Different effects of lipolytic hormones and phosphodiesterase inhibitors on cyclic 3′,5′-AMP levels in isolated fat cells

Summary Cyclic AMP levels of isolated fat cells of rats were increased about 50-fold by noradrenaline (1 M) and isoprenaline (1 M) within 4 min of incubation and had declined markedly after 10 min. The effect of a variety of lipolytic hormones on cyclic AMP levels was dose-dependent over a wide range of concentrations, ACTH and glucagon being most potent on a molar basis. Among the adrenergic compounds tested isoprenaline elicited the grea test response. Glucagon, in maximally effective concentrations, caused only half the increase in cyclic AMP values produced by other hormones. This pattern was largely paralleled by the lipolytic effects of the hormones as measured by glycerol production. Various inhibitors of cyclic AMP phosphodiesterase had only small effects on cyclic AMP accumulation. Methylxanthines caused only a 3- to 5-fold elevation of cyclic AMP levels at concentrations which induced maximal lipolytic effects. Papaverine (0.5 mM) and phentolamine (0.1 mM) had virtually no effect and did not potentiate hormone effects on cyclic AMP production. However, theophylline (1 mM) caused a nearly tenfold increase in the effects of isoprenaline when added to 100 000 cells per ml in the medium. At a concentration of 20 000 fat cells per ml isoprenaline alone increased cyclic AMP levels 300-fold and the addition of theophylline had no further stimulatory effect. Our results suggest that lipolysis induced by hormones is mainly mediated by an accumulation of cyclic AMP, whereas methylxanthines must have additional effects. The potentiation of lipolytic hormones by methylxanthines cannot be attributed to an inhibition of phosphodiesterase alone, but seems to be due mainly to their antagonism with an inhibitory factor, which is produced by fat cells and released into the incubation medium.     

A simple method for sampling murine pulmonary and cardiac tissues for analysis of cyclic nucleotide levels

Summary A simple method for the rapid removal and freezing of mouse cardiac and pulmonary tissues is described. Samples thus obtained were judged to be suitable for valid estimation of in vivo levels of cyclic AMP and cyclic GMP based on the following findings: (a) the samples could be obtained and frozen in the very short time period of a few seconds; (b) no indication of adverse effects of the collection procedure was found upon examination of chemical indicators of energy metabolism; (c) the apparent rates of change of cyclic AMP and cyclic GMP levels during the first seconds after tissue isolation could produce small, but acceptable errors; and (d) dose-dependent elevations of pulmonary cAMP levels consistent with known effects in vitro were found after in vivo administration of isoproterenol.Abbreviations cAMP adenosine-3,5-cyclic monophosphate - cGMP guanosine-3,5-cyclic monophosphate     

Stimulation of adenosine 3′,5′-monophosphate formation in guinea-pig cerebral cortical slices in a calcium free medium

Summary In guinea-pig cerebral cortical slices cyclic AMP concentrations increase during incubation with histamine+noradrenaline. After 10 min of incubation the levels of cyclic AMP start to decline. When calcium ions are omitted from the incubation medium, cyclic AMP levels do increase to a greater extent under the same conditions and do not drop during 30 min incubation. In the presence of calcium ions cyclic AMP synthesis can not be elicited by noradrenaline alone. In calcium-free Krebs-Ringer solution a pronounced effect of noradrenaline on cyclic AMP levels is observed. This effect of noradrenaline is shown to be mediated by a classical -type receptor. 5-Hydroxytryptamine, prostaglandin E ₁ and dopamine do not significantly enhance cyclic AMP formation in guinea-pig brain slices in either the presence in, or the absence of calcium ions from the incubation medium. Under depolarizing conditions of incubation the stimulatory effect of ouabain or 125 mM K⁺ is blocked in a calcium-free medium, while with the depolarizing agent veratridine no significant reduction of cyclic AMP formed during incubation in a calcium-free medium is obtained.     

