and calcium current activation.

Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells.

Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels, depolarization, and Ca2+ influx in B cells. Mouse islets were used to investigate whether glucose can still regulate insulin release when it cannot control ATP-sensitive K+ channels. Opening of these channels by diazoxide (100-250 mumol/liter) blocked the effects of glucose on B cell membrane potential (intracellular microelectrodes), free cytosolic Ca2+ (fura-2 method), and insulin release, but it did not prevent those of high K (30 mmol/liter). K-induced insulin release in the presence of diazoxide was, however, dose dependently increased by glucose, which was already effective at concentrations (2-6 mmol/liter) that are subthreshold under normal conditions (low K and no diazoxide). This effect was not accompanied by detectable changes in B cell membrane potential. Measurements of 45Ca fluxes and cytosolic Ca2+ indicated that glucose slightly increased Ca2+ influx during the first minutes of depolarization by K, but not in the steady state when its effect on insulin release was the largest. In conclusion, there exists a mechanism by which glucose can control insulin release independently from changes in K(+)-ATP channel activity, in membrane potential, and in cytosolic Ca2+. This mechanism may serve to amplify the secretory response to the triggering signal (closure of K(+)-ATP channels--depolarization--Ca2+ influx) induced by glucose.

Leucine and tissue distribution of bulky and small neutral amino acids in rats: dissociation between transport and insulin-mediated effects.

The mechanism of the observed decrease in the plasma concentration of several amino acids in the presence of high levels of Leu has remained unexplained. In the present study a decrease in the plasma concentration of Ile, Val, Phe, Tyr, Met, Ala, Pro and Gly was observed after the intraperitoneal injection of Leu to weanling rats. Decreases in net intracellular concentrations in muscle accompanied the decrease in plasma of all of these amino acids except Pro and Gly. An increase in the distribution ratio muscle/plasma was observed exclusively for Gly after administration of Leu or of a non-insulinogenic transport system L analogue. Diazoxide suppressed the Leu-induced decreases in plasma and muscle intracellular concentrations of Ile and Val as well as of Pro in plasma. An increase in the distribution ratio liver/plasma was observed for Pro and Gly in the absence but not in the presence of diazoxide. All the above changes were statistically significant. Hence insulin probably mediates Leu effects, promoting an increased utilization of Ile and Val in muscle and of Pro in liver. A more direct effect of Leu appears to be involved in the apparent increased utilization of Phe, Tyr and Ala in the same tissue. Gly depletion in plasma can be explained by its trapping by inhibitory action of Leu on the exodus of Gly through transport system L.

Demonstration of glucose inhibition of insulin release in the presence of diazoxide.

beta-Cell-rich pancreatic islets from ob/ob mice were taken for measurements of insulin release in response to glucose after culture in RPMI 1640 medium. The stimulatory effect of 20 mmol/l glucose was converted into an inhibition when the medium was supplemented with 400 mumol/l diazoxide. Glucose inhibition of insulin release was observed when the islets had been cultured in the presence of 1 or 20 mmol/l glucose in media either containing or lacking Ca2+. The data provide further evidence for an inhibitory component in the action of glucose on insulin release, suggesting that glucose stimulation of the Ca2+ efflux is essential for the appearance of this inhibition.

Modification by diazoxide of canine renal vascular responsiveness to endogenous vasoconstrictors

AbstractDiazoxide‐induced modification of renal vascular responses to norepinephrine and angiotensin II was studied in dogs. Diazoxide was infused directly into the renal artery in order to minimize the effect of high plasma protein binding and to obtain maximum exposure of the test vessel to the administered drug. During the 30‐min infusion period, renal blood flow showed an initial, transitory increase followed by a progressive decline which paralleled the induced fall in systemic blood pressure. In the presence of diazoxide, renal vasoconstrictor responses to intravenous and intraarterial norepinephrine and intravenous angiotensin II were significantly attenuated. In contrast, the renal vasoconstrictor response to intraarterial angiotensin II was not significantly modified by diazoxide. This differential inhibition by diazoxide of renal vasoconstriction to norepinephrine or angiotensin II may warrant consideration when these pressor agents are being used to overcome the excessive hypotension resulting from inadvertent diazoxide overdosage.

Diazoxide and D600 inhibition of insulin release. Distinct mechanisms explain the specificity for different stimuli.

