Morphine modulation of plasmodial-antigens-induced colony-stimulating factors production by macrophages

Morphine abuse is known to cause immunosuppression and enhanced host susceptibility to malaria. We studied the effect of morphine on the Plasmodium berghei total-parasite-antigens soluble in culture medium (P.b.SA)-induced production of colony-stimulating factors (CSFs) by mouse peritoneal macrophages, in vitro. Morphine exerted a concentration-dependent biphasic modulatory effect; at 1 x 10(-4)-1 x 10 x 10(-6) M it slightly inhibited, whereas at 1 X 10(-8)-1 x 10(-10) M it augmented the production of CSFs. However, at 1 x 10(-12) M concentration the augmenting effect of morphine was significantly (p<0.05) diminished. Selective agonists of delta- (DPDPE) and mu- (DAGO) opioid receptors also respectively, inhibited and augmented the production of CSFs. The CSFs appear to be synthesized de novo as cycloheximide (50.0 microg/ml) completely inhibited their production. Naloxone ( 1 x 10(-5) M) lacked any effect on the inhibitory effect of morphine; however, at 1 x 10(-3) M it exerted partial blocking effect. Conversely, at 1 x 10(-5) M naloxone significantly (p<0.05) blocked the augmenting effect of morphine. These results suggest that morphine via opioid receptors, in a concentration-dependent biphasic manner, modulated the P.b.SA-induced de novo production of CSFs by macrophages, in vitro.     

Leishmania donovani amastigote component-induced colony-stimulating factor production by macrophages: modulation by morphine

The neuroimmunomodulatory effects of opiates during microbial infections are now well known; however, not much is known during leishmaniasis. Here, we report the effects of morphine on purified approximately 12-kDa component of Leishmania donovani amastigote antigen (LDAA-12)-induced colony-stimulating factor (CSF) production by mouse peritoneal macrophages (PMs) in vitro. Low concentrations (1 x 10(-9) and 1 x 10(-11) M) of morphine significantly (P < 0.05) augmented the production of CSFs, whereas high concentrations (1 x 10(-3) and 1 x 10(-5) M) inhibited CSF production. Morphine exerted a similar concentration-dependent biphasic effect on the LDAA-12-induced elaboration of granulocyte (G)-macrophage (M)-CSF (GM-CSF) and M-CSF by PMs in their conditioned medium, as quantified by using enzyme-linked immunosorbent assay. Furthermore, selective agonists of mu-(DAGO) and delta-(DPDPE) opioid receptors also, respectively, augmented and inhibited the production of CSFs. Pretreatment of PMs with naloxone (1 x 10(-5) M) significantly (P < 0.05) blocked the augmenting effect of morphine. In contrast, at 1 x 10(-5) M, naloxone lacked any effect on the inhibitory effect of morphine; however, its 100-fold higher concentration partially blocked it. This study, apparently for the first time, demonstrates that morphine, via surface opioid receptors, biphasically modulates the LDAA-12-induced CSF production by PMs, in vitro. These results thus show the implications of opiate abuse on the outcome of therapeutic interventions in areas where both visceral leishmaniasis and drug abuse are rampant.     

Catecholamine effects on testicular testosterone production in the gonadally active and the gonadally regressed adult golden hamster