Adenosine receptor activation by adenine nucleotides requires conversion of the nucleotides to adenosine

Summary Adenine nucleotides cause adenosine receptor-mediated increases in cyclic AMP in the VA13 human fibroblast line. Levels of adenosine accumulated in the medium are insufficient to account for the responses to adenine nucleotides. Since rapid conversion of the nucleotides to adenosine by 5-nucleotidase in the vicinity of the receptor might account for the responses, six experimental methods were developed to distinguish between local conversion and direct action of the nucleotides. Results of all six methods favored local conversion. (1) 5-Nucleotidase inhibitors blocked the accumulations of cyclic AMP elicited by AMP, ADP, and ATP, but did not affect the response to adenosine. The most potent inhibitor of both conversion of AMP and response to AMP was ,-methylene-ADP (APCP). (2) Adenosine deaminase blocked the responses to AMP, ADP, ATP, and adenosine-containing coenzymes. (3) Theophylline, a specific competitive adenosine antagonist, was an insurmountable inhibitor of the increases in cyclic AMP caused by AMP, ADP, and ATP. The insurmountability was presumably due to substrate sataration of the converting enzyme 5-nucleotidase. (4) Although ADP and ATP had partial agonist-like dose-response curves, they did not inhibit the response to adenosine. (5) Nine cell lines which responded to adenosine were tested for response to AMP. Cell lines with high levels of 5-nucleotidase had large responses to AMP, those with intermediate levels of 5-nucleotidase had large or intermediate responses to AMP, and those with low 5-nucleotidase levels did not respond to AMP. (6) Inhibition of the uptake of labelled adenosine was used as an indicator of unlabelled adenosine concentrations near the cell membrane. Unlabelled AMP inhibited uptake nearly as effectively as unlabelled adenosine. APCP reversed the inhibition by AMP but not the inhibition by adenosine.The adenosine receptor is concluded to be an enity distinct from adenine nucleotide receptors.Submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy in Neurosciences, University of California, San Diego. Supported by NIMH DA-00265 and PHS RR 05665. The author has been a NSF Graduate Fellow. An abstract of this material has been published (Bruns 1977)     

Stimulation of adenylate cyclase of guinea pig gastric mucosa by histamine,sodium fluoride and 5′-guanylylimidodiphosphate and inhibition by histamine H1- and H2-receptor antagonists in vitro

Summary There are experimental data indicating that cyclic AMP is involved in the regulation of gastric acid secretion in various mammalian species. In a broken cell preparation of guinea pig gastric mucosa the effects of some stimulants of gastric acid secretion on the activity of adenylate cyclase were studied. The basal adenylate cyclase activity was 483±43 pmoles cyclic AMP/mg proteinx10 min. The activity could be stimulated by histamine maximally 5-fold, by sodium fluoride (NaF) maximally 20-fold and by 5-guanylylimidodiphosphate (GMP-PNP) maximally 10-fold. Neither pentagastrin nor carbachol were able to stimulate the adenylate cyclase. Stimulants of adrenergic - or -receptors (phenylephrine, isoproterenol) were also ineffective.The activation of the adenylate cyclase by histamine was inhibited by the histamine H₁-receptor antagonists diphenhydramine and mepyramine as well as by the histamine H₂-receptor antagonist metiamide. On the other hand, the stimulatory action of NaF or GMP-PNP could be antagonized only by high concentrations of dipenhydramine or mepyramine while metiamide showed no antagonizing effect in this respect. Thus this preparation can be used as a tool to determine the activity and specificity of histamine H₂-receptor antagonists.     

Effect of phenothiazine neuroleptic drugs and tricyclic antidepressants on phosphodiesterase activity in rat cerebral cortex

The activity of phosphodiesterase (PDE) of rat cerebral cortex following the administration in vitro and in vivo of various concentrations of neuroleptic phenothiazine drugs and tricyclic antidepressive drugs has been investigated. It has been shown that PDE activity is inhibited by phenothiazine neuroleptic drugs (fluphenazine > trifluperazine > thioproperazine > chlorpromazine = thioridazine). Tricyclic antidepressants nortriptyline, chlorimipramine, protiptyline, imipramine and desipramine at a concentration of 10^–3 M caused 60–80% inhibition of PDE activity. It has also been found that the investigated phenothiazine compounds inhibit the high affinity PDE activity more than the PDE activity of low affinity to the substrate.The results obtained suggest that the mechanism of the neuroleptic action of phenothiazine drugs is partially connected with their influence on cyclic 3,5-AMP metabolism.Supported by Polish Academy of Sciences, 09.4.1.5.     