The mechanisms by which diazoxide and D600 affect insulin release have been compared in experiments using isolated rat islets. Diazoxide (20–400 (JLM) and D600 (1–50 μM) produced a dose-dependent inhibition of glucose-stimulated release. Diazoxide also inhibited the insulinotropic effect of leucine and related substances (ketoisocaproate and BCH), but not that of potassium or of arginine and other cationic amino acids. Diazoxide suppressed glucose and leucine stimulation of Ca uptake in islet cells, but had no effect on the stimulation by potassium and arginine. By contrast, D600 suppressed the effect of all these agents on both Ca uptake and insulin release. Theophylline partially antagonized the inhibitory effect of D600 on release, in the presence of diazoxide, theophylline was much less effective, except when combined with cationic amino acids. Diazoxide inhibition of glucose-induced release was prevented by phentolamine, but hot by dihydroergotamine and yohimbine, two other blpckers of a-adrenergic receptors. Epinephrine abolished the insulinotropic effect of arginine alone or with theophylline. Diazoxide increased 86Rb+ efflux from islet cells, whereas D600 and epinephrine decreased it. The acceleration of efflux by diazoxide was inhibited by D600 and phentolamine, but not by epinephrine or dihydroergotamine. It thus appears that the effects of diazoxide on B-cells are not due to activation of α-adrenergic receptors. The results suggest that, in contrast to the direct blockade of Ca channels by D600, the blockade of these channels by diazoxide is secondary to the hyperpolarization of the B-cell membrane. Since the latter results from an increase in K permeability, the inhibitory effects of diazoxide are restricted to stimulators that depolarize the B-cell membrane by decreasing its K permeability (glucose, leucine, and related substances) and do not affect the stimulation by K and cationic amino acids, which depolarize by other mechanisms.

Relaxation of isolated middle cerebral artery induced by diazoxide.

The effect of diazoxide on isometric tension of goat middle cerebral arteries was investigated both under resting conditions and under contraction produced by 10(-6) M serotonin. In addition, the inhibitory action of diazoxide was tested against contractile effects induced by different experimental interventions such as electrical field stimulation, norepinephrine, tyramine, histamine, and KCl. Diazoxide caused dose-dependent relaxation of cerebral arteries which was more pronounced when the vessels were previously contracted. High doses of diazoxide (10(-3) M) inhibited significantly the contraction induced by electrical field stimulation and all the vasoactive agents used. This inhibitory effect of diazoxide was greater for those drugs that directly or indirectly act through alpha-adrenergic receptor stimulation. Tritium release induced by electrical field stimulation of cerebral arteries previously labelled with 3H-norepinephrine was not affected in the presence of diazoxide. We conclude that diazoxide has a dilatory effect on goat brain vessels due to direct relaxation of smooth muscle together with a possible blockade of the alpha -adrenergic receptors. This effect might explain the maintenance of cerebral blood flow observed in vivo during diazoxide-induced arterial hypotension.

Effect of Benzothiadiazine Derivatives on Cyclic Nucleotide Phosphodiesterase and on the Tension of the Aortic Strip

Diazoxide and chlorothiazide (0.1--1.5 mM) had a dose-dependent inhibitory effect on the rate of cAMP and cGMP hydrolysis determined in a 500-g supernatant of rat aorta homogenates; both compounds were weaker inhibitors of cAMP and cGMP hydrolysis than theophylline. cAMP and cGMP content of the aorta did not change in the presence of diazoxide or chlorothiazide; diazoxide, however, further increased the isoproterenol-induced rise in cAMP, while chlorothiazide did not. Both benzothiadiazines decreased the maximum tension of the aortic strip induced by serotonin, phenylephrine or potassium. Diazoxide was a stronger and chlorothiazide a weaker inhibitor of the contractile response than theophylline. Comparison of the biochemical and functional effects of diazoxide and chlorothiazide indicates that the inhibitory effect of these compounds on cyclic nucleotide phosphodiesterase does not by itself explain their vasodilating effect.

Pancreatic Pancreatic β-Cell Replication: Relation to Insulin Secretion*

The relationship between beta-cell replication and insulin release was investigated utilizing neonatal rat pancreatic monolayer cell cultures. Glucose-induced insulin release was either inhibited using diazoxide or mannoheptulose, or potentiated using theophylline. The corresponding effects on the frequency of beta-cell replication were determined by incubating cultures with [3H]thymidine and estimating the frequency of beta-cell labeling in aldehyde thionine-stained radioautographs. beta-Cell replication and insulin release were shown to be dissociable processes in two ways. First, in the presence of diazoxide (1-100 microgram/ml), insulin release was inhibited by as much as 86%, while the frequency of beta-cell replication was not reduced. Second, in the presence of theophylline (1 mM), insulin release was increased by 23%, while beta-cell replication was inhibited. Finally, the inhibition of both beta-cell replication and insulin release by mannoheptulose (5.5 mM) indicated that glucose utilization may be important for both of these beta-cell processes.

Dynamics of Insulin Release and Microtubular‐Microfilamentous System

Abstract. A model is proposed to account for the phasic nature of insulin release. It is suggested that one modality of secretion results from the mobilization of secretory granules already located in the close vicinity of the microfilamentous cell web. This corresponds to a fraction of the initial secretory response to a high glucose concentration, the residual release of insulin provoked by glucose in the presence of diazoxide, and the total release of insulin evoked by sulphonylurea at a low glucose concentration. A second modality of release, which is characteristic of the late phase of glucose‐induced insulin secretion, is thought to correspond to the mobilization of secretory granules transported to the periphery of the β‐cell along oriented microtubular pathways. Whether only one or both modalities of release are initiated by a given insulinotropic stimulus may depend on the magnitude and duration of the changes induced by secretagogues in the insular handling of calcium.

