Potentiation of the insulin-releasing capacity of tolbutamide by thiols: Studies on the isolated perfused pancreas

Summary It has been suggested that the islet thiol redox status plays a role in the regulation of -cell sensitivity in response to insulin secretagogues. Employing the isolated perfused rat pancreas, the effect of reduced glutathione (1 mM) and L-cysteine (5 mM) on insulin release induced by tolbutamide (0.2 mg/ml), glucose (5.6 and 11.1 mM) and tolbutamide (0.1 mg/ml) in the presence of 5.6 mM glucose was studied.In the absence of glucose or in the presence of 5.6 mM of glucose neither glutathione nor L-cysteine stimulated the release of insulin. Reduced glutathione potentiated the secretion induced by glucose (11.1 mM) during the first and the second phase. L-Cysteine potentiated only the first phase of glucose-induced insulin release, whereas the second phase was depressed. Both of the tested thiols potentiated the insulin secretory action of either tolbutamide (0.2 mg/ml) alone or tolbutamide (01. mg/ml) in the presence of glucose (5.6 mM).The data suggest that supplementation of thiols to the pancreatic -cells perse cannot initiate the insulin secretory process. It is also suggested that GSH and L-cysteine increase the sensitivity of -cells to the stimulatory action of tolbutamide and/or glucose.This study was reported previously and published as an abstract (Abdel-hamid and Ammon 1980)     

Effects of propylthiouracil and methylthiouracil on cyclic AMP and ion movements in rat pancreatic islets

Summary Propylthiouracil and methylthiouracil have been shown to potentiate glucose-induced insulin secretion from rat pancreatic islets: the effect of methylthiouracil being less pronounced than that of propylthiouracil. In this study the effects of these substances on cAMP levels, ⁸⁶Rb⁺ efflux, ⁴⁵Ca²⁺ net uptake, and ⁴⁵Ca²⁺ efflux were tested in isolated rat islets in order to obtain information on their possible mechanism of action. Propylthiouracil and to a lesser extent methylthiouracil increased islet cyclic AMP in a concentration-related manner. Maximum increases at the highest concentrations tested were 261% and 190% respectively. In the presence of 3 mM glucose propylthiouracil and methylthiouracil led to a decrease in the ⁸⁶Rb efflux rate. With 5.6 mM glucose, both thiourea derivatives produced an increase in the ⁸⁶Rb⁺ efflux rate which was independent of the presence or absence of calcium in the medium. Propylthiouracil and methylthiouracil augmented the ⁴⁵Ca²⁺ efflux rate in the presence as well as in the absence of external calcium at various glucose concentrations. Propylthiouracil did not change, and methylthiouracil only slightly augmented, ⁴⁵Ca²⁺ net uptake into the isolated islets. It is suggested that the synergistic effect of propylthiouracil and methylthiouracil on glucose-induced insulin release is at least in part due to an increase in islet cAMP levels. Whether the two substances have additional direct effects on ionic fluxes which contribute to their insulinotropic action or whether the observed changes in ion movements are secondary to the elevation of cAMP levels remains to be unclear and needs further investigation. Send offprint requests to H. P. T. Ammon at the above address     

The effects of glibenclamide on rat islet radioactive nucleotide efflux,ATP contents and respiratory rates

In order to assess the effects of sulphonylurea on islet function, changes in radioactive nucleotide efflux, ATP contents and oxygen uptake of low or high glucose cultured rat islets in response to glibenclamide were determined. It was observed that: (1) low glucose cultured islets displayed a prompt increase in radioactive efflux in response to glibenclamide when perfused in the prescence of 1.7, 5.6 and 11.1 mM glucose. the response was most prominent in the presence of 5.6 mM glucose. (2) High glucose cultured islet did not respond to glibenclamide with increased radioactive efflux in the presence of 11.1 mM glucose, in contrast to the effects in the presence of 1.7 or 5.6 mM glucose. The sulphonylurea-induced changes could be delayed by adding glibenclamide in parallel with a decrease in glucose from 20 to 1.7 mM. (3) No effeets of glibenclamide on radioactive nucleotide efflux from either low or high glucose cultured islets could be observed in the absence of extracellular Ca²⁺. (4) Glibenclamide decreased the islet ATP content of low glucose cultured islets in the presence of 5.6 mM glucose. On the contrary, the ATP content of high glucose cultured islets was increased by glibenclamide with 1.7 mM glucose present. (5) Islet respiration was increased by adding glucose in both high or low glucose cultured islets, although respiration in the presence of both 5.6 and 11.1 mM glucose was higher in high glucose cultured islets. (6) The addition of glibenclamide decreased oxygen uptake of low glucose cultured islets in the presence of 5.6 and 11.1 mM glucose and that of high glucose cultured islets at all glucose concentrations tested (1.7, 5.6 and 11.1 mM). It is concluded that glibenclamide-induced changes in radioactive nucleotide efflux may reflect metabolic and ionic changes of importance to islet functions.     

