Inhibition of catecholamine release by tolbutamide and other sulfonylureas.

Tolbutamide and other sulfonylureas inhibited spontaneous and nicotine-induced release of catecholamines from the perfused cat adrenal gland and nicotine-induced release of [3-H]norepinephrine from isolated guinea pig hearts. Of the sulfonylureas tested, the order to potency of this inhibitory effect paralled the hypoglycemic action. These results raise the possibility that the inhibition of the sympathoadrenal system may contribute in part to the hypoglycemic action of sulfonylureas.

In vitro effects of a sulfonylurea on insulin action in adipocytes. Potentiation of insulin-stimulated hexose transport.

The mechanism(s) by which the oral sulfonylurea, tolazamide, exerts its extrapancreatic hypoglycemic effects was studied using rat epididymal adipose tissue maintained 20-44 h in the presence or absence of the drug. Insulin binding, hexose transport and glucose metabolism were compared in adipocytes isolated from the cultured tissue. In contrast to earlier reports that suggested that sulfonylureas alter the binding of insulin, neither receptor number nor affinity were changed by tolazamide treatment. The uptake of the glucose analogs 2-deoxyglucose and 3-0-methylglucose in the absence of insulin (i.e., basal) was also unchanged. However, exposure to tolazamide resulted in a potentiation of the stimulatory effects of insulin by approximately 30% at each hormone concentration assayed (0.4-40 ng/ml). This potentiation was dependent on the tolazamide concentration (0.003-0.30 mg/ml), with a maximal effect observed at therapeutic levels. A tolazamide analog hypoglycemic activity in vivo was found not to enhance either basal or insulin-stimulated uptake in vitro. Conversion of 0.1-5.0 mM glucose to CO2 and total lipids in the presence of insulin was also potentiated by tolazamide treatment. The inability of the drug to directly stimulate basal glucose uptake was paralleled by its lack of effect on glucose metabolism. At 50 mM glucose, where transport is no longer rate-limiting, tolazamide did not potentiate metabolism in the absence or the presence of insulin. These studies demonstrate that tolazamide in vitro alters postreceptor insulin action without influencing the receptor, and suggests insulin-stimulated hexose transport as the cellular process responsible for the hypoglycemic effect of sulfonyureas in adipose tissue.

Coordinate regulation of glucose transporter function, number, and gene expression by insulin and sulfonylureas in L6 rat skeletal muscle cells.

The extrapancreatic actions of sulfonylureas on the glucose transport system were studied in the L6 line of cultured rat skeletal muscle cells. Insulin (10(-7) M) increased 2-deoxyglucose uptake in differentiated L6 myotubes by 30-40% after 8 h of incubation. The sulfonylurea tolazamide (0.6 mg/ml, 22 h) had no effect on glucose uptake in the absence of insulin, but increased insulin-stimulated 2-deoxyglucose uptake twofold. The total cellular content of glucose transporters was assessed with a monoclonal anti-transporter antibody by a solid-phase ELISA method. Insulin (8 h) increased the quantity of glucose transporters, with a maximal twofold increase at 10(-7) M and a dose-response curve similar to that for insulin stimulation of glucose uptake. In spite of its lack of effect on glucose uptake, tolazamide alone (0.6 mg/ml) increased the cellular content of transporters by 70%. The effects of insulin and tolazamide on transporter gene expression were studied with probes derived from Hep G2 glucose transporter cDNA. Insulin increased the transporter mRNA level 1.7-fold, tolazamide increased it 1.5-fold, and the combination of insulin and tolazamide increased transporter mRNA 3-fold. It is concluded that sulfonylureas, together with insulin, enhance glucose uptake in L6 skeletal muscle cells by increasing the number of functioning glucose transport molecules. The long-term regulation of the glucose transport system in skeletal muscle by insulin and sulfonylureas in vivo may involve similar changes in transporter function, number, and gene expression.

The effect of sulfonylurea drugs on rabbit myocardial contractility, canine purkinje fiber automaticity, and adenyl cyclase activity from rabbit and human hearts.