Several lines of evidence support a role of testicular innervation and peripheral catecholamines in the control of male gonadal function, particularly before puberty. It was therefore of interest to compare the effects of catecholamines on androgen production during the periods of gonadal activity and quiescence in a seasonally breeding species. We have examined direct effects of epinephrine (EPI), norepinephrine (NE), the beta-adrenergic agonist isoproterenol (ISO), and the alpha-adrenergic agonist phenylephrine (PHE) on testicular testosterone (T) production in hamsters with gonadal regression induced by 12 wk exposure to short photoperiod (SD) and in gonadally active hamsters maintained in long photoperiod (LD). Fragments of decapsulated testes were incubated with various combinations of these catecholamines (10(-5)-10(-9) M), human chorionic gonadotropin (hCG; 3.1 mIU/ml), the beta-receptor antagonist propranolol (10(-5) M) and the alpha-l-receptor antagonist prazosin (10(-5) M), for 6 h. In the incubations of testes from LD hamsters, the accumulation of T in the medium was stimulated by hCG but not affected by either catecholamine. However, EPI, NE, and PHE at 10(-5) M, but not ISO, augmented the stimulation of T by hCG. In sharp contrast to these findings, T production by the regressed testes of SD animals was stimulated by EPI (at 10(-8)-10(-5) M), NE (at 10(-6)-10(-5) M), and PHE (at 10(-6)-10(-5) M) in a dose-related manner, but unaffected by ISO. These stimulatory effects were prevented by prazosin, but not by propranolol. Moreover, 10(-5) M of EPI, NE, and PHE augmented the stimulatory effect of hCG on T production. We conclude that the seasonal transition from gonadal activity to quiescence in the adult golden hamster is accompanied by a major increase in the responsiveness of testicular steroidogenesis to catecholamines acting via the alpha-1-adrenoreceptor and that catecholamines can modulate Leydig cell response to gonadotropins in this species. These findings could be related to up-regulation of the alpha-1-receptor in the testis of the SD animal and suggest that catecholamines may be involved in the regulation of the testis during physiological suppression of gonadotropin release and during stress.     

Effect of serotonin (5-HT) and other monoamines on murine macrophages: modulation of interferon-gamma induced phagocytosis

We have previously shown that serotonin (5-HT) suppresses interferon-gamma (IFN-gamma)-induced Ia expression. In the present report, we show that 5-HT as well as other monoamines, histamine and dopamine, modulate IFN-gamma-induced phagocytosis in murine bone marrow macrophages. The effect of 5-HT on IFN-gamma-induced phagocytosis varied according to the concentration of IFN-gamma to which the macrophages were exposed. At low concentrations of IFN-gamma, 5-HT augmented phagocytosis, whereas at high concentrations of IFN-gamma, 5-HT suppressed phagocytosis. At both low and high IFN-gamma concentrations the response to 5-HT was dose-related and occurred at physiologic concentrations; the half-maximal effect was 6 X 10(-7) M and 3 X 10(-7) M for low and high IFN-gamma concentrations, respectively. Both histamine and dopamine also augmented IFN-gamma (1 U/ml) induced phagocytosis, at half-maximal augmenting concentrations of 7 X 10(-8) M and 4 X 10(-7) M, respectively. The 5-HT effects were blocked by the 5-HT antagonists spiperone, ketanserin, LY53857, mCPP, and PAPP, but not by the histamine antagonists pyrilamine, chlorpheniramine, or cimetidine. Histamine augmentation of IFN-gamma-induced phagocytosis was blocked by the H1 antagonists pyrilamine and chlorpheniramine, but not by the H2 antagonist cimetidine. The dopamine effect was blocked by spiperone and pyrilamine, both of which have been shown to block dopaminergic effects in other systems. This data provides functional evidence that at least part of the modulation of IFN-gamma-induced phagocytosis by 5-HT occurs through a 5-HT receptor-mediated mechanism, and 5-HT, dopamine, and histamine modulate IFN-gamma-induced phagocytosis independently through their respective receptors.     

Modulation of bovine adrenal gland secretion by etorphine and diprenorphine

Retrograde perfusion was used to investigate the effect of an opiate agonist and an opiate antagonist on the release of catecholamines and [Met5]-enkephalin immunoreactive material (ME-IRM) from bovine adrenal glands. Etorphine (5 X 10(-7) M) inhibited the spontaneous outflow of ME-IRM by approximately 10 percent but had no significant effect on the spontaneous catecholamine release. Acetylcholine (ACh, 5 X 10(-5) M) or 1,1-dimethyl-4-phenylpiperazinium (DMPP, 5 X 10(-5) M) stimulated release of ME-IRM and catecholamines was significantly decreased by the addition of etorphine. Diprenorphine (5 X 10(-7) M) had no significant effect on the spontaneous outflow of either ME-IRM or catecholamines. Diprenorphine reversed the inhibition of the DMPP-stimulated release caused by etorphine. After submaximal stimulation of the gland with DMPP (1 X 10(-5) M), a further stimulation of release of ME-IRM and catecholamines was observed after the addition of diprenorphine alone, i.e., in the absence of etorphine. These results provide further evidence supporting the contention that opiates modulate the secretion of catecholamines and ME-IRM from the adrenal gland.     

