Concentrations Of Tolbutamide Research Articles

Glucose Regulation of Insulin Secretion Independent of the Opening or Closure of Adenosine Triphosphate-Sensitive K+ Channels in Cells

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the β-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on[ Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mm K+, i.e. without pharmacological action on K+-ATP channels,[ Ca2+]i and insulin secretion were already elevated at 3 mm glucose. High glucose (20 mm) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence[ Ca2+]i and insulin secretion at 3 mm glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and β-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between[ Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells.

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.

A minimum of fuel is necessary for tolbutamide to mimic the effects of glucose on electrical activity in pancreatic beta-cells.

Glucose stimulation of pancreatic beta-cells triggers electrical activity (slow waves of membrane potential with superimposed spikes) that is best monitored with intracellular microelectrodes. Closure of ATP-sensitive K+ channels underlies the depolarization to the threshold potential and participates in the increase in electrical activity produced by suprathreshold (>7 mM) concentrations of glucose, but it is still unclear whether this is the sole mechanism of control. This was investigated by testing whether blockade of ATP-sensitive K+ channels by low concentrations of tolbutamide is able to mimic the effects of glucose on mouse beta-cell electrical activity even in the absence of the sugar. The response to tolbutamide was influenced by the duration of the perifusion with the low glucose medium. Tolbutamide (25 microM) caused a rapid and sustained depolarization with continuous activity after 6 min of perifusion of the islet with 3 mM glucose, and a progressive depolarization with slow waves of the membrane potential after 20 min. In the absence of glucose, the beta-cell response to tolbutamide was a transient phase of depolarization with rare slow waves (6 min) or a silent, small, but sustained, depolarization (20 min). Readministration of 3 mM glucose was sufficient to restore slow waves, whereas an increase in the glucose concentration to 5 and 7 mM was followed by a lengthening of the slow waves and a shortening of the intervals. In contrast, induction of slow waves by tolbutamide proved very difficult in the absence of glucose, because the beta-cell membrane tended to depolarize from a silent level to the plateau level, at which electrical activity is continuous. Azide, a mitochondrial poison, abrogated the electrical activity induced by tolbutamide in the absence of glucose, which demonstrates the influence of the metabolism of endogenous fuels on the response to the sulfonylurea. The partial repolarization that azide also produced was reversed by increasing the concentration of tolbutamide, but reappearance of the spikes required the addition of glucose. It is concluded that inhibition of ATP-sensitive K+ channels is not the only mechanism by which glucose controls electrical activity in beta-cells.

Effects of Sho-saiko-to on the pharmacokinetics and pharmacodynamics of tolbutamide in rats.

Although Sho-saiko-to (Xiao Chai Hu Tang), a major Chinese traditional medicine, is frequently prescribed with other synthetic or biotechnological drugs for the treatment of various chronic diseases, there is a dearth of information about interactions between sho-saiko-to and co-administered drugs. This paper reports the effects of Sho-saiko-to on the pharmacokinetics and glucose responses of a sulphonylurea hypoglycaemic agent, tolbutamide, after their oral administration in rats. After oral administration of tolbutamide (50 mg kg(-1)) with or without Sho-saiko-to extract powder (300 mg kg(-1)) to male Sprague-Dawley rats cannulated in the jugular vein, plasma tolbutamide and glucose levels were periodically measured. Co-administration of Sho-saiko-to tended to elevate the plasma tolbutamide concentration in the absorption phase. A two-compartment lag-time model was found to describe the plasma tolbutamide concentration-time data. The maximum concentration of tolbutamide was significantly increased and time to reach the maximum concentration was reduced to about 70% by co-administration with Sho-saiko-to. There was no significant change in area under the curve or in the elimination half-life of tolbutamide. The extent of the lowering effect of tolbutamide on plasma glucose levels was increased up to 0.75 h and decreased after 5 h after co-administration of Sho-saiko-to. In conclusion, these studies suggest that sho-saiko-to slightly hastens the gastrointestinal absorption of tolbutamide. Furthermore, it is considered that elevation of the gastrointestinal absorption rate by Sho-saiko-to might potentiate the hypoglycaemic effect of this sulphonylurea in the early period after oral administration.

Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic beta-cells by both direct and indirect mechanisms.