Fluoxetine-induced toxicity results in human placental glutathione S-transferase-π (GST-π) dysfunction

Context: The antidepressant drug fluoxetine (FLU) is considered in the group of selective serotonine re-uptake inhibitors. Its distribution in brain and binding to human brain glutathione S-transferase-π (GST-π) have been shown. FLU can cross blood brain barrier and placenta, accumulate in fetus and may cause congenital malformations. Objective: To elucidate the interaction of placental GST-π with FLU. Materials and methods: First, concentration-dependent inhibition of human placental GST-π was evaluated by using different FLU concentrations and then 0.3125, 0.625, 1.25, 2.5 and 5?mM FLU concentrations were chosen and tested while keeping GSH concentration constant and 1-chloro-2,4-dinitrobenzene (CDNB) concentration varied and vice versa. The data were evaluated with different kinetic models and Statistica 9.00 for Windows. Results: The V_m, at variable [CDNB] (142?±?16?U/mg protein) was 3 times higher than the V_m obtained at variable [GSH] (49?±?4?U/mg protein). On the other hand, the K_m for CDNB was ～10 times higher than the K_m for GSH (1.99?±?0.36?mM versus 0.21?±?0.06?mM). The IC₅₀ value for FLU was 8.6?mM. Both at constant [CDNB] and variable [GSH] and at constant [GSH] and variable [CDNB] the inhibition types were competitive with the K_i values of 5.62?±?4.37 and 8.09?±?1.27?mM, respectively. Conclusion: Although the K_i values obtained for FLU in vitro are high, due to their uneven distribution, long elimination time and inhibitory behavior on detoxification systems, it may cause defects in adults but these effects may be much more severe in fetus and result in congenital malformations.     

Role of calcium and cyclic adenosine 3′,5′-monophosphate in the antilipolytic effect of tolbutamide and glibenclamide

Summary Tolbutamide (1 mM) and glibenclamide (0.1 mM) reduced lipolysis due to adrenaline, noradrenaline, isoprenaline, glucagon, and ACTH, whereas theophylline-induced glycerol production was only marginally affected. Furthermore, the effect of dibutyryl cyclic AMP on lipolysis was markedly decreased by low concentrations of both drugs. The antilipolytic action of glibenclamide and, to a smaller degree, of tolbutamide was highly dependent on the calcium concentration in the incubation medium used after preparation of fat cells in the absence of calcium. In the presence of isoprenaline as a lipolytic agent the antilipolytic effect of glibenclamide was progressively enhanced by raising the calcium concentration up to 1.2 mM. Howver, further elevation of calcium to 2.4 mM resulted in a loss of antilipolytic effect. Basal cyclic AMP levels of isolated fat cells were increased twofold by glibenclamide or tolbutamide. In contrast to the effect of glibenclamide, cyclic AMP levels in the presence of isoprenaline were further increased by tolbutamide. Low K _m cyclic AMP phosphodiesterase of fat cells was inhibited by tolbutamide as well as by glibenclamide. The results indicate that the antilipolytic action of sulfonylurea drugs is closely related to changes in calcium metabolism but independent of cyclic AMP levels.     

Metabolism of acrylamide to glycidamide and their cytotoxicity in isolated rat hepatocytes: protective effects of GSH precursors