Dynamics of Insulin Release and Microtubular‐Microfilamentous System

Abstract. The effect of colchicine upon glucose‐induced insulin release by the isolated perfused rat pancreas was examined in order to characterize the participation of the β‐cell microtubular apparatus in the biphasic insulin secretory response to glucose. After a short pretreatment (25 min.) with a low colchicine concentration (2.10−5 M), both the early and late phases of glucose‐induced insulin secretion were markedly enhanced. As either the concentration of colchicine or the length of the pretreatment period with this mitotic spindle‐inhibitor were increased, the facilitating effect faded out and partial inhibition of insulin release was observed. However, colchicine, even at high concentration, markedly enhanced the residual early release of insulin evoked by glucose in the presence of diazoxide (0.05 mg/ml). These findings suggest that (i) a fraction of the early phase of insulin secretion completely escapes from the inhibitory effect of colchicine and may correspond, therefore, to a modality of hormonal release which does not involve the oriented intracellular translocation of secretory granules; and (ii) extensive disruption of the microtubular apparatus is associated with inhibition of insulin release, especially during the late phase of the secretory response to glucose.

Dynamics of insulin release and microtubular-microfilamentous system. III. Effect of colchicine upon glucose-induced insulin secretion.

Abstract. The effect of colchicine upon glucose‐induced insulin release by the isolated perfused rat pancreas was examined in order to characterize the participation of the β‐cell microtubular apparatus in the biphasic insulin secretory response to glucose. After a short pretreatment (25 min.) with a low colchicine concentration (2.10‐5M), both the early and late phases of glucose‐induced insulin secretion were markedly enhanced. As either the concentration of colchicine or the length of the pretreatment period with this mitotic spindle‐inhibitor were increased, the facilitating effect faded out and partial inhibition of insulin release was observed. However, colchicine, even at high concentration, markedly enhanced the residual early release of insulin evoked by glucose in the presence of diazoxide (0.05 mg/ml). These findings suggest that (i) a fraction of the early phase of insulin secretion completely escapes from the inhibitory effect of colchicine and may correspond, therefore, to a modality of hormonal release which does not involve the oriented intracellular translocation of secretory granules; and (ii) extensive disruption of the micro‐tubular apparatus is associated with inhibition of insulin release, especially during the late phase of the secretory response to glucose.

Dynamics of insulin release and microtubular-microfilamentous system. V. A model for the phasic release of insulin.

Abstract. A model is proposed to account for the phasic nature of insulin release. It is suggested that one modality of secretion results from the mobilization of secretory granules already located in the close vicinity of the microfilamentous cell web. This corresponds to a fraction of the initial secretory response to a high glucose concentration, the residual release of insulin provoked by glucose in the presence of diazoxide, and the total release of insulin evoked by sulphonylurea at a low glucose concentration. A second modality of release, which i3 characteristic of the late phase of glucose‐induced insulin secretion, is thought to correspond to the mobilization of secretory granules transported to the periphery of the β‐cell along oriented microtubular pathways. Whether only one or both modalities of release are initiated by a given insulino‐tropic stimulus may depend on the magnitude and duration of the changes induced by secretagogues in the insular handling of calcium.

Effects of Some Modifiers of Insulin Secretion on Insulin Biosynthesis

Insulin release is not a prerequisite for insulin synthesis in the pancreatic islets. Glucose is a strong stimulus for both synthesis and release of insulin. This stimulatory effect on insulin synthesis (and release) is enhanced by caffeine and dibutyryl cyclic AMP. Glucose—stimulated synthesis of insulin proceeds unabated even in the presence of diazoxide or of epinephrine or in the absence of calcium ion all of which are reported to inhibit insulin release. While the omission of magnesium ion from the incubation medium is reported not to affect the release of insulin stimulated by glucose, it nevertheless greatly depresses the biosynthesis of insulin. A small increase in insulin synthesis is noted in the presence of citrate. However, citrate does not enhance the effect of a submaximal concentration of glucose on synthesis. (Endocrinology 92: 735, 1973)

Diazoxide blood levels in man

Blood diazoxide levels were measured in six adult human males at various times after i.v. drug administration. On the basis of these results, the diazoxide half-life in blood was determined as 28.0 ± 8.3hr. The presence of diazoxide in blood was verified by thin-layer chromatography (TLC) and by its U.V. spectrum. This communication describes a simple spectrophotometric quantitative method and a useful TLC method for determination of diazoxide in biological fluids.

The effect of diazoxide and chlorothiazide on glucose uptake in vitro

The glucose uptake of rat hemidiaphragms and epididymal fat pads has been measured in the presence of diazoxide and chlorothiazide. The combination of these compounds consistently reduced the basal glucose uptake of both tissues. The insulin responsiveness of the tissues was apparently unchanged by these benzothiazides. Neither tolbutamide in vitro nor a high KCl incubation medium reversed the effect of these compounds on basal glucose uptake. It is concluded that at least a part of the hyperglycemia induced by diazoxide and chlorothiazide is the result of impaired glucose uptake by peripheral tissues.