1. In non-excitable cells, the depletion of intracellular Ca2+ stores triggers Ca2+ influx by a process called capacitative Ca2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 microM) influences Ca2+ influx in electrically excitable pancreatic beta-cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse beta-cells or in whole islets loaded with fura-2. 2. The membrane was first held hyperpolarized by diazoxide, an opener of ATP-sensitive K+ (KATP) channels, in the presence of 4.8 mM K+. Alternating between Ca(2+)-free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (approximately 14 nM) in clusters of beta-cells. A larger rise (approximately 65 nM), resistant to the blockade of voltage-dependent Ca2+ channels by D600, occurred when extracellular Ca2+ was readmitted after emptying intracellular Ca2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca2+ entry in beta-cells. 3. When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K+ in the presence of diazoxide, [Ca2+]i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca2+]i, but had little (at 10 mM K+) or no effect on the plateau level. This confirms that the capacitative Ca2+ entry is small. 4. In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mM glucose (in 4.8 mM K+) induced [Ca2+]i oscillations by promoting Ca2+ influx through voltage-dependent Ca2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca2+]i. Similar results were obtained from whole islets perifused with a medium containing > or = 6 mM glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca2+ entry, probably because of a potentiation of Ca2+ influx through voltage-dependent Ca2+ channels. 5. This potentiating effect of thapsigargin did not result from an acceleration of cell metabolism since the drug did not affect glucose-induced changes in NAD(P)H fluorescence. It is also unlikely to involve the inhibition of KATP channels because thapsigargin steadily elevated [Ca2+]i in cells in which [Ca2+]i oscillations persisted in the presence of a maximally effective concentration of tolbutamide. 6. In conclusion, the emptying of intracellular Ca2+ stores in beta-cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose-induced Ca2+ influx through voltage-dependent Ca2+ channels.

Selegiline induces dopamine release through ATP-sensitive potassium channels in the rat caudate-putamen in vitro

We used superfusion chambers to investigate the role of ATP-sensitive potassium (K ATP) channels in dopamine (DA) release elicited by the monoamine oxidase inhibitor selegiline in the rat caudate-putamen in vitro. Selegiline (R[−]-deprenyl), but not the S[+] enantiomer, concentration-dependently induced increases in extracellular concentrations of DA, with a maximal increase to 185% in comparison to basal outflow at 0.1 mM selegiline. Since in our experimental conditions exclusive MAO inhibition does not lead to an enhancement of extracellular DA levels, the effect of selegiline on DA levels seems not to be related to MAO inhibition. Butanedione (0.1 mM), a specific K ATP channel blocker, also significantly enhanced extracellular DA levels in the rat caudate-putamen to approx. 260%. Selegiline only led to an additional increase of DA outflow, when added to submaximal concentrations of butanedione or tolbutamide, implying that selegiline is acting on identical sites. When the K ATP channel opener cromakalim was added to the incubation medium, basal as well as butanedione-enhanced DA levels markedly decreased to about 40% when compared to baseline values. Selegiline-activated DA release was also antagonized by cromakalim. The selegiline effect was neither modulated by preincubation with the uptake inhibitor nomifensine nor by the DA agonist quinpirole and antagonist sulpiride. In conclusion these results suggest that selegiline is able to modulate K ATP channels in the caudate-putamen of the rat in vitro resulting in an enhancement of striatal DA release.

Contribution of ATP-sensitive potassium channels to hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro.