Characteristics of β-endorphin-induced histamine release from rat serosal mast cells Comparison with neurotensin,dynorphin and compound 48/80

Summary Rat peritoneal mast cells were exposed to the neurohormone and basic opioid peptide -endorphin. -Endorphin induced a dose-dependent release of histamine from the mast cells. A significant histamine release was found at 5 mol/l of -endorphin and maximal release (35% of total) at 20 mol/l. The histamine release process was very rapid and terminated within 30 s at 37°C, and in this sense is very similar to the histamine release induced by compound 48/80 or neurotensin. The histamine release was temperature-dependent showing an optimum release around 30°C, and it was independent of available extracellular calcium, but was inhibited in the presence of high extracellular calcium concentrations. Naloxone, only in very high concentrations (10 mmol/l), inhibited the release, and the very same concentration also inhibited the neurotensin — as well as the compound 48/80-induced histamine release. Cromoglycate and benzalkoniumchloride, a 48/80 antagonist, both produced a progressive dose-dependent inhibition of -endorphin-, neurotensin- as well as compound 48/80-induced histamine release. Taken together, the findings indicate that the opioid peptide -endorphin induces a selective, energy-dependent release of histamine from peritoneal rat mast cells. The pattern of release has much in common with that of compound 48/80 and other basic peptides, such as neurotensin and substance P. In addition this pattern of release is similar to that induced by dynorphin. Send offprint requests to Anita Sydbom at the above address     

Presence of functionally active β-adrenoceptors in rat mast cells

Summary The correlation between the binding of a -adrenoceptor antagonist, (–)[³H]-dihydroalprenolol (DHAP), and the adrenergic inhibition of histamine release by acetylcholine and by compound 48/80 was studied with isolated purified rat mast cells and in rat mast cell crude membrane fractions.Acetylcholine-evoked histamine release was inhibited by catecholamines, in the order isoprenaline > adrenaline > noradrenaline. Pretreatment of cells with (–)alprenolol antagonized the inhibitory effect of isoprenaline on acetylcholine-induced histamine release.40/80-evoked histamine release was bocked by isoprenaline at significantly higher concentrations than those required to inhibit cholinergic histamine release. The inhibitory effect of isoprenaline was equally antagonized by preincubating mast cells with (–)alprenolol.Specific binding sites for DHAP have been demonstrated in rat mast cell membranes. The specific binding of DHAP was inhibited by adrenoceptor agonists and antagonists according to the stereospecificity of these compounds.A close correlation between the binding-inhibitory potency of various adrenergic compounds and the data obtained in the pharmacological experiments was found, thus indicating the presence of -adrenoceptors in rat mast cells.     

Effect of six virustatic nucleoside analogues on the development of fetal rat thymus in organ culture

The effects of the virustatic agents zidovudine (azidothymidine, AZT) 23-dideoxycytidine (ddC), 23-dideoxyinosine (ddI), acyclovir (ACV), ganciclovir (GCV), and vidarabine phosphate (VP) on the in vitro development of thymic lobes of 17-day-old rat fetuses were tested in an organ culture system. The virustatics were added to the medium for a culture period of 7 days. All nucleoside analogues inhibited the proliferation and differentiation of lymphatic cells. However, differences were observable with respect to the potency of the six drugs to interfere with thymic development. Compared to untreated controls, reduction in the number of thymocytes was significant at concentrations of 30 M AZT and ddI. In the case of ACV, GCV, VP, and ddC concentrations as low as 10 M were sufficient to cause a significant reduction, ddC being the most potent derivate. Increasing concentrations of the nucleoside analogues led to a dose-dependent further inhibition of cell proliferation. At a concentration of 30 M flow cytometry revealed a decrease in the relative number of double positive CD4⁺ CD8⁺ and single positive CD4⁺ CD8^– cells but an increase in the relative number of CD4-CD8⁺ cells. At the same concentration the expression of the CD5 antigen was reduced by the antimetabolites, indicating that maturation of the thymocytes was inhibited. Distribution of the forward light scatter, a cell size-related parameter, showed that the formation of small thymocytes was reduced by the nucleoside analogues. Light and electron microscopic investigations indicated cytotoxic effects of the drugs on the thymocytes, whereas the epithelium was only slightly affected.     