Long-term clinical studies have associated tolbutamide therapy with an increased incidence of cardiovascular deaths. The effects of this and other sulfonylurea drugs on contractility and rate of isolated rabbit atria, automaticity of isolated dog Purkinje fibers, and adenyl cyclase activity in particulate preparations of rabbit and human hearts were studied. At concentrations that are attained clinically, tolbutamide (10 mg/100 ml) increased contractility of driven rabbit atria to 124+/-5% of control, acetohexamide (3.9 mg/100 ml) to 140+/-5%, chlorpropamide (8.3 mg/100 ml) to 139+/-6%, and tolazamide (3.1 mg/100 ml) to 119+/-6%. These effects were accentuated in the presence of 2.5 x 10(-4) M theophylline and were not blocked by 1 x 10(-5) M propranolol. Adenyl cyclase was activated by each of these drugs at concentrations below those which increase contractility. The drugs also increased the rate and slope of phase 4 depolarization in spontaneously beating Purkinje fibers, but did not alter the spontaneous rate of isolated rabbit atria. Since inotropic and chronotropic stimulation can be deleterious in some clinical settings, these findings may be of significance in interpretation of cardiovascular mortality data.

Selective unresponsiveness of pancreatic beta-cells to acute sulfonylurea stimulation during sulfonylurea therapy in NIDDM.

Patients with non-insulin-dependent diabetes mellitus (NIDDM) who have chronic hyperglycemia lose acute incremental insulin responses to glucose but are able to briskly respond to other beta-cell secretagogues. To investigate whether this is a defect specific for glucose or represents a more general phenomenon, we measured the insulin responses to acute intravenous tolbutamide in 10 obese patients with NIDDM both before and during sulfonylurea therapy with tolazamide. Comparable glycemia was achieved with oral dextrose 2 h before intravenous testing. To assess beta-cell responsiveness to a nonsulfonylurea secretagogue, 1 mg glucagon was administered intravenously during tolazamide therapy. In seven patients, the mean peak insulin increment 5 or 10 min after intravenous tolbutamide was 54 +/- 11 microU/ml when not receiving tolazamide (0.14 +/- 1.3 microU/ml) with tolazamide (P less than .001), even though serum insulin responded rapidly to intravenous glucagon. In four patients tested for reversibility of their refractoriness to intravenous tolbutamide during chronic tolazamide therapy, the mean peak insulin increment 1 wk after discontinuing tolazamide was 79 +/- 22 microU/ml. A relatively rapid development of refractoriness was documented in four patients who were tested only 12 h after beginning tolazamide therapy; the mean peak insulin increments 5-10 min after intravenous tolbutamide were undetectable (-0.5 microU/ml), yet responses to intravenous glucagon were evident. In these NIDDM patients, exposure of pancreatic beta-cells to sustained levels of sulfonylureas induces a reversible state of refractoriness to acute stimulation with sufonylureas but not to another secretagogue.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation of insulin action by a sulfonylurea in primary cultures of hepatocytes from normal and diabetic rats.

Although sulfonylureas have been used extensively in the treatment of non-insulin-dependent (type II) diabetes, controversy exists as to whether these agents act primarily by increasing insulin secretion or by enhancing insulin action. To determine whether sulfonylureas potentiate insulin action in the liver, we evaluated the ability of the sulfonylurea tolazamide to affect insulin-sensitive lipogenesis utilizing primary cultures of hepatocytes prepared from both normal and nonketotic streptozotocin-diabetic rats. Hepatocytes were cultured for 16 h in serum-free media with no additions, tolazamide alone (0.3 mg/ml), or insulin (10(-10) to 10(-7)M) in the absence and presence of tolazamide. Following culture, lipogenesis and specific insulin binding were assessed. Dose-dependent increases in lipogenesis were found in hepatocytes from both normal and diabetic rats after the chronic exposure to insulin. In hepatocytes from diabetic rats, the basal and the maximal insulin-stimulated rates of lipogenesis were only 27% and 13% of normal, respectively, establishing this as a model of hepatic insulin resistance. In the presence of tolazamide, significant potentiation of insulin action was found in hepatocytes from normal and diabetic rats although hepatocytes from diabetic animals remained relatively resistant to insulin when compared with those from nondiabetic animals. While exposure to tolazamide increased insulin responsiveness in both groups of cells, no changes in insulin sensitivity (ED50) were observed. Tolazamide significantly increased insulin binding (12%) in hepatocytes from normal rats cultured in the absence of insulin, but no alterations in insulin binding were found under incubation conditions in which tolazamide potentiated insulin action. These results give the first direct evidence for an insulin-dependent action of a sulfonylurea on the liver from both normal and diabetic rats and indicate that the enhancement of insulin responsiveness occurs through postbinding mechanisms.

Differential activities of tolbutamide, tolazamide, and glyburide in vitro on rabbit myocardial membrane Ca2+-transporting ATPase activity.