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxicity and prevention. Metabolism and cytotoxicity of AA and its epoxide glycidamide (GA) were studied in the hepatocytes freshly isolated from male Sprague–Dawley rats. The isolated hepatocytes metabolized AA to GA. The formation of GA followed Michaelis-Menten kinetic parameters yielded apparent K _m = 0.477 ± 0.100 and 0.263 ± 0.016 mM, V _max = 6.5 ± 2.1 and 26.4 ± 3.0 nmol/h/10⁶ cells, and CL_int = 14 ± 5 and 100 ± 12 μl/h/10⁶ cells for the hepatocytes from untreated and acetone-treated rats, respectively. There were lower K _m and marked increases in V _max (fourfold) and in CL_int (sevenfold) in acetone-treated rat hepatocytes. The data suggest that CYP2E1 played a major role in metabolizing AA to more toxic GA. Both AA and GA induced a concentration- and time-dependent glutathione (GSH) depletion of the hepatocytes. From decreasing rates of GSH contents in hepatocytes, the parameters of glutathione S-transferase (GST) in hepatocytes to AA and GA were calculated to be K _m = 1.4 and 1.5 mM, V _max = 21 and 33 nmol/h/10⁶ cells, and CL_int = 15 and 23 μl/h/10⁶ cells, respectively. GA 1.5-times more readily depleted GSH content than AA. GA decreased the viability of hepatocytes at 3 mM, but AA did not. These data indicate that GA is more toxic than AA as assessed by intracellular GSH depletion and loss of viability of hepatocytes. GSH precursors such as N-acetylcysteine and methionine provided significant anti-cytotoxic effects on the decrease of GSH content and cell viability of hepatocytes induced by GA and AA.     

Effects of db cAMP on tyrosine hydroxylase activity of ganglia and nerve endings

Summary Preincubation of intact superior cervical ganglia or nictitating membrane for 2 h with dibutyryl cyclic AMP (db cAMP) increased the hydroxylation of tyrosine. This effect was not blocked by the protein synthesis inhibitor, cycloheximide. The K _m of tyrosine hydroxylase for the substrate, tyrosine, and for the cofactor, reduced pteridine, were decreased by db cAMP. There were no changes in the V _max of the enzyme. The inhibitory potency of noradrenaline on the hydroxylation of tyrosine was also decreased. Thus an inductive effect may be ruled out. The activation of the enzyme was only observed when the tissues were preincubated with the db cAMP and not when the cyclic nucleotide was added to the isolated enzyme.Preincubation of cervical ganglia for 4 h with db cAMP increased activity of decarboxylase and monoamine oxidase in tissue homogenates without changing the tyrosine hydroxylase activity.     

Interaction of cyclophosphamide and its metabolites with adenosine 3',5'-monophosphate binding proteins.

The sensitivity for recognition of cyclophosphamide and its metabolites by adenosine 3',5'-monophosphate (cAMP) specific proteins has been investigated. A 4-hydroxyl substituent in the 1,3,2-oxazaphosphorine ring is required for inhibition of cAMP binding to both cAMP phosphodiesterase and the regulatory subunit of the cAMP protein kinase holoenzyme. Binding to the latter causes an activation of the kinase and results in a dissociation into regulatory and catalytic subunits. The inhibitor constant K_i, for the inhibition of cAMP binding (0.19 mM) correlates well with that for inhibition of the low K_m form of the phosphodiesterase (0.19mM). In both cases inhibition is of the competitive type. Although 4-ketocyclophosphamide resembles 4-hydroxycyclophosphamide in electron donating properties it is inactive with respect to binding to cAMP specific sites. This probably results from the difference in conformation of the rings of these two compounds.     

