Effect of different classes of antibiotics on amniotic prostaglandin E release.

Our purpose was to investigate the effects of different classes of antibiotics, namely beta-lactamines, aminoglicosides, tetracyclines, macrolides, on amniotic prostaglandin E release to clarify their role in the treatment of premature labor. The effects of these antibiotics were tested also in combination with ampicillin, whose antiprostaglandinergic action had been demonstrated previously. Ceftriaxone and gentamicin significantly and reversibly inhibit both basal and arachidonic acid- or oxytocin-stimulated prostaglandin E release from amnion, although to a different extent. On the contrary, tetracycline and erythromycin do not influence