At clinically achievable concentrations (10(-9) to 5 X 10(-6) M), tolbutamide and tolazamide are in vitro inhibitors of Ca2+-transporting ATPase activity in sarcolemma-enriched rabbit myocardial membranes (sulfonylurea IC50, 10(-7) M). Thyroid hormone stimulation of this calcium pump-associated enzyme in vitro has been previously reported; in our study, this hormonal action was shown to be inhibited by tolbutamide and tolazamide. In contrast to these two sulfonylureas, glyburide (up to 5 X 10(-6) M) had no effect on basal or thyroid hormone-stimulable Ca2+-ATPase activity in vitro. Studies of binding of radiolabeled purified calmodulin to heart membranes showed that tolbutamide and tolazamide inhibited this interaction, whereas glyburide had no effect on calmodulin binding. Addition of purified calmodulin (5-40 ng/micrograms membrane protein) to myocardial membranes incubated with 10(-7) M tolbutamide or tolazamide restored Ca2+-ATPase activity and thyroid hormone responsiveness of the enzyme. Inhibition by tolbutamide and tolazamide of myocardial sarcolemmal Ca2+-ATPase is a mechanism by which these two sulfonylureas may at least transiently raise resting sarcoplasmic Ca2+ concentration. This effect of sulfonylureas on Ca2+-ATPase is not expressed in the presence of the benzamide side chain of glyburide. The inhibitory action of certain sulfonylureas on Ca2+-ATPase is mediated by interference of the agents with the binding of calmodulin to cardiac membranes.

Sulfonylurea effects on target tissues for insulin.

We have examined the nonpancreatic actions of sulfonylureas on multiple aspects of insulin responsiveness in two target tissues for insulin, liver and fat. In vivo administration of tolazamide and glipizide reduced significantly the postabsorptive serum glucose levels in rats without altering the levels of insulin. This was consistent with extrapancreatic sites of drug action. The number and affinity of hepatic insulin receptors was not different from those of control rats. Using a tissue culture system for rat adipose tissue, a 20-h treatment with sulfonylureas markedly potentiated insulin action in fat cells. The primary augmentation was at the level of insulin-stimulated glucose transport. Again, there was no alteration of the insulin receptors located on the adipose tissue. Furthermore, consistent with the lack of an influence on insulin-induced receptor loss after in vitro treatment with sulfonylureas, the in vivo administration of these agents did not alter the transglutaminase activity in rat hepatic tissue. The data demonstrate that sulfonylureas potentiate the responsiveness of the target tissues for insulin. Thus, these hypoglycemic agents probably act by correcting some of the cellular lesions associated with the insulin resistance in type II diabetes mellitus.

Hepatic insulin resistance in non-insulin-dependent diabetes mellitus and the effects of a sulfonylurea in potentiating insulin action.

We have utilized primary cultures of rat hepatocytes to study insulin resistance in the liver of nonketotic streptozotocin-diabetic animals. Diabetes mellitus is associated with insulin resistance with regard to hepatic lipogenesis. This resistance is profound at serum glucose levels above 400 mg/dl and, below that, inversely related to the serum glucose. The insulin resistance can be reversed by in vivo treatment of animals with insulin, indicating that the resistance to insulin is secondary to the diabetic state. However, the in vitro treatment of primary cultures of hepatocytes with insulin, a variety of glycolytic intermediates, or a combination of the two does not reverse the resistance to insulin. Thus, in contrast to cells from fasted animals, in vivo factors other than insulin are important to the recovery of hepatic insulin responsiveness. With regard to the mechanism of this insulin resistance, insulin binding is normal to increased, suggesting postbinding mechanisms. To further define the mechanism(s) of insulin resistance, we evaluated the ability of insulin to release the putative second messenger of insulin action from a liver particulate fraction. Insulin reproducibly and significantly enhanced the release of mediator from the liver particulate fraction of control animals, but the particulate fraction from diabetic animals was resistant to this effect of insulin. Insulin treatment of animals restored the ability of liver particulate fractions to release mediator in response to insulin. These data support the concept that alterations at or near the plasma membrane can be responsible for or accompany the insulin resistance observed in the liver in diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged sulfonylurea administration decreases insulin resistance and increases insulin secretion in non-insulin-dependent diabetes mellitus: evidence for improved insulin action at a postreceptor site in hepatic as well as extrahepatic tissues.