To investigate the mechanism of generation of the hypoxia-induced hyperpolarization (hypoxic hyperpolarization) in hippocampal CA1 neurons in rat tissue slices, recordings were made in current-clamp mode and single-electrode voltage-clamp mode. Superfusion with hypoxic medium produced a hyperpolarization and corresponding outward current, which were associated with an increase in membrane conductance. Reoxygenation produced a further hyperpolarization, with corresponding outward current, followed by a recovery to the preexposure level. The amplitude of the posthypoxic hyperpolarization was always greater than that of the hypoxic hyperpolarization. In single-electrode voltage-clamp mode, it was difficult to record reproducible outward currents in response to repeated hypoxic exposure with the use of electrodes with a high tip resistance. The current-clamp technique was therefore chosen to study the pharmacological characteristics of the hypoxic hyperpolarization. In 60-80% of hippocampal CA1 neurons, glibenclamide or tolbutamide (3-100 microM) reduced the amplitude of the hypoxic hyperpolarization in a concentration-dependent manner by up to approximately 70%. The glibenclamide or tolbutamide concentrations producing half-maximal inhibition of the hypoxic hyperpolarization were 6 and 12 microM, respectively. The chord conductance of the membrane potential between -80 and -90 mV in the absence of glibenclamide (30 microM) or tolbutamide (100 microM) was 2-3 times greater than that in the presence of glibenclamide or tolbutamide. In contrast, the reversal potential of the hypoxic hyperpolarization was approximately -83 mV in both the absence and presence of tolbutamide or glibenclamide. In approximately 40% of CA1 neurons, diazoxide (100 microM) or nicorandil (1 mM) mimicked the hypoxic hyperpolarization and pretreatment of these drugs occluded the hypoxic hyperpolarization. When ATP was injected into the impaled neuron, hypoxic exposure could not produce a hyperpolarization. The intracellular injection of the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate lithium salt reduced the amplitude of the hypoxic hyperpolarization. Furthermore, application of dinitrophenol (10 microM) mimicked the hypoxic hyperpolarization, and the dinitrophenol-induced hyperpolarization was inhibited by either pretreatment of tolbutamide or intracellular injection of ATP, indicating that the hypoxic hyperpolarization is highly dependent on intracellular ATP. It is therefore concluded that in the majority of hippocampal CA1 neurons, exposure to hypoxic conditions resulting in a reduction in the intracellular level of ATP leads to activation of ATP-sensitive potassium channels with concomitant hyperpolarization.

Interaction of a herbomineral preparation D-400, with oral hypoglycaemic drugs

D-400, a herbomineral preparation has proven antidiabetic activity in experimental models as well as in clinical trials. The possibility of concomitant use of this drug with sulphonylureas was explored in animal models. D-400 has been investigated for its interaction with oral hypoglycaemic agents namely, tolbutamide and glibenclamide in alloxan-induced diabetic rabbits. Administration of D-400 at a dose of 1 g/kg for 15 days significantly elevated plasma tolbutamide and glibenclamide concentrations with simultaneous reduction of blood glucose. Plasma tolbutamide and glibenclamide concentrations were significantly lowered after withdrawal of D-400 treatment. Elevation of plasma concentration of tolbutamide was observed only for the first 4 h after which it declined towards normal levels and no significant difference between D-400 treated and control group was observed at the end of 8 h. Significant elevation of plasma glibenclamide levels was observed at 2, 4 and 8 h with D-400 treatment. Incubation of D-400 with tolbutamide in plasma resulted in a significant increase in free tolbutamide levels.

Effects of tolbutamide on vascular ATP-sensitive potassium channels in humans. Comparison with literature data on glibenclamide and glimepiride.

Sulfonylurea (SU) derivatives exert their hypoglycemic effect by blockade of adenosine-5'-triphosphate-sensitive potassium (KATP) channels in the beta-cell of the pancreas. Interestingly, KATP channels also occur in the cardiovascular system, where they are thought to play an important role in cardioprotective mechanisms against ischemia. We have recently shown that the classical second generation SU-derivative glibenclamide is able to block vascular KATP channels in man, whereas the newly developed second generation derivative glimepiride was devoid of this property. The aim of this study was to determine whether the first generation SU derivative tolbutamide has KATP channel blocking properties in humans. In a group of 12 healthy male non-smoking volunteers, we investigated whether therapeutic concentrations of tolbutamide were able to inhibit the forearm vasodilation in response to the infusion of the KATP channel opening drug diazoxide into the brachial artery. Changes in forearm blood flow were recorded by venous occlusion mercury-in-silastic strain-gauge plethysmography. Diazoxide alone increased the forearm blood flow ratio dose-dependently by ultimately 691 +/- 198%. A second diazoxide infusion in the presence of tolbutamide revealed a comparable vasodilator response with a percentage increase in forearm blood flow ratio of ultimately 542 +/- 111%. This response did not differ from the vasodilator response to diazoxide alone. The present study shows that therapeutic concentrations of tolbutamide are not able to attenuate the vasodilation caused by the KATP channel opener diazoxide in man. When compared with published data on second generation SU derivatives, tolbutamide shows an intermediate position between glibenclamide (with significant blockade of vascular KATP channels) versus glimepiride (with no blockade at all). It remains to be determined whether these acute effects of SU derivatives on pharmacological opening of forearm vascular KATP channels can be extrapolated to the chronic effects of these drugs on ischemia-mediated opening of myocardial KATP channels during treatment of NIDDM patients.

Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).

Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells.

Rat pancreatic AR42J cells possess exocrine and neuroendocrine properties. Activin A induces morphological changes and converts them into neuron-like cells. In activin-treated cells, mRNA for pancreatic polypeptide (PP) but not that for either insulin or glucagon was detected by reverse transcription-PCR. About 25% of the cells were stained by anti-PP antibody. When AR42J cells were incubated with betacellulin, a small portion of the cells were stained positively with antiinsulin and anti-PP antibodies. The effect of betacellulin was dose dependent, being maximal at 2 nM. Approximately 4% of the cells became insulin positive at this concentration, and mRNAs for insulin and PP were detected. When AR42J cells were incubated with a combination of betacellulin and activin A, approximately 10% of the cells became insulin positive. Morphologically, the insulin-positive cells were composed of two types of cells: neuron-like and round-shaped cells. Immunoreactive PP was found in the latter type of cells. The mRNAs for insulin, PP, glucose transporter 2, and glucokinase, but not glucagon, were detected. Depolarizing concentration of potassium, tolbutamide, carbachol, and glucagon-like peptide-1 stimulated the release of immunoreactive insulin. These results indicate that betacellulin and activin A convert amylase-secreting AR42J cells into cells secreting insulin. AR42J cells provide a model system to study the formation of pancreatic endocrine cells.

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.

Supramaximal decrease of sulphonylurea-induced accumulation of sodium in pancreatic islets

Sodium was measured in β-cell-rich pancreatic islets of mice under steady state conditions or after 6 min of exposure to I mm ouabain. The islet content of sodium increased when 100,μ M tolbutamide or 1 μ M glipizide were added to an albumin-containing (1 mg ml −1) medium, but remained unaffected at 10-fold higher concentrations. Both sulphonylurea compounds promoted the uptake of Na + in the presence of ouabain. Whereas tolbutamide was stimulatory at 10μ M or above in a medium containing 10 mg ml − albumin, only 0.1 μ M was required in the absence of albumin. In the latter situation there was a reduction of the stimulatory action with increase of the tolbutamide concentration from 100 to 1000 μ M. The inhibitory component in the sulphonylurea action on the Na + uptake was particularly impressive with glipizide, maximal stimulation being reached at 10 μ M in the presence of 1 mg ml − albumin. Diazoxide (400 μ M) modified the glipizide action on Na + uptake, making 1000 μ M stimulatory instead of 1 μ M. The latter concentration of glipizide became inhibitory after removal of K +. Glipizide stimulated the Na + uptake both at low and high concentrations in a medium deficient in Ca 2+ or when the cotransport of Na +, K + and Cl − was blocked by 20 μ M bumetanide. The observation that the sulphonylurea-induced islet accumulation of sodium is diminished at supramaximal concentrations reinforces existing arguments for additional effects of high concentrations of hypoglycemic sulphonylureas.

Tolbutamide, but not glyburide, affects the excitability and contractility of unfatigued frog sartorius muscle

The goal of this study was to characterize the effects of tolbutamide and glyburide, two known K ATP channel blockers, on intact, unfatigued sartorius muscle fibres of the frog, Rana pipiens. Tetanic contractions were elicited by field stimulation with 200 ms long train of pulses (0.5 ms, 6 V, 140 Hz). Resting and action potentials were measured using conventional microelectrodes. At pH o 7.2(extracellular pH), the tetanic force was unaffected by 0.5 mM and 1.0 mM tolbutamide, but at 2.0 mM it decreased by 15.5±1.0%. The effect of tolbutamide on the tetanic force was significantly greater at pH o 6.4: all three tolbutamide concentrations caused a significant decrease in tetanic force, being 62.3±9.4% at 2 mM. In the presence of tolbutamide a large number of fibres became unexcitable at pH o 6.4, but not at pH o 7.2. Glyburide at 10 μM, on the other hand, caused a 5–7% decrease in tetanic force at both pH o 6.4 and 7.2, but no further decreases in tetanic force were observed when the glyburide concentration was increased up to 100 μM. Unlike tolbutamide, glyburide did not affect the excitability of muscle fibres, but significantly prolonged the repolarization phase of action potentials, especially at pH o 6.4. We suggest that several of the tolbutamide effects reported in this study cannot be accounted for by a direct effect on K ATP channels, and that the large decrease in membrane excitability and muscle contractility in the presence of tolbutamide must seriously be taken into consideration when this channel blocker is used to study the physiological role of K ATP channels in intact muscle fibres.