Effects of methacholine,histamine and atropine on pulmonary guanosine-3′, 5′-monophosphate levels in hypersensitive mice

Summary Pulmonary levels of cGMP and cAMP in mice sensitized to methacholine and histamine with b. pertussis were examined to determine whether sensitization could be the result of an alteration in the metabolism of these cyclic nucleotides. The results presented show that in sensitized mice, methacholine raised cGMP to levels that were about double those produced without sensitization. In analogous experiments, histamine raised cGMP by approximately 100% in sensitized mice without producing significant increases in nonsensitized groups. Atropine completely blocked the cGMP rises produced by methacholine but did not eliminate those produced by histamine, thus indicating that cholinergic, but not the histaminergic elevation of cGMP involves activation of muscarinic receptors. The influence of pertussis on cAMP appeared to be opposite in direction from cGMP, i.e., a small but significant drop in cAMP levels was found following methacholine administration to sensitized, but not to nonsensitized mice. It was concluded that pertussis sensitization increases the responsiveness of the pulmonary guanylate cyclase-cGMP system to methacholine and histamine, and that the altered patterns of cGMP accumulation may contribute to the biochemical mechanism of sensitization.     

Pharmacokinetics of 2′,3′-dideoxyadenosine in dogs

The pharmacokinetics of 2,3-dideoxyadenosine (ddAdo) and 2-3-dideoxyinosine (ddIno) were determined after intravenous bolus administration and long-term intravenous infusion of ddAdo in dogs. ddAdo was rapidly deaminated to ddIno and ddAdo plasma concentrations were only a fraction of ddIno concentrations. The total body clearance of ddAdo exceeded the literature value for the cardiac output of the dog, indicating an extremely rapid metabolism, and the existence of extrahepatic metabolism. Urinary excretion of unchanged ddAdo was a minor route of elimination ( 1%). The pharmacokinetics of ddIno was determined assuming complete conversions of ddAdo to ddIno. ddIno elimination was dose-dependent with total body clearance ranging from 4 to 55 ml/min/kg in individual animals. The plasma half-life was approximately 30 min after most routes of administration, but increased to approximately 60 min in two animals receiving a large intravenous dose of 500 mg/kg. ddIno penetrated into the cerebrospinal fluid to a limited extent, reaching concentrations of 3–11% of those in plasma. Urinary excretion of unchanged ddIno accounted for approximately 20% of the administered dose of ddAdo, while uric acid and hypoxanthine were minor urinary metabolites.Concentrations exceeding the in vitro minimal viral inhibitory concentration (2.4 g/mL) could be safely maintained in plasma for a 10-day period. Infusions which gave cerebrospinal fluid concentrations of 12 to 17 Lg/mL resulted in dose limiting myelosuppression and intestinal toxicity, after less than 10 days of infusion. Orally administered ddAdo was absorbed as ddIno, with bioavailabilities ranging from 28 to 93% in experiments where no emesis occurred. These studies indicate the rapid in vivo conversion of ddAdo to ddIno, and support the selection of ddIno over ddAdo for further drug development.     

Disposition of 2′, 3′-dideoxyadenosine and 2′, 3′-dideoxyinosine in mice

Summary Mice were dosed with [³H]2,3-dideoxyadenosine ([³H]ddA) in three procedures: intravenously, intraperitoneally, and interperitoneally following a dose of 2 -deoxycoformycin (dCF). For mice dosed intravenously, the content of radioactivity in plasma and tissue samples were essentially constant after 30 min. Of the radioactivity in plasma and brain samples collected between 30 min and 24 hr, more than 94% was present as ³H₂O, indicating that most of the tritium from [³H]ddA had exchanged with water. No intact ddA was detected, and the deamination product, 2,3 -dideoxyinosine (ddI), was present only transiently. In the urine, the major radioactive material was [³H]ddI. Also detected were ³H₂O and small amounts of [³H]hypoxanthine and [³H]ddA. Following intraperitoneal doses to mice, levels of radioactivity in plasma, liver, and kidney increased to a maximum by 15–30 min after dosing but dropped to essentially constant levels thereafter, again indicating that the tritium had exchanged with water. At 5, 15, and 30 min after dosing, ddI was the major radioactive component in plasma. Only small amounts of ddA were present. When dCF was administered 24 hr prior to intraperitoneal [³H]ddA, levels of radioactivity in plasma, liver, and kidney reached a maximum at 30 to 60 min after dosing and decreased to essentially constant levels thereafter. The dCF transiently inhibited the deamination of ddA to ddI, since, in plasma, [³H]ddA was the main radioactive component at 5 and 15 min after dosing. Comparison of HPLC assays based on radioactivity detection and UV absorbance showed that they were equivalent for measuring ddA and ddI in samples derived from dosed mice. Therefore, exchange of tritium must have occurred at a metabolic step beyond ddI.For mice dosed intravenously and orally with unlabeled ddI, there was evidence of a saturated process. Nevertheless, for the high and low intravenous doses of ddI, the percent of dose excreted in the urine as unchanged drug was the same.     