PURIFICATION AND PROPERTIES OF RAT SKIN ACID PHOSPHATASES

Four acid phosphatases (APases I-IV) were purified from Triton X-100 extracts of rat skin. APase I appeared as a high-molecular weight, most likely lysosomal lipo(glyco)-protein with a pI of 4.3. APase I, extractable as a Triton-protein complex with a mol.wt. of 2 10⁶, hydrolyzed α-naphthyl phosphate (α-NP; K_m= 0.18 mM) and p-nitrophenyl phosphate (PNP; K_m= 0.96 mM) at an almost equal rate at pH 5.0. Mo₇O₂₄^6-inhibited noncompetitively (K_i= 0.15 μM). APase II had a pI of 7.8–8.0 and a mol.wt. of 104000 ± 4000. APase II is likely lysosomal and at pH 5.0 it hydrolyzed most rapidly PNP (K_m= 0.057 mM), α-NP (K_m= 0.18 mM) and phenyl phosphate (PP; K_m= 0.41 mM). Mo₇O₂₄^6- (K_i=2.5 nM) inhibited competitively. APase III, also of lysosomal origin, showed a pI of 5.3–5.5 and a mol.wt. of 87000 ± 3000. This enzyme, at pH 5.0, hydrolyzed most rapidly α-NP (K_m= 0.16 mM), pyridoxal 5-phosphate (PLP; K_m= 0.41 mM), PNP (K_m= 0.16 mM) and PP (K_m= 0.16 mM). Mo₇O₂₄^6- inhibition (K_i= 0.75 μM) was of a mixed type. p-Chloromercuribenzoate (PCMB; K_i= 2.0 μM) inhibited noncompetitively. A pI of 5.6–6.2 and a mol.wt. of 14000 ± 400 was obtained for the cytoplasmic APase IV. This enzyme, at pH 5.0, hydrolyzed most rapidly PNP (K_m= 0.16 mM), PP (K_m= 0.81 mM) and riboflavin 5′-phosphate (RP; K_m= 0.20 mM). Mo₇O₂₄^6- inhibited competitively (K_i= 8.5 μM) but PCMB (K_i= 0.32 μM) showed a mixed type of inhibition. PLP may be a natural substrate of APase III and RP that of APases II and IV. The activity of APases II and III may depend on histidyl and SH-groups, that of APase I on histidyl groups, and SH-groups may be necessary for the activity of APase IV. All these APases seem to have an active tyrosyl and carboxyl residue. The pH dependence of the catalyses, inhibition characteristics and anatomic location showed APases I-IV to be true skin acid phosphatases.     

Influence of leucine on intestinal baclofen absorption as a model compound of neutral α-aminoacids

The inhibitory effect of the essential α-aminoacid L-leucine on the intestinal absorption of the antispastic drug baclofen was examined by means of an in situ rat gut perfusion technique. When 0.5 mM baclofen solutions were perfused in the presence of increasing concentrations of the aminoacid (5–100 mM), the apparent absorption rate constant of the drug decreased as the initial leucine concentration increased. Higher leucine concentrations, however, did not completely abolish the absorption of the drug (at 100 mM of leucine, only 76% inhibition was observed). The interaction can be mathematically described as a complete competitive inhibition with a second component, K = 0.35 (±0.08)h^?1, K_i = 0.25 (±0.09)mM, AIC= ?97.02. In the light of some of the absorption features of the drug, however, the residual absorption of baclofen in the presence of high leucine concentrations should be attributed to another transport system not used by leucine. Apparent parameters characterizing absorption of leucine in the presence of baclofen (0.5mM) were V_m=61.02 (±5.46)mM h^?1; K_m=8.04 (±0.89)mM, and AIC=62.25. The results indicate that baclofen and leucine share some carriers in the intestinal absorption processes. Since leucine is an essential dietary aminoacid, and therefore a normal food component, this finding could be relevant in preventing interactions that would lead to a reduced oral bioavailability during baclofen therapy.     

Adenosine 3',5'-cyclic monophosphate phosphodiesterase in guinea-pig lung--properties and effect of adrenergic drugs

The hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP) by adenosine 3',5'-monophosphate phosphodiesterase (PDE) was studied in whole homogenates and cell fractions of guinea-pig lung. Approximately 60 per cent of the activity of the whole homogenate was contained in the 10,000 g supernatant. This cell fraction contained PDE with two separate affinities for cAMP: PDE I with low affinity. K_m ? 3.1 × 10^?4 M; V_max ? 90 nmoles cAMP hydrolyzed/mg protein/20 min and PDE II with high affinity K_m ? 5.7 × 10^?5 M; V_max ? 35 nmoles cAMP hydrolyzed/mg protein/20 min. Both forms required magnesium which could be replaced by manganese and cobalt but not by copper. Aminophylline and theophylline inhibited both PDE forms. Calcium only inhibited PDE I but not PDE II. Adrenergic drugs inhibited PDE I and at 0.1 mM decreased in potency in the following order: isoproterenol  epinephrine > norepinephrine > methoxamine. Butoxamine was ineffective on PDE I. Only norepinephrine inhibited PDE II but not to the same extent as its effect on PDE I. Chelation of added magnesium and endogenous calcium was not responsible for the inhibition of PDE I by the adrenergic drugs used. Propranolol (0.1 mM) and phentolamine (0.05 to 0.001 mM) alone did not effect PDE I. Propranolol (0.1 mM) failed to reverse the inhibitory effect of isoproterenol (0.1 mM) and epinephrine (0.1 mM). Thus a β-adrenergic mechanism could not explain the inhibitory effect of these agents. Phentolamine (0.05 to 0.001 mM) reversed the inhibition by epinephrine bitartrate but not by epinephrine hydrochloride. These data are inconclusive to support an α-adrenergic mechanism for the inhibition of cAMP phosphodiesterase by epinephrine.     