Evidence that imidazol(id)ine- and sulphonylurea-based antagonists of cromakalim act at different sites in the rat thoracic aorta.

1. Ring segments of rat thoracic aorta were suspended in organ baths to record isometric tension. Tissues were precontracted with K+ (20 mmol/L), and full concentration-relaxation curves constructed to cromakalim (0.01-30 mumol/L) in the absence and presence of increasing concentrations of glibenclamide, glipizide, tolbutamide (the sulphonylureas), alinidine (an imidazolidine), phentolamine (an imidazoline), and chlorpromazine (the phenothiazine derivative). Whereas the active sulphonylureas, glibenclamide and glipizide, displayed classical competitive antagonism, the remaining compounds (alinidine, phentolamine and chlorpromazine) caused shifts in the cromakalim concentration-effect curves associated with a reduction in the slope and maximum response. 2. A single concentration of each antagonist was selected and the shift in the concentration-effect curve determined. The possibility that sulphonylurea and imidazol(id)ine antagonists act at different sites was tested using the concentration-ratio method for combined antagonists described by Paton and Rang (1965). The combination of alinidine and phentolamine (collectively called imidazol(id)ines) at a number of different concentrations (10-30 mumol/L) resulted in a concentration-ratio to cromakalim which was additive, suggesting a common site of action. Similar results were obtained when examining the interaction between two sulphonylurea compounds (glibenclamide and glipizide). However, the interaction between sulphonylurea (glibenclamide) and imidazol(id)ine (alinidine) produced concentration-ratios which were multiplicative, suggesting a different or additional site of action for compounds from these two groups. Results indicated that chlorpromazine was able to block cromakalim via an action at the same site where alinidine and phentolamine act.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioavailability study of tolbutamide β-cyclodextrin inclusion compounds, solid dispersions and bulk powder

Tolbutamide PEG 6000 solid dispersions as well as tolbutamide β-cyclodextrin complexes were prepared with a view to increasing the bioavailability of this poorly soluble drug. Absolute and relative bioavailabilities were determined by comparison with the administration of a commercial solution of the drug. The study was carried out in rabbits ( n = 5 per dosage form). The aqueous solution of tolbutamide (Dolipol ®) was administered either intravenously (10 mg/kg) or orally (20 mg/kg). Bulk powder, comelt, coprecipitate and solid complex of tolbutamide were administered orally at a dose of 20 mg/kg. Plasma tolbutamide concentrations were measured by an HPLC method. Our results indicate that the absorption of tolbutamide is not increased in comparison with either bulk powder or a solution of the drug. However, there are obvious differences in the kinetics of absorption: indeed, tolbutamide is absorbed rapidly from the complex and the bulk powder. The process of absorption is much slower for the other dosage forms. Finally, even if the quantitative part of bioavailability is not modified, complexation with cyclodextrins could be interesting in order to increase the kinetic process of absorption of poorly soluble drugs.

Determination of tolbutamide hydroxylation in rat liver microsomes by high-performance liquid chromatography: Effect of psychoactive drugs on in vitro activity

A simplified HPLC method for tolbutamide metabolism to hydroxytolbutamide has been used to screen sixty psychoactive drugs for their ability to inhibit rat liver microsomal tolbutamide hydroxylation. One-step extraction with diethyl ether was followed by reconstitution and isocratic HPLC analysis with a binary mobile phase (ammonium phosphate:methanol, 45:55, v/v). Nanogram amounts of hydroxytolbutamide formation were estimated with UV detection at 240 nm. Hydroxytolbutamide formation was linear with incubation times of 40 – 120 min, but specific activity increased with increases in microsomal protein (0.15 – 1.10 mg). A differential inhibitory response was demonstrated for tolbutamide and debrisoquine hydroxylation to 5 psychoactive drugs, suggesting that tolbutamide hydroxylation is not dependent on P4502D1. Sixty psychoactive drugs, or drug metabolites, (at 33 μM) were then co-incubated with tolbutamide (at 2.5 and 10.2 μM). Tolbutamide hydroxylation was refractory (<25% inhibition) to twenty-four of the drugs and only mildly inhibited (25–50% inhibition) by twenty-eight. Two compounds, trans-3-methylfentanyl and flurazepam, produced >50% inhibition that was independent of tolbutamide concentration. Five of the drugs (methadone, chlorpheniramine, meperidine, 6-monoacetylmorphine and methylphenidate), however, caused greater than 50% inhibition in a competitive manner which suggests these drugs may share an affinity for the substrate binding site for tolbutamide.