To determine whether long-term sulfonylurea therapy ameliorates glucose homeostasis in patients with NIDDM predominantly by improving insulin secretion or by improving insulin action, we evaluated changes in fasting plasma glucose concentrations, intravenous glucose tolerance, glucose-stimulated insulin secretion, facilitation of glucose disposal by exogenous insulin, and erythrocyte insulin receptor binding before and after prolonged (congruent to 4 mo) administration of tolazamide to 18 patients with NIDDM. Before tolazamide administration, 15 patients had decreased insulin secretion (50 +/- 31 vs 577 +/- 176 microU/ml X 10 min in nondiabetic subjects, P less than 0.05) and insulin resistance (Km 166 +/- 31 vs 58 +/- 3 microU/ml in nondiabetic subjects, P less than 0.05; Vmax 7.3 +/- 0.6 vs 9.8 +/- 0.2 mg/kg/min in nondiabetic subjects, P less than 0.05), whereas the other three patients had comparably impaired insulin secretion (56 +/- 52 microU/ml X min) but were not insulin resistant (Km 70 +/- 6 microU/ml; Vmax 10.8 +/- 0.6 mg/kg/min). The insulin-resistant patients had fasting hyperinsulinemia (19 +/- 4 vs 11 +/- 1 microU/ml in nondiabetic subjects, P less than 0.05), decreased erythrocyte insulin receptor binding (4.8 +/- 0.4 vs 5.8 +/- 0.3%/1.6 X 10(9) cells in nondiabetic subjects, P less than 0.05), and impairment in both insulin-induced suppression of glucose production (Km 97 +/- 31 vs 21 +/- 7 microU/ml in nondiabetic subjects, P less than 0.05), and insulin-induced stimulation of glucose utilization (Km and Vmax 176 +/- 29 microU/ml and 5.8 +/- 0.7 mg/kg/min vs 50 +/- 2 microU/ml and 9.1 +/- 0.6 mg/kg/min in nondiabetic subjects, both P less than 0.05). The nonresistant patients were not hyperinsulinemic (12 +/- micU/ml), had normal insulin receptor binding (5.9 +/- 0.5%/1.6 X 10(9) cells), and were less hyperglycemic than the insulin-resistant patients (128 +/- 11 vs 181 +/- 12 mg/dl, P less than 0.05). After tolazamide administration, both the early phase of glucose-induced insulin secretion (56 +/- 52 vs 141 +/- 68 microU/ml . 10 min) and insulin binding (5.9 +/- 0.5 vs 7.0 +/- 0.5%/1.6 X 10(9) cells) increased in all three nonresistant patients, but there was no consistent improvement in fasting hyperglycemia (128 +/- 11 vs 130 +/- 24 mg/dl), intravenous glucose tolerance (Kivgtt 0.77 +/- 0.18 vs 0.89 +/- 0.29%/min), or facilitation of glucose disposal by insulin (Km 70 +/- 5 vs 64 +/- 5 microU/ml; Vmax 10.8 +/- 0.6 vs 10.1 +/- 0.2 mg/kg/min).(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of tolazamide on basal ketogenesis, glycogenesis, and gluconeogenesis in liver obtained from normal and diabetic rats.

The effect of tolazamide on in vitro rates of gluconeogenesis, ketogenesis, and glycogenesis was determined in liver tissue from fasted normal and diabetic rats. Hormones were not added to the incubation mixture. Two concentrations of the drug were tested, one of which was therapeutic (40 micrograms/ml) and other immoderately elevated (400 micrograms/ml). Neither drug concentration affected hepatic glycogen synthesis. However, the low dose of tolazamide inhibited ketogenesis in the diabetic liver by 39% and in the control liver by 32%; oxidative CO2 production from palmitate was reduced in parallel with ketogenesis. The drug did not alter ketogenesis in isolated intact mitochondria. Similarly, this same therapeutic dose curtailed hepatic gluconeogenesis only in control liver (74% inhibition); this reaction was unaltered by this drug concentration in the explants derived from the diabetic rats. The logarithmically higher dose inhibited hepatic gluconeogenesis in both control and diabetic liver tissue by 56% and 51%, respectively. Hence, possibly acting at a postreceptor site, therapeutic concentrations of tolazamide can decrease rat hepatic in vitro gluconeogenesis and ketogenesis.

Extrapancreatic effects of sulfonylureas. Potentiation of insulin action through post-binding mechanisms.