Segmental vascular responses to voltage-gated calcium channel potentiation in rat lung.

We determined lung vascular responses to voltage-gated Ca2+ channel potentiation with BAY K 8644 (BAY). We anesthetized 46 rats (Sprague-Dawley; halothane and pentobarbital) and then excised and perfused their lungs at constant blood flow of 25 +/- 2 (SE) ml.kg-1.min-1 at constant airway and left atrial pressures of 5 and 6 cmH2O, respectively. Pulmonary arterial pressure (Ppa) increased from 13.3 +/- 0.3 cmH2O at baseline to 17.3 +/- 1.3 cmH2O after BAY (2.8 x 10(-6) M; n = 5; P less than 0.01). As determined by micropuncture, arteriolar and venular (Pven) pressures did not change. Increase of perfusate Ca2+ (16 x 10(-3) M; n = 8) similarly increased Ppa. NG-mono-methyl-L-arginine (2 x 10(-4) M), an inhibitor of endothelium-derived relaxing factor, augmented the pressor effect of BAY when given after (n = 4) but not before (n = 4) BAY (P less than 0.01). Prior cyclooxygenase blockade with indomethacin (5 mg/kg; n = 5) attenuated the Ppa response to BAY (P less than 0.01). None of these agents changed Pven. To confirm vasoactivity in veins, we induced smooth muscle depolarization with KCl (20 x 10(-3) M; n = 6) and receptor-mediated responses with histamine (3 x 10(-4) M; n = 7). Both of these agents increased Pven markedly (P less than 0.01). We interpret that, in rat lung, BAY causes arterial but not venous constriction, because the venous segment differs from the arterial with regard to Ca2+ channel potentiation.     

Neuroendocrine integrative mechanisms in mammalian pineal gland: effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.     

The effects of furosemide on adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate and corticosterone production stimulated by adrenocorticotropin in monolayer cultured rat adrenal cells

Furosemide has been reported to have a suppressive effect on ADH-, PTH- and adrenaline-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) production, but the effect on adrenocorticotropin (ACTH) action has not yet been elucidated. In the present study, therefore, the effects of furosemide on cAMP and also on guanosine 3':5'-cyclic monophosphate (cGMP) and corticosterone, stimulated by ACTH in monolayer cultured rat adrenal cells, were investigated. The intra- and extracellular cAMP stimulated by ACTH was dose-dependently suppressed by furosemide within the concentration range of 10(-3) M to 3 X 10(-3) M, and the suppressive effect of the drug was accompanied with decreased corticosterone production. However, non-stimulated basal corticosterone production was not influenced by the drug even at 3 X 10(-3) M. A similar suppressive effect of dibutyryl cAMP-stimulated corticosterone production by 3 X 10(-3) M furosemide was observed. The intracellular cAMP bound to its binding protein in sonicated adrenal cell extract was also suppressed in a very similar dose-dependent manner to total cAMP. However, though the effect on corticosterone production was also observed when the calcium concentration in the loading medium was changed, the magnitude of the effectiveness (percent of control) was relatively constant at each calcium concentration, suggesting that furosemide may not affect the site(s) at which calcium acts. Intracellular cGMP, on the other hand, was increased by 10(-3) M to 3 X 10(-3) M of furosemide, suggesting an intensifying effect of furosemide on guanylate cyclase activity. Dibutyryl cGMP-stimulated corticosterone production was also increased at the same concentration range. These results indicated that furosemide may act not only on adenylate cyclase but also on the additional step(s) to suppress the resultant corticosterone production. In contrast to the effects of furosemide on such cAMP-mediated processes, this drug treatment appeared to enhance cGMP-mediated corticosterone production.     