Therapeutical concentrations of tolbutamide, glibenclamide, gliclazide and gliquidone at different glucose levels: in vitro effects on pancreatic A- and B-cell function

In the classical model of isolated perfused rat pancreas four commonly used sulfonylureas — tolbutamide, glibenclamide, gliquidone and gliclazide — were investigated at therapeutical concentrations at three different glucose levels (with 0, 2.22 and 5 mmol/l glucose surrounding) and in the presence of a metabolic stimulus with glucose at 8.33 mmol/l. All the sulfonylureas stimulated the B-cell function. Tolbutamide, gliquidone and gliclazide produced a prompt biphasic hormone release while glibenclamide induced a delayed monophasic insulin secretion. In all cases the amount of insulin released depended on the metabolic condition. As the environmental glucose levels fell, the sulfonylureas' stimulatory effect on the B-cell function decreased. At the therapeutical concentrations we tested, no sulfonylurea influenced A-cell activity whether directly or indirectly via an insulin-mediated paracrine inhibition of glucagon release.

Glyburide and tolbutamide induce desensitization of insulin release in rat pancreatic islets by different mechanisms

Insulin secretion was studied in rat pancreatic islets after 24-h exposure to various glyburide or tolbutamide concentrations. Glucose-induced insulin release was significantly (P < 0.05) reduced in islets cultured with 0.1 microM glyburide or 100 microM tolbutamide (2098 +/- 187, 832 +/- 93, and 989 +/- 88 pg/islet.h in control, glyburide-exposed, and tolbutamide-exposed islets, respectively). When glyburide-treated islets were stimulated with glyburide or tolbutamide, insulin release was also impaired compared to that in control islets (P < 0.05). In contrast, tolbutamide-exposed islets showed an impaired response to tolbutamide, but a normal response to glyburide. To investigate the mechanism of the sulfonylurea-induced impairment of insulin secretion, we measured insulin release and Rb+ efflux (a marker of the K+ channel activity) in a perifusion system and islet Ca2+ uptake under static conditions. Insulin release in response to 16.7 mM glucose increased in control islets from 9.4 +/- 1.1 to 131 +/- 19 pg/islet.min (first phase secretion peak). Simultaneously, the fractional 86Rb+ efflux declined from 0.015 +/- 0.002% to 0.006 +/- 0.001% (change in decrement, -63.5%). Glucose-induced insulin release in glyburide- and tolbutamide-treated islets was significantly reduced (first phase peak, 22.1 +/- 5 and 39.7 +/- 8 pg/islet.min, respectively; P < 0.05), and the fractional 86Rb+ efflux decrement was -21 +/- 6% for glyburide (P < 0.005 vs. control islets) and -65 +/- 4% (not different from control) for tolbutamide. When glyburide- or tolbutamide-exposed islets were stimulated with the corresponding sulfonylurea, insulin release was impaired compared to that in control islets (P < 0.05), but, again, 86Rb+ efflux was impaired (P < 0.05) only in glyburide-exposed islets. When 45Ca2+ uptake was studied, the increase in glucose concentration from 2.8 to 16.7 mM increased calcium uptake in control islets from 1.76 +/- 0.58 to 7.27 +/- 1.36 pmol/islet.2 min (n = 4). Preexposure to 0.1 microM glyburide did not change calcium uptake at a glucose concentration of 2.8 mM (1.44 +/- 0.45 pmol/islet.2 min) but significantly reduced calcium uptake stimulated by 16.7 mM glucose (3.21 +/- 0.35 pmol/islet.2 min; n = 4; P < 0.005 compared to control islets). In contrast, preexposure to 100 microM tolbutamide did not change either basal or glucose-stimulated calcium uptake (1.44 +/- 0.45 and 6.90 +/- 0.81 pmol/islet.2 min, respectively; n = 4). These data show that in vitro chronic exposure of pancreatic islets to the sulfonylureas glyburide and tolbutamide impairs their ability to respond to a subsequent glucose or sulfonylurea stimulation.