Pieces of rat epididymal fat tissue were maintained in a biochemically defined medium for 20 to 44 hours in either the absence or presence of a sulfonylurea at levels known to be effective in humans. Prolonged exposure of adipocytes to sulfonylureas did not influence the number of insulin receptors or their affinity to insulin or the ability of insulin to induce receptor loss (down-regulation). Also, the sulfonylureas did not influence the basal uptake of the D-glucose analogs 2-deoxyglucose and 3-O-methylglucose. However, exposure to these drugs resulted in a potentiation of the stimulatory effects of insulin on hexose transport at submaximal and maximally effective concentrations of insulin. The average potentiation was approximately 30%. In addition, sulfonylureas enhanced stimulation of hexose uptake by the insulin mimickers, hydrogen peroxide and vitamin K5. These oxidants are known to manifest insulin-like actions subsequent to insulin binding. Under conditions in which glucose transport was rate limiting, the conversion of glucose to carbon dioxide and the total lipids mirrored the findings of hexose uptake. However, at a glucose concentration of 50 mM, at which hexose transport is no longer rate limiting, sulfonylureas did not potentiate metabolism in th absence or presence of insulin. These results may help to explain the hypoglycemic action of the drug in view of the recent finding that a postreceptor deficit is present in noninsulin-dependent diabetes mellitus.

Metabolic fate of tolazamide in man and in the rat.

The metabolic fate of tolazamide, 1-(hexahydroazepin-1-yl)-3-p-tolylsulfonylurea (1), was studied in man and in the rat using tritium-labeled 1. The metabolites were isolated in crystalline form from urine for structure determination. The crystal structure and final molecular structure of one of these, 1-(4-hydroxyhexahydroazepin-1-yl)-3-p-tolylsulfonylurea (5), were determined using single-crystal X-ray techniques. Following oral administration of tritiated tolazamide to male humans, 85% of the radioactivity was excreted in urine during a 5-day period. In addition to being excreted in urine unchanged, tolazamide was metabolized to 1-(hexahydroazepin-1-yl)-3-p-(carboxyphenyl)sulfonylurea (2), p-toluenesulfonamide (3), 1-(hexahydroazepin-1-yl)-3-p-(hydroxymethylphenyl)sulfonylurea (4), 1-(4-hydroxyhexahydroazepin-1-yl)-3-p-tolylsulfonylurea (5) and a labile, unidentified metabolite 6 by man. The relative amounts of these materials excreted in 0-24-h urine collections from eight subjects averaged 7, 17, 26, 10, 25, and 15% for 1-6, respectively. In the female rat, 79% of an orally administered dose of tritiated tolazamide was excreted in urine during a 5-day period as 1-4. The relative amounts of these materials excreted during the 24-h period following administration of tolazamide were 10, 5, 5, and 80% for 1-4, respectively.

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells.

1. The pharmacological properties of K(ATP) channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg(-1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues.

Inhibition of ATP-activated potassium channels exerts pressor effects and improves survival in a rat model of severe hemorrhagic shock.

Potassium channels closed by increases in intracellular ATP levels (KATP channels) have been described in vascular smooth muscle cells and other cell types. These channels are responsive to the metabolic state of the cells, and can be opened by a decrease in intracellular ATP levels and intra- or extracellular acidosis. Hemorrhagic shock is associated with early vasomotor paralysis as well as with early derangements in the intracellular metabolic status. Here we have tested whether activation of KATP channels contributes to the vasodilatation and early mortality in a rat model of severe hemorrhagic shock. In anesthetized rats hemorrhaged to a mean arterial blood pressure (MAP) of 35 mmHg, inhibition of KATP channels with glibenclamide or tolazamide (10 mg/kg i.v. bolus injection followed by an infusion of 10 mg/kg/h for 60 min), rapidly increased MAP and improved survival rate. The same dose of the KATP channel inhibitors did not cause a significant increase of MAP in animals not subjected to hemorrhage. The approach of inhibition of KATP channel activation in hemorrhagic shock is worthy of further investigations to determine whether it may represent a novel approach for early resuscitation during hemorrhage.

Potentiation of glucose-stimulated insulin release by tolazamide and paradoxical absence of glucose facilitation (Staub effect) in non-insulin-dependent diabetes.