Interactions of opiates and prostaglandins E with regard to cyclic AMP in striatal tissue of rats in vitro

Summary The effects of prostaglandins E on the concentration of cyclic AMP (cAMP) and a possible antagonism of opiates vs. prostaglandins E were studied in homogenates and in slices of rat striata in vitro.In homogenates, PGE₁ or PGE₂ did not affect the synthesis of cAMP. Morphine slightly lowered the cAMP synthesis, in presence or absence of PGE₁ or PGE₂.In slices, PGE₂ significantly elevated the cAMP concentrations, either in presence or in absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Morphine, met-enkephalin and levorphanol, but not dextrorphan, antagonized this rise of cAMP. The effect of morphine was antagonized by naloxone. Adenosine or an elevation of K⁺-ions raised the cAMP concentrations, and PGE₂ induced a further increase. In presence of elevated K⁺-ions or adenosine, however, morphine did not antagonize the PGE₂-induced rise of cAMP concentration. It is suggested that under some experimental conditions described in the literature, endogenous activators of cAMP formation, e. g. adenosine, might mask the inhibitory effect of opiates on stimulation of cAMP synthesis induced by prostaglandins E.     

Kinetic studies on toluene metabolism in ethanol- and phenobarbital-induced rat liver microsomes in vitro

In vitro metabolism of toluene was investigated at substrate concentrations of 0.03–6.25 mM in liver microsomes from control and ethanol- and phenobarbital (PB)-treated rats. Three metabolites, benzylalcohol (BA),o- andp-cresol, were measured by high-performance liquid chromatograph. BA was the main metabolite of toluene, whereaso- andp-cresol contributed only 1.1–1.5% and 1.7–2.8% of total metabolites, respectively, in microsomes from control rats. Ethanol treatment showed little effect on the percentages of three metabolites, but PB increased the percentages ofo- andp-cresol to as high as 5.5% and 8.0%, respectively, following the increase in toluene concentration. There were two different isozymes with different K_m involved in the side-chain hydroxylation of toluene in microsomes from control and ethanol-treated rats. One had a low K_m value (0.13–0.17 mM) and could be greatly induced with ethanol treatment. The other was a high K_m isozyme (0.60–0.87 mM). PB-induced isozyme showed a similar K_m value to that of the high K_m isozyme existing in microsomes from control and ethanol-treated rats. Two isozymes were involved in the formation ofp-cresol in microsomes of control rats; the low-K_m type had a similar value (0.15 mM) to the low isozyme of BA formation, but the high K_m isozyme had a larger value (2.04 mM) than the high isozyme of BA. Only one enzyme responsible foro-cresol formation was detected in microsomes of control rats, and had a similar K_m (2.11 mM) to that of the high K_m isozyme ofp-cresol. The high K_m and low V_mak values of isozymes of aromatic hydroxylation account for the minor proportion of cresols. Ethanol and PB treatments enhanced the metabolism of toluene, but they differ in that the ethanol-inducible isozyme metabolized toluene at a faster rate at low substrate concentrations, whereas PB did so at high toluene concentrations. The possible involvement of subtypes of cytochrome P450, and the significance of the results in biological monitoring of toluene exposure are discussed.     

Activation and inhibition of lipolysis in isolated fat cells by various inhibitors of cyclic AMP phosphodiesterase

Summary The effects of methylxanthines, papaverine, dipyridamole and imipramine on lipolysis and phosphodiesterase activity of rat adipose tissue were investigated. Lipolysis in isolated fat cells was stimulated by theophylline and caffeine whereas papaverine, dipyridamole and imipramine had no substantial effect on the basal lipolytic rate. Lipolysis induced by noradrenaline was potentiated by theophylline, but blocked by papaverine, dipyridamole and imipramine at concentrations between 0.02 to 0.2 mM. These agents also depressed lipolysis induced by theophylline and dibutyryl cyclic AMP and reduced the lipolytic activity of homogenates of adipose tissue. The activity of phosphodiesterase assayed over a wide range of substrate concentrations revealed two different Michaelis constants. Both types of phosphodiesterase were inhibited by theophylline, papaverine, dipyridamole and imipramine in a competitive manner, the low K _m enzyme being more sensitive for inhibition than the high K _m enzyme. On both types of phosphodiesterase papaverine and dipyridamole proved to be 10 to 100 times more potent inhibitors than theophylline and imipramine. To explain the antilipolytic effect of phosphodiesterase inhibitors it is assumed that they do not only affect substrate binding of cyclic AMP to phosphodiesterase but also displace cyclic AMP from the binding site on protein kinase, thus acting as inhibitors of the activation process within the lipolytic system.     