Presynaptic alpha-adrenoceptor mediated inhibition of the neurogenic cholinergic contraction in the isolated intestinal bulb of the carp (Cyprinus carpio)

The effects of norepinephrine, epinephrine and clonidine on neurogenic cholinergic contraction were examined in the presence of a beta-adrenoceptor blocking agent, carteolol (5 X 10(-6) M), in the isolated intestinal bulb of the carp. Norepinephrine, epinephrine (10(-9)-10(-6) M) and clonidine (10(-8)-10(-5) M) inhibited the contraction induced by low frequency (2 or 5 Hz) transmural stimulation (TMS) without inhibiting the contraction induced by acetylcholine (ACh, 6 X 10(-8)-4 X 10(-7) M). Methoxamine (10(-4) M) and phenylephrine (10(-4) M) showed no such inhibitory effect on the TMS-induced contraction. The inhibitory effects of catecholamines and clonidine were decreased by phentolamine (5.4 X 10(-6) M) and yohimbine (10(-7)-10(-6) M) but not by prazosin (7 X 10(-7)-10(-6) M). Nicotine (10(-6)-10(-4) M) and serotonin (3 X 10(-8)-3 X 10(-6) M) caused contraction of the intestinal bulb indirectly by releasing endogenous ACh. This contraction was inhibited by norepinephrine, epinephrine and clonidine in a concentration-dependent manner. The present results suggest that catecholamines and clonidine inhibit cholinergic transmission via the activation of a presynaptic alpha-adrenoceptor (presumably of alpha-2 type) located on the cholinergic nerve terminals innervating the smooth muscle of the intestinal bulb of the carp.     

Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage

The beta-adrenergic receptor was characterized on BCG-activated rat peritoneal macrophage membranes by radio-ligand binding studies. Saturable binding with [125I]iodocyanopindolol (125I-ICYP) was demonstrated. With Scatchard analysis, rat macrophages demonstrate approximately 1000 receptors per cell with a Kd of 5 X 10(-11) M for 125I-ICYP. Competition curves with (-) and (+) propranolol at concentrations below 10(-6) M confirmed stereospecificity. The potency of various ligands to compete for 125I-ICYP binding sites followed the order: propranolol greater than isoproterenol greater than epinephrine greater than norepinephrine with apparent Kd of 2.0 X 10(-9), 3.9 X 10(-7), 1.0 X 10(-5), and 2.5 X 10(-5) M, respectively. Isoproterenol-stimulated adenylate cyclase activity was two-fold above basal activity. The potential physiologic significance of a beta-adrenergic receptor on rat peritoneal macrophages was suggested by a dose-dependent decrease in phagocytosis of soluble, model immune complexes (aggregated gamma-globulin) by macrophages incubated with metaproterenol. We conclude that the rat macrophage has a beta-adrenergic receptor and that catecholamines may thereby modulate macrophage function.     

Clotting of fibrinogen. 1. Scanning calorimetric study of the effect of calcium

The denaturation temperature Td and the enthalpy of thermal denaturation delta Hd of the D nodules of fibrinogen increase 12-13 degrees C and 40%, respectively, when fibrinogen is clotted by thrombin in the presence of 10(-3) M calcium ion. The rate of change of Td and delta Hd is first order in thrombin concentration. In the absence of calcium, little change in Td is observed, but the increase in delta Hd still occurs. The shift in Td as a function of logarithm of calcium concentration is sigmoid, with a half-point at 2.5 X 10(-5) M calcium for human and 6.0 X 10(-5) M calcium for bovine fibrinogens, suggesting that the shift is due to binding of calcium at the high-affinity binding sites of fibrin. The Td of the D nodule of native fibrinogen also increases, but not as much, on addition of calcium. This increase in Td is also sigmoid with log calcium, with a half-point of 1.6 X 10(-3) M calcium for human and 3.2 X 10(-3) M calcium for bovine fibrinogens, and appears to be due to binding of calcium to the low-affinity binding sites of fibrinogen. At calcium concentrations greater than 10(-4) M, traces of factor XIII in the bovine fibrinogen preparation become activated and cause cross-linking of the fibrin gel. But the changes in Td and delta Hd still occur when factor XIIIa is inactivated by iodoacetamide, and the rate of the changes is not altered by addition of large amounts of factor XIIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Sympathetic control of the forearm blood flow in man during brief isometric contractions