Acceleration of glucose tolerance after repetitive intravenous glucose loads (Staub-Traugott effect) has not previously been tested in non-insulin-dependent diabetes (NIDDM). Six overt, untreated subjects were administered three hourly IV glucose tolerance tests (GTT). The glucose disappearance rate (K) changed very little between loads, indicating a suppressed Staub effect. However, insulin levels increased with each load. The characteristic loss of early phase insulin release after the first intravenous glucose injection was recovered with the third injection. After four months of tolazamide treatment the fasting plasma glucose fell from 180 +/- 19 to 134 +/- 13 mg/dL. Despite dramatic potentiation of glucose-stimulated insulin release and further improvement of early phase insulin release, K values again failed to progressively rise. This paradox occurred even in three additional subjects tested after two years of tolazamide treatment, suggesting that tolazamide may not ameliorate the postreceptor defects that impede the expression of the Staub effect. The applicability of the glucose facilitatory effect to the treatment of NIDDM might be limited to subjects in whom adjunctive measures have reestablished effective tissue responsiveness to endogenous insulin.

Extrapancreatic action of sulfonylureas: hypoglycemic effects are not dependent on altered insulin binding or inhibition of transglutaminase.

It has been proposed by others that sulfonylureas exert their extrapancreatic hypoglycemic effects by increasing insulin binding through inhibition of receptor-mediated hormone internalization. In this study, we examined the possibility that the drugs act by inhibiting transglutaminase, an enzyme thought important in the internalization process. For ten days, male rats were fed pulverized chow containing either no drug, glipizide (5 mg/kg initial body wt/d), or tolazamide (75 mg/kg initial body wt/d). Prior to sacrifice, the six-hour fasting level of serum glucose was significantly reduced from 96 mg/100 ml in the control rats to 81 and 42 mg/100 ml in the glipizide- and tolazamide-treated rats, respectively. In contrast, the serum level of insulin was similar for all groups. The activity of transglutaminase in the postnuclear fraction of liver homogenate also was the same for all experimental groups. The specific binding of labeled insulin to purified liver plasma membranes was examined over a broad range of insulin concentrations; once again, there was no difference between experimental groups. Thus, the hypoglycemia caused by sulfonylurea administration could not be attributed to increases in insulin binding, inhibition of transglutaminase activity, or enhanced insulin levels. These data support our previous suggestion, based on in vitro studies, that sulfonylureas act predominately on processes beyond the binding portion of the insulin receptor.

Factors moderating the effect of oral sulfonylureas on free water clearance.

The effects of the antidiuretic agent chlorpropamide and the diuretic agent tolazamide on solute-free water clearance (CH2O) were compared in noninsulin-dependent diabetic patients undergoing water diuresis. Hyperglycemia (fasting serum glucose above 200 mg/dl) obscured the effects of these two sulfonylureas on CH2O. Thiazide or ethacrynic acid enhancement of chlorpropamide antidiuresis was also blunted by hyperglycemia and attendant osmotic diuresis. Thus, the low incidence of symptomatic hyponatremia during chlorpropamide treatment of diabetic patients may be explained by persistent hyperglycemia in such patients.

Heterogeneity of the inhibitory influence of sulfonylureas on prostanoid-induced smooth muscle contraction.

In addition to their hypoglycemic influence, sulfonylureas have been reported to inhibit prostanoid-induced vasoconstriction. Using isometric tension measurements we investigated whether this inhibitory influence is exerted by different sulfonylureas in various types of blood vessels from different species and in other types of smooth muscle cells. It was found that in addition to glibenclamide and tolbutamide also gliclazide (1 mM) and tolazamide (1 mM) block contractions induced by prostaglandin F2alpha and the thromboxane A2 mimetic U-46619 in rat aorta, but not the contractions elicited by norepinephrine, serotonin or high potassium. Glibenclamide (10 microM) inhibits the prostaglandin F2alpha- and U-46619-induced contractions on rat tail, femoral and renal interlobar arteries and on bovine retinal and ciliary arteries, but not those on aorta and carotid artery from guinea pigs and on human subcutaneous arteries. Glibenclamide (10 microM), tolbutamide (1 mM), tolazamide (1 mM) and gliclazide (1 mM) all block contractions induced by U-46619, but not those induced by carbachol, on rat intrapulmonary bronchioles. However, prostanoid-induced contractions of guinea-pig trachea and main bronchi are not influenced by glibenclamide (10 microM). From these results it is concluded that the ability of sulfonylureas to block prostanoid-induced contractions is shared by all sulfonylureas tested, that this is not limited to vascular smooth muscle cells and that it shows a heterogeneity, that might be linked to interspecies differences.