Cyclic nucleotide phosphodiesterases of human and rat gastric mucosa

Summary Cyclic adenosine-3,5-monophosphate (cAMP) phosphodiesterases (PDE) were partially purified from human and rat gastric mucosa. Drugs known to affect the cyclic nucleotide system and/or gastric secretion were tested for effects on the PDE-activities from both species.In rat gastric mucosa PDE-activity can be detected in the 100 000×g sediment (K _m =8.3 M; V _max=3.2 nmoles cAMP hydrolyzed/mg protein x min) and the cytoplasma (K _m =5.6 M; V _max=2.6 nmoles cAMP hydrolyzed/mg protein x min).The most effective inhibitors of the particle-bound activity are papaverine (K _i =4 M, non-competitive) and 3-isobutyl-1-methylxanthine (K _i=14 M, competitive). There was only a modest competitive inhibition by theophylline (K _i =495 M). PDE-activity in the cytoplasma was inhibited competitively by these three drugs (papaverine: K _i =6.5 M; 3-isobutyl-1-methylxanthine: K _i =37 M; theophylline: K _i =152 M.In human gastric mucosa PDE-activity can be detected in the particular fraction (K _m =23.9 M; V _max=1.2 nmoles cAMP hydrolyzed/mg protein x min), and the soluble fraction (K _m =12.1 M; V _max=2.4 nmoles cAMP hydrolyzed/mg protein x min).PDE-activity in the 100 000×g sediment was inhibited by papaverine (K _i =5.6 M, non-competitive), 3-isobutyl-1-methylxanthine (K _i =16 M, non-competitive), theophylline (K _i =165 M, non-competitive), and N₆-2-O-dibutyryl-cAMP (K _i =746 M, competitive).Inhibition in the 100 000×g supernatant was noncompetitive with 3-isobutyl-1-methylxanthine (K _i =7.1 M and papaverine (K _i =8.5 M), but competitive with N₆-2-O-dibutyryl-cAMP (K _i =170 M), and theophylline (K _i =225 M). This study indicates that PDE-activities of the two species are qualitatively similar, but quantitative differences exist.     

Effect of methylene blue and thiol oxidants on pancreatic islet GSH/GSSG ratios and tolbutamide mediated insulin release in vitro

Summary Methylene blue (an oxidant of NADPH), diamide (an oxidant of glutathion-SH [GSH]) and tertbutyl hydroperoxide (a substrate of glutathione peroxidase) significantly decreased the GSH content of pancreatic rat islets and decreased their GSH/GSSG ratio. They also significantly depressed the single peak insulin response to tolbutamide by the isolated perfused pancreas as well as its synergistic action with glucose in isolated pancreatic islets.These results suggest that the effect of tolbutamide alone and its synergistic action with glucose could depend on the islet NADPH and GSH. In addition it appears that augmentation of tolbutamide action by glucose in insulin release is mediated by the provision of additional NADPH and GSH through glucose metabolism.     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.     

Structural isomerization of synephrine influences its uptake and ensuing glutathione depletion in rat-isolated cardiomyocytes