2,2′,3′,4,4′,5,5′-Heptachlorobiphenyl (PCB 180) — on its toxicokinetics,biotransformation and porphyrinogenic action in female rats

The toxicokinetics and biotransformation of 2,2,3,4,4,5,5-heptachlorobiphenyl, as well as its influence on the activity of microsomal and cytosolic enzymes and on the porphyrin pathway in the liver were studied in female rats following oral treatment with 7 mg/kg every other day for 3 months. One day after cessation of treatment the concentration of the compound in liver, spleen, CNS and blood was 100–500 times and in the trachea it was only 5 times less than in the adipose tissue. The daily excretion with the feces and urine amounted to 35 and 1.5 g, respectively. In both excreta, heptachlorobiphenylol was identified as a metabolite. The biotransformation rate was estimated to be about 5%. Investigations of the liver revealed increases in the relative liver weight, total cytochrome P-450 content, O-deethylation of 7-ethoxycoumarin and in the activity of glutathione S-transferases. Disturbances of the hepatic porphyrin pathway were not detected. Only at the end of a post-dosing period of 12 months did the hepatic uroporphyrinogen decarboxylase show diminished activity. Only one of these animals with diminished enzyme activity showed drastically elevated porphyrins. In these animals, the fecal and urinary porphyrins did not differ from controls. At no time did heptachlorobiphenyl influence the urinary excretion of delta-aminolevulinic acid and porphobilinogen. The results indicate 1) that this congener shows expected toxicokinetics with the exception of being accumulated in the trachea and 2) that this congener induces disturbances of the hepatic porphyrin pathway several months after cessation of treatment.     

Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria     

Evidence for two separate vasoconstriction-mediating nucleotide receptors,both distinct from the P2X-receptor,in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides

Summary Uridine 5-triphosphate- (UTP-) and adenosine 5-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure.Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5-O-(3-thio)triphosphate (ATPS), and ,-imido adenosine 5-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, , -methylene-ATP and ,-methylene-ATP up to 10^–3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP UMP = CTP; that of the purine nucleotides was: ATPS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = , -methylene-ATP = ,-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to ,-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10^–3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10^–3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10^–3 to 2 × 10^–3 mol/l.It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P₂-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P₂-receptor which is distinct from the known P₂-subtypes.Send offprint requests to I. v. Kügelgen at the above address     

Effects of pentagastrin and carbachol on the gastric histamine level in α-fluoromethylhistidine-treated mice and rats

Summary The gastric mucosal histamine level in mice increased by about 80% and 100% after fasting for 24 and 48 h, respectively. In non-fasted mice, -fluoromethylhistidine (-FMH), a specific histidine decarboxylase inhibitor, significantly decreased the histamine level, the reduction amounting to 35% and 49%, 2 h and 4 h after treatment, respectively. In mice fasted for 24 h, a significant decrease of 42% was observed 4 h after treatment. However, in mice fasted for 48 h, no significant decrease was seen even 4 h after -FMH treatment. Therefore, the histamine-releasing effect of re-feeding and drugs on the gastric mucosa was examined in vivo, using animals fasted for 48 h and subsequently treated with -FMH. Food given simultaneously with -FMH to 48-h fasted mice significantly decreased the histamine level 4 h later. Pentagastrin and carbachol administered alone (0.25–2.0 mg/kg, i.p.) had no significant effect on the histamine level. However, the combined treatment with these drugs significantly decreased the histamine level. In rats fasted for 48 h and treated with -FMH, pentagastrin (0.25 and 0.5 mg/kg, i.p.) but not carbachol (0.125 – 0.5 mg/kg, i. p.) caused a significant decrease in the mucosal histamine level. In contrast to mice, the effect of the combined treatment with pentagastrin and carbachol was not synergistic in rats. These findings suggest that gastrin acts synergistically with acetylcholine in the histamine release from the gastric mucosa in mice, whereas such synergism may not occur in rats. Send offprint requests to K. Saeki     

