Studies on the activity of phorbol myrystate acetate on the human polymorphonuclear leukocytes.

Phorbol myrystate acetate (PMA) activates nitroblue tetrazolium reduction in human polymorphs. The activation is inhibited by dibutyryl cyclic AMP, theophylline and phenylbutazone, but is not influenced by hydrocortisone in vitro, nor is it inhibited by leukocytes from patients treated with prednisone. Peptide analogues of Tuftsin also had no effect on this stimulatory activity. We conclude that the action of PMA on the nitroblue tetrazolium reduction is mediated through cyclic nucleotides.     

Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin

Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999     

Phosphorylation of cockroach antennal polypeptides: effects of second messengers and pheromonal blend

In insect antennal extracts, Schleicher et al.¹ showed that protein kinase C (PKC) inhibitors abolish the transience of pheromone-induced rapid inositol trisphosphate responses, which suggests that pheromonal signals act on phosphorylation of specific proteins. To confirm this hypothesis, we studied the effects of second messengers and a pheromonal blend on phosphorylation of antennal proteins in the cockroachPeriplaneta americana. Proteins from adult male antennae were phosphorylated in vitro in the presence of [³²P] triphosphate, then separated by SDS-polyacrylamide gel electrophoresis. Numerous phosphopolypeptides were visualized. The presence of Ca⁺⁺/calmodulin in the incubation medium resulted in increased phosphorylation of polypeptides with molecular weights of 38, 48, 51, 54 and 58 kDa. Stimulation of PKC by addition of Ca⁺⁺ phosphatidylserine (PS)/phorbol myristate acetate (PMA) resulted in the appearance of three phosphopolypeptides of 36, 70 and 120 kDa. In the presence of cyclic adenosine monophosphate, two new major polypeptides of 46 and 42 kDa appeared; the latter polypeptide also appeared in the presence of cyclic guanosine monophosphate. Comparison with polypeptide composition of tissue from the cerci, leg, brain and fat body showed that the 36 and 48 kDa polypeptides were specific to antennae, whereas the 120 kDa polypeptide was also present in the adult brain. When antennae are subjected to pheromonal stimulation for 16 seconds prior to homogenization, in vitro phosphorylation of the 120, 70, 64 and 38 kDa polypeptides was inhibited, whereas phosphorylation of the 58, 54, 51 and 48 kDa polypeptides was strongly stimulated. It is noteworthy that a 107 kDa polypeptide was observed only after pheromonal stimulation by Ca⁺⁺/PS/PMA. Our findings suggest that Ca⁺⁺-and PKC-dependent protein phosphorylation systems play an important role in the transduction of pheromonal signals in antennae of male cockroachP. americana. We speculate that specific phosphoproteins may modulate sensitivity and signal amplification during the olfactory transduction process.     

Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin) on cyclic nucleotide phosphodiesterases in human platelets

Summary The effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin), an inhibitor of platelet aggregation, on cyclic nucleotide metabolism was investigated. AD6 inhibited selectively human platelet cyclic GMP phosphodiesterase, which was separated from cyclic AMP phosphodiesterase by DEAE-cellulose chromatography. Addition of AD6 to washed platelets increased cyclic GMP levels significantly in two minutes. These results could be useful in elucidating the action of AD6 on intact platelets.     

Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylme tho xy coumarin) on cyclic nucleotide phosphodiesterases in human platelets

The effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmeth oxy coumarin), an inhibitor of platelet aggregation, on cyclic nucleotide metabolism was investigated. AD6 inhibited selectively human platelet cyclic GMP phosphodiesterase, which was separated from cyclic AMP phosphodiesterase by DEAE-cellulose chromatography. Addition of AD6 to washed platelets increased cyclic GMP levels significantly in two minutes. These results could be useful in elucidating the action of AD6 on intact platelets.     

Inhibition of prostaglandin-induced cyclic AMP accumulation in the rat anterior pituitary by alrestatin.

Alrestatin at 25-1 X 10(-4) M inhibited the accumulation of cyclic AMP induced by prostaglandin E2, but not theophylline, in the rat anterior pituitary in vitro. Somatostatin, at lower concentrations, inhibited both; maximal inhibition of the prostaglandin effect was greater with alrestatin. As cyclic AMP is considered to be a mediator in induced-hormonal release, it appears from the present findings that alrestatin may be of potential use in altering hormonal release.     

Regulation of calcitonin release from the 6.23 rat C-cell line by cyclic nucleotide analogues and pharmacological mediators.

Calcitonin release from 6.23 rat medullary thyroid carcinoma C-cells was stimulated by dibutyryl cyclic AMP and inhibited by dibutyryl cyclic GMP in concentration dependent fashion. Histamine, isoproterenol, prostaglandin E2 and Bay K 8644 stimulated calcitonin release, while acetylcholine and serotonin had no significant effect on CT release.     

Regulation of calcitonin release from the 6.23 rat C-cell line by cyclic nucleotide analogues and pharmacological mediators

Calcitonin release from 6.23 rat medullary thyroid carcinoma C-cells was stimulated by dibutyryl cyclic AMP and inhibited by dibutyryl cyclic GMP in concentration dependent fashion. Histamine, isoproterenol, prostaglandin E₂ and Bay K 8644 stimulated calcitonin release, while acetylcholine and serotonin had no significant effect on CT release.     

Adenosine promoted accumulation of adenosine 3',5'-monophosphate in rabbit vagus nerve.

Desheathed rabbit vagus nerve has been found to form cyclic AMP when incubated with adenosine. This accumulation of cyclic AMP is inhibited by theophylline but not by antiadrenergic agents, anticholinergic agents or local anaesthetics. Depolarizing media are not able to promote cyclic AMP accumulation in this preparation.     

Effect of sodium butyrate in combination with X-irradiation, chemotherapeutic and cyclic AMP stimulating agents on neuroblastoma cells in culture.

Sodium butyrate, X-irradiation, chemotherapeutic agents and cyclic AMP-stimulating agents cuased reduction in the cell number (due to cell death and reduction in cell division) when added individually to mouse neuroblastoma cells in culture. However, the combination of sodium butyrate with X-irradiation, chemotherapeutic and cyclic AMP-stimulating agents produced a greater reduction in the cell number than that produced by the individual agents.     

Effect of sodium butyrate in combination with X0irradiation,chemotherapeutic and cyclic AMP stimulating agents on neuroblastoma cells in culture

Summary Sodium butyrate, X-irradiation, chemotherapeutic agents and cyclic AMP-stimulating agents caused reduction in the cell number (due to cell death and reduction in cell division) when added individually to mouse neuroblastoma cells in culture. However, the combination of sodium butyrate with X-irradiation, chemotherapeutic and cyclic AMP-stimulating agents produced a greater reduction in the cell number than that produced by the individual agents.     

Inhibition of prostaglandin-induced cyclic AMP accumulation in the rat anterior pituitary by alrestatin

Summary Alrestatin at 25–1×10^–4 M inhibited the accumulation of cyclic AMP induced by prostaglandin E₂, but not theophylline, in the rat anterior pituitary in vitro. Somatostatin, at lower concentrations, inhibited both; maximal inhibition of the prostaglandin effect was greater with alrestatin. As cyclic AMP is considered to be a mediator in induced-hormonal release, it appears from the present findings that alrestatin may be of potential use in altering hormonal release.The author acknowledges the technical assistance of Miss C. Pilapil.     

Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

To investigate the roles of adenosine A1 and A2 receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N6-cyclohexyl adenosine; selective adenosine A1 receptor agonist and 5'-N-ethylcarboxamine adenosine; selective adenosine A2 receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5'-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10(-3) M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N6-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10(-4) M and 10(-3) M; furthermore, 10(-3) M N6-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A1 receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.     

The behavior of alloxan in the Hill reaction

Summary Hill activity induced by alloxan is characterized by non-stoichiometric oxygen evolution and cyclic electron flow due to autoxidation of dialuric acid and alloxantin, the reduction products of alloxan.     

Effects of several preoperative medications on fat cell lipolysis, and activity of adipose tissue cyclic AMP phosphodiesterase.

Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a 'clinical dose', omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 microgram ml-1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compatible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.     

Beta-adrenergic-stimulated adenylate cyclase activity in normal and EBV-transformed lymphocytes

Beta-adrenergic-associated cyclic AMP accumulation was studied in intact lymphocytes before and after transformation with Epstein-Barr virus into immortal cell lines. Although a marked reduction in isoproterenol-stimulated cyclic AMP synthesis was observed in transformed cells, forskolin-stimulated cyclic AMP accumulation was preserved. A parallel loss of 125-iodocyanopindolol binding sites suggests that the reduction in beta-adrenergic-stimulated AMP synthesis is due to receptor down-regulation.     

Beta-adrenergic-stimulated adenylate cyclase activity in normal and EBV-transformed lymphocytes

Summary Beta-adrenergic-associated cyclic AMP accumulation was studied in intact lymphocytes before and after transformation with Epstein-Barr virus into immortal cell lines. Although a marked reduction in isoproterenol-stimulated cyclic AMP synthesis was observed in transformed cells, forskolin-stimulated cyclic AMP accumulation was preserved. A parallel loss of¹²⁵-iodocyanopindolol binding sites suggests that the reduction in beta-adrenergic-stimulated AMP synthesis is due to receptor down regulation.     

Effect of sodium butyrate in combination with prostaglandin E1 and inhibitors of cyclic nucleotide phosphodiesterase on human amelanotic melanoma cells in culture

Summary Sodium butyrate and cyclic AMP-stimulating agents (prostaglandin E₁, papaverine, theophylline, and RO20-1724) caused reductions in the cell number (primarily due to reduction in cell division) when added individually to human melanoma cells in culture. However, the combination of sodium butyrate with one of the cyclic AMP-stimulating agents produced a marked reduction in cell number (primarily due to cell death).Supported in part by BRSG grant RR-05357 awarded by Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health. We thank Marianne Gaschler for her technical help.     

Ethanol influence on insulin secretion from isolated rat islets

This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.     

Effect of sodium butyrate in combination with prostaglandin E1 and inhibitors of cyclic nucleotide phosphodiesterase on human amelanotic melanoma cells in culture.

Sodium butyrate and cyclic AMP-stimulating agents (prostaglandin E1, papaverine, theophylline, and RO20-1724) caused reductions in the cell number (primarily due to reduction in cell division) when added individually to human melanoma cells in culture. However, the combination of sodium butyrate with one of the cyclic AMP-stimulating agents produced a marked reduction in cell number (primarily due to cell death).