Experiments were performed to assess the possible neurally mediated constriction in active skeletal muscle during isometric hand-grip contractions. Forearm blood flow was measured by venous occlusion plethysmography on 5 volunteers who exerted a series of repeated contractions of 4 s duration every 12 s at 60% of their maximum strength of fatigue. The blood flows increased initially, but then remained constant at 20-24 ml X min(-1) X 100 ml(-1) throughout the exercise even though mean arterial blood pressure reached 21-23 kPa (160-170 mm Hg). When the same exercise was performed after arterial infusion of phentolamine, forearm blood flow increased steadily to near maximal levels of 38.7 +/- 1.4 ml X min(-1) X 100 ml(-1). Venous catecholamines, principally norepinephrine, increased throughout exercise, reaching peak values of 983 +/- 258 pg X ml(-1) at fatigue. Of the vasoactive substances measured, the concentration of K+ and osmolarity in venous plasma also increased initially and reached a steady-state during the exercise but ATP increased steadily throughout the exercise. These data indicate a continually increasing alpha-adrenergic constriction to the vascular beds in active muscles in the human forearm during isometric exercise, that is only partially counteracted by vasoactive metabolites.     

Effect of atrial natriuretic peptide on renin release in rat isolated glomeruli

Effects of atrial natriuretic peptide (ANP) on renin release in isolated rat glomeruli were investigated. ANP suppressed renin release by 25% at 5 x 10(-8) M when glomeruli were incubated in a medium containing 1.26 mM calcium (p = 0.0019). When glomeruli were incubated in a calcium free medium containing 2 mM EGTA, ANP suppressed stimulated renin release significantly at 5 x 10(-8) and 5 x 10(-9) M by 25% (p = 0.0204, and p = 0.0101, respectively). These results indicate that ANP suppresses renin release in a dose dependent manner, probably through a calcium independent process.     

Inhibition of dihydropteridine reductase by catecholamines and related compounds

Catecholamines and related compounds, such as dopamine, 5- or 6-hydroxydopamine, N-methyldopamine, tyramine, octopamine, norepinephrine and epinephrine, inhibit human liver dihydropteridine reductase (NADH:6,7-dihydropteridine oxidoreductase, EC 1.6.99.10) noncompetitively with Ki values ranging from 7.0 X 10(-6) - 1.9 X 10(-4)M (I50 values = 2.0 X 10(-5) - 2.0 X 10(-4)M). The tyrosine analogs alpha-methyltyrosine and 3-iodotyrosine are weak inhibitors of this enzyme (I50 greater than 10(-3)M). The inhibitory effect of catecholamines is slightly decreased by O-methylation of one hydroxyl group, but is essentially abolished by total methylation. The inhibitory strength of the catecholamines and related compounds tested against this enzyme can be arranged in the following order: dopamine, 6-hydroxydopamine, 5-hydroxydopamine, N-methyldopamine greater than tyramine, 3-O-methyldopamine, 4-O-methyldopamine much greater than epinephrine, 3-O-methylepinephrine, norepinephrine, octopamine less than tyrosine much less than alpha-methyltyrosine, 3-iodotyrosine much less than homoveratrylamine. These results suggest that dopamine, norepinephrine and epinephrine may serve as physiological regulators of mammalian dihydropteridine reductase.     

Calcium modulation of inositol 1,4,5-trisphosphate-induced calcium release from neuroblastoma x glioma hybrid (NG108-15) microsomes

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.     