In vivo release of neuronal histamine in the hypothalamus of rats measured by microdialysis

Summary Using an in vivo intracerebral microdialysis method coupled with an HPLC-fluorometric method, we investigated the extracellular level of endogenous histamine in the anterior hypothalamic area of urethaneanaesthetized rats. The basal rate of release of endogenous histamine in the anterior hypothalamic area measured by this method was 0.09 + 0.01 pmol/20 min. When the anterior hypothalamic area was depolarized by infusion of 100 mM K⁺ through the dialysis membrane or electrical stimulation at 200 A was applied through an electrode implanted into the ipsilateral tuberomammillary nucleus, histamine release increased to 175% and 188%, respectively, of the basal level. These increases were completely suppressed by removal of extracellular Ca²⁺. The basal release of histamine was also suppressed after infusion of 10^–6 M tetrodotoxin or i.p. administration of 100 mg/kg of -fluoromethylhistidine. On the other hand, 3-fold increase in the basal release was observed after i. p. administration of 5 mg/kg thioperamide. These results clearly indicate that both the basal and evoked release of histamine measured by our method are of neuronal origin. Send offprint requests to T. Mochizuki at the above address     

Adrenal cortex adenylate cyclase

Summary 5-guanylyl-imidodiphosphate [Gpp(NH)p], a GTP analogue, increases both basal and ACTH_1–24-stimulated adenylate cyclase (EC 4.6.1.1.) activities in rat adrenal particulate fractions and bovine adrenal cortex.In the absence of 5-guanylyl-imidodiphosphate ACTH_1–24 evoked a considerable response of adenylate cyclase in bovine adrenal cortex membranes. The rat adrenal particulate preparation, however, demonstrated only small increases upon ACTH_1–24 addition unless 5-guanylyl-imidodiphosphate was present.GTP imitated the effects of Gpp (NH)p on ACTH_1–24-stimulated rat adrenal adenylate cyclase, but only during the first minutes of incubation. Thereafter, the rate of cyclic AMP production rapidly declines. When Gpp (NH)p and GTP were given together, GTP inhibited the effects of Gpp (NH)p after a prolonged incubation. To explain the transient nature of the GTP effect and the inhibition of the Gpp (NH)p stimulation, it is suggested that GTP itself is an activator of adrenal cortex adenylate cyclase. The subsequent inhibition might be due to a rapidly accumulating product of GTP; alternatively it might be connected, in a still unknown way, to the hydrolysis of the terminal phosphate of GTP. These findings indicate the presence of regulatory guanyl nucleotide sites for adrenal cortex adenylate cyclase.     

Hemmung von Adenyl-Cyclase-Präparationen aus der Rattenniere durch Calciumionen und verschiedene Diuretica