Synephrine is a natural compound, frequently added to ephedra-free dietary supplements for weight-loss, due to its effects as a nonspecific adrenergic agonist. Though only p-synephrine has been documented in plants, the presence of m-synephrine has also been reported in weight-loss products. The use of synephrine in dietary supplements was accompanied by reports of adverse effects, especially at the cardiovascular level. It is well known that the imbalance in cardiac glutathione levels can increase the risk of cardiomyopathy. The present work aimed to study the role of organic cation-mediated transport of m- and p-synephrine and the possibility that p- and m-synephrine induce intracellular changes in glutathione levels in calcium-tolerant freshly isolated cardiomyocytes from adult rat. After a 3 h incubation with 1 mM p- or m-synephrine, the intracellular content of synephrine was measured by gas chromatography/ion trap-mass spectrometry (GC/IT-MS); cell viability and intracellular glutathione levels were also determined. To evaluate the potential protective effects of antioxidants against the adverse effects elicited by m-synephrine, cells were pre-incubated for 30 min with Tiron (100 μM) or N-acetyl-cysteine (NAC) (1 mM). To assess the influence of α₁-adrenoceptors activation in glutathione depletion, a study with prazosin (100 nM) was also performed. The results obtained provide evidence that organic cation transporters OCT3 and OCT1 play a major role in m- and p-synephrine-mediated transport into the cardiomyocytes. The importance of these transporters seems similar for both isomers, although p-synephrine enters more into the cardiomyocytes. Furthermore, only m-synephrine induced intracellular total glutathione (GSHt) and reduced glutathione (GSH) depletion. NAC and Tiron were able to counteract the m-synephrine-induced GSH and GSHt decrease. On the other hand, the incubation with prazosin was not able to change m-synephrine-induced glutathione depletion showing that this effect is independent of α₁-adrenoceptor stimulation. In conclusion, both positional isomers require OCT3 and OCT1-mediated transport to enter into the cardiomyocytes; however, the hydroxyl group in the p-position favours the OCT-mediated transport into cardiomyocytes. Furthermore, the structural isomerization of synephrine influences its toxicological profile since only m-synephrine caused GSH depletion.     

Effect of tolbutamide on aminophylline-, 3,5-AMP-dibutyrate-or glucagon-induced insulin release from pancreatic islets after impairment of pyridine nucleotide metabolism caused by 6-aminonicotinamide (6-AN)

Summary The effect of tolbutamide on pyridine nucleotides and insulin secretion stimulated by aminophylline, 3,5-AMP-dibutyrate or glucagon was studied in pancreatic islets of rats previously treated with 6-aminonicotinamide (6-AN), an inhibitor of pyridine nucleotide synthesis.After being incubated for 60 min in a Krebs-Ringer-Bicarbonate-Buffer in the absence of glucose, pancreatic islets of rats i.p. injected with 35 mg/kg of 6-AN 6 hrs before pancreas removal contained about 30% less NADP and NADPH than did islets of control rats. No changes of NAD or NADH were observed in islets of 6-AN-treated animals. Addition of 16.5 mM glucose led to an increase of NADH, NADPH and a decrease of NADP in islets of both groups of animals; NAD levels remained unchanged. In vitro addition of tolbutamide to islets of control rats did not affect the levels of NADPH or NADP in the presence of 5.5 mM glucose. When 16.5 mM glucose were present, a decrease of NADPH and an increase of NADP was obvious. No effect of tolbutamide on insular NADPH or NADP was observed in islets of rats previously treated with 6-AN be it in the presence of 5.5 or 16.5 mM glucose.In islets of 6-AN-treated rats insulin release in response to aminophylline or 3,5-AMP-dibutyrate in the presence of 5.5 mM glucose was significantly depressed, when compared to islets of untreated controls. Addition of tolbutamide increased insulin release due to aminophylline, 3,5-AMP-dibutyrate or glucagon from islets of controls. Tolbutamide alone was without effect. In islets of 6-AN-treated rats aminophylline, 3,5-AMP-dibutyrate or glucagon stimulated insulin release only when tolbutamide was present.Our data suggest that there is no direct interference of tolbutamide with pyridine nucleotides of pancreatic islets, and that tolbutamide increases the secretory response of the -cell to aminophylline, 3,5-AMP-dibutyrate or glucagon when insulin release due to these agents is inhibited during decrease of insular NADP and NADPH, caused by 6-AN.Supported by the Deutsche Forschungsgemeinschaft.     

Inhibition of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase of human erythrocytes by purine and pyrimidine nucleotides.

Orotidine-5′-phosphate decarboxylase of human hemolysates exhibits triphasic kinetics with K_m values of 33, 1.7 and 0.082 μM. Inhibition of this enzyme at low OMP concentrations (<3 μM) by several naturally occurring purine and pyrimidine nucleotides was investigated. No significant inhibition was observed with IMP, GMP, TMP, ADP, and TTP at 5 mM. Inhibition constants for CMP, AMP, and dAMP were 31 μM, 0.11 mM and 0.21 mM, respectively. The results are discussed in relation to inhibition by nucleotides of orotate phosphoribosyltransferase, previously measured with a method which depends on orotidine-5′-phosphate decarboxylase activity.