Lipoxygenase-induced lipid peroxidation of isolated cardiac microsomes modulates their calcium-transporting function

We demonstrated previously that products of linoleic and arachidonic acids, arising from enzymatic or non-enzymatic oxidation, inhibit ATP-dependent calcium accumulation into and promote release of calcium from vesicles derived from sarcoplasmic reticulum of guinea-pig heart. In the present study, direct enzymatic peroxidation of cardiac membrane lipids was performed and the effect on calcium transport was examined. Vesicles were preincubated at 37 degrees C with soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) for up to 1 h prior to the initiation of calcium accumulation. The extent of membrane peroxidation was assessed by monitoring the production of malondialdehyde. Pretreatment of vesicles with lipoxygenase for 40 and 60 min markedly depressed calcium accumulation. The lipoxygenase-induced suppression of calcium transport was completely antagonized by nordihydroguaiaretic acid (1 microM), not at all by indomethacin (1 microM), and only partially by 5,8,11,14-eicosatetraynoic acid (0.3 microM). Low concentrations of calcium (10(-5)-5 X 10(-5) M) enhanced, and a high concentration (10(-3) M) inhibited lipoxygenase-induced peroxidation of membrane lipids. The calcium-accumulating ability of the vesicles was inversely related to the extent of membrane peroxidation. The vesicles which showed the highest degree of peroxidation in the presence of 5 X 10(-5) M calcium, accumulated the lowest amount of calcium. In contrast, calcium at 10(-3) M suppressed lipid peroxidation, resulting in higher calcium uptake than in vesicles peroxidized in the absence of calcium. Thus, calcium transport is depressed in microsomes undergoing lipoxygenase-induced peroxidation, a process which in turn is modulated by calcium.     

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes

The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 . 10(-3)-1 . 10(-4) M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 . 10(-4) M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 . 10(-4)-1 . 10(-5) M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 . 10(-6) M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.     

Naturally occurring opioid peptides modulate H+-production by isolated rat parietal cells

The rationale for the present study was to determine the effects of naturally occurring opioid peptides on H+-production by isolated rat parietal cells as indirectly measured by [14C]-aminopyrine uptake. In crude preparations (18 to 25% parietal cells) and in enriched (80 to 90%) parietal cell fractions stimulation by submaximal histamine- or dibutyryl cAMP-concentrations (10(-6)-10(-4) mol/l) was augmented by 20-30% in the presence of methionine-enkephalin (Met-Enk) and Met-Enk Arg6Phe7 (10(-7) to 10(-5) mol/l). This augmentation was blocked by the opiate receptor antagonist (-)naloxone (10(-6) mol/l) suggesting specificity of the action of Met-Enk and Met-Enk Arg6Phe7. At 10(-6) mol/l (-)naloxone did not exert nonspecific toxic effects. Yet, even in the absence of exogenous opioids, histamine-induced H+-production was inhibited by 3 X 10(-5) or 10(-4) mol/l (-)naloxone. Since similar inhibition occurred with (+)naloxone, an inactive stereoisomer which does not interact with opiate receptors, effects of (-)naloxone at concentrations above 10(-5) mol/l must be considered nonspecific. We conclude that Met-Enk and Met-Enk Arg6Phe7 have no effect on basal, but augment stimulated H+-production by a direct effect on the parietal cells. At nontoxic concentrations (-)naloxone antagonizes this augmentation indicating that it is mediated by specific opiate receptors on the parietal cells.     

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+-uptake by cultured vascular smooth muscle cells derived from rat aorta

We have recently shown the presence of receptors for 1,25-dihydroxyvitamin D3 and that 1,25-dihydroxyvitamin D3 stimulates Ca-ATPase in vascular smooth muscle cells presumably via receptor mediated mechanism. These data suggest that the sterol may directly be involved in the regulation of cellular calcium homeostasis. To further define action of vitamin D in smooth muscle cells, we studied effect of the sterol on cellular uptake of calcium. 1,25-dihydroxyvitamin D3 stimulated 45Ca2+ uptake by cultured cells, A7r5, derived from fetal rat aorta, when the cells were incubated with the sterol for 18 hr. The effect was dose-dependent at 10(-10) to 10(-9) M, and three orders of magnitude higher concentration of 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 was needed to obtain similar effects. Furthermore, the effect of 1,25-dihydroxyvitamin D3 was abolished by cycloheximide (10(-5) M), a protein synthesis inhibitor. These data clearly suggest that 1,25-dihydroxyvitamin D3 may directly regulate cellular calcium homeostasis in vascular smooth muscle cells presumably via receptor mediated mechanism.