Summary Antidiuretic hormone (ADH) increases the permeability to water of certain epithelial membranes. This effect, found in the urinary bladder of the toad and in the distal tubules and the collecting ducts of kidney, is mediated intracellularly by adenosine 35-monophosphate (Ado-35-P). Calcium ions and the diuretic ethacrynic acid are known to inhibit the ADH-induced increase in water permeability of the toad bladder. In adenyl cyclase preparations from rat renal cortex and medulla, the influence of these substances as well as of other diuretics added in vitro has been studied. Adenyl cyclase activity has been determined, excepted as noted, by measuring Ado-35-P formed from 1 mM ¹⁴C-ATP in the presence of 10 mM Mg⁺⁺, an ATP regenerating system, and 5 mM unlabeled Ado-35-P to reduce the enzymatic degradation of the labeled Ado-35-P.Calcium ions reduced the rate of Ado-35-P formation by particles from renal cortex and medulla when the activity was measured in the presence of either Mg⁺⁺ or Mn⁺⁺. With 10 mM Mg⁺⁺, 1 mM Ca⁺⁺ decreased adenyl cylase activity by about 50%. Activities of cortical adenyl cyclase stimulated by parathyroid hormone, thyrocalcitonin or ADH and of medullary adenyl cyclase stimulated by ADH were also reduced by about 50% in the presence of 1 mM Ca⁺⁺. The inhibition was independent of the ATP concentration, but was influenced by the Mg⁺⁺ content of the incubation medium.Adenyl cyclase activities of cortical and medullary membrane preparations were reduced by about 50% by 0.2 mM ethacrynic acid. The extent of this inhibition was essentially the same whether the enzymatic activity was determined in the absence or presence of stimulating hormones. The inhibitory action of ethacrynic acid was partially prevented by simultaneous addition of dithioerythritol (DTE). A derivative of ethacrynic acid, L 589420-0-2, also inhibited renal adenyl cyclase, but its action was not influenced by the addition of DTE. Adenyl cyclase from both parts of the kidney was inhibited by about 90% by 0.2 mM mersalyl. This action was almost completely prevented by the addition of 1 mM DTE. The pharmacological significance of adenyl cyclase inhibition by these diuretics is still uncertain since the role of Ado-35-P in the regulation of sodium transport is as yet unclear.Other diuretics, hydrochlorothiazide, furosemide, mefruside, amiloride, and the non-diuretic benzothiadiazine, diazoxide, had essentially no effect on cortical and medullary adenyl cyclase preparations when they were added in 0.1–0.5 mM concentration.The methylxanthines, theophylline and caffeine, which are known to inhibit nucleoside 35-monophosphate phosphodiesterase, reduced the rate of Ado-35-P formation. The unstimulated and the hormone-stimulated adenyl cyclases were inhibited to the same extent by theophylline. When adenyl cyclases was stimulated by fluoride, however, we found only a very small inhibition by theophylline. Inhibition of the medullary adenyl cyclase was greater than that of the enzyme prepared from renal cortex. At a concentration of 1 mM these methylxanthines significantly inhibited the medullary enzyme, but the inhibition became asymptotic at about 50% when concentrations up to 20 mM were used. Therefore, it is likely that inhibition by these substances varies in different cell types and tissues.Instead of phosphodiesterase inhibitors, unlabeled Ado-35-P can be used in the assay of adenyl cyclase activity to reduce the degradation of enzymatically formed labeled Ado-35-P. This addition, though, can also influence adenyl cyclase activity. In a medullary enzyme preparation 0.2 mM Ado-35-P reduced the adenyl cyclase activity by 13%, 5 mM Ado-35-P by 35%.

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Abkürzungen Ado-35-P Adenosin-35-monophosphat - Guo-35-P Guanosin-35-monophosphat - ADH antidiuretisches Hormon, Vasopressin - PTH Parathormon - TCT Thyreocalcitonin - DTE Dithioerythrit - EDTA Äthylendiamintetraessigsäure Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Über einen Teil der Ergebnisse wurde auf der 11. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft berichtet (Jakobs et al., 1970). Einige der vorliegenden Ergebnisse sind der Inauguraldissertation von K. H. J. (Medizinische Fakultät der Universität Heidelberg, 1971) entnommen.     

Percutaneous Absorption of 2′,3′-Dideoxyinosine in Rats

This study explored the topical route for administering of 2,3-dideoxyinosine (ddI), a nucleoside analog used for treating patients with acquired immunodeficiency syndrome. A dose of ddI (180 mg/kg) dispersed in ~1 g ointment base was applied, with or without occlusion, to the back of high follicular density (HFD) and low follicular density (LFD) rats. The systemic ddI clearance was determined using a concomitant administration of an intravenous tracer dose of [³H]ddI. At 24 hr, the experiment was terminated and skin sections at the application site were removed. After topical application, average plateau plasma levels of about 0.6 µg/ml were achieved within 1 to 2 hr and maintained for 24 hr. Occlusion gave a more uniform plasma profile but did not increase the bioavailability. The systemic bioavailability in HFD and LFD rats was about the same at 33%. In addition, a depot of about 16% of the dose was recovered by rinsing the application area and extracting the drug from the excised application site. These data indicate that about 50% of the dermal dose penetrated the skin barrier in 24 hr. The similar bioavailability in the HFD and LFD rats further suggests an unimportant role for the transfollicular absorption route for ddI. The effect of a mixture of penetration enhancers, Azone and propylene glycol (5:95), was studied in HFD rats. Coadministration of ddI with the enhancers did not increase the ddI bioavailability. However pre-treatment and coadministration with the enhancers significantly increased the bioavailability to 62%, which is a conservative estimate because the plasma drug level was still at a plateau when the experiment was terminated at 24 hr. In summary, the transdermal bioavailability of ddI exceeded the 15% oral bioavailability found in previous studies by more than 3 folds and was further increased by the pretreatment with absorption enhancers. These data indicate the topical route as an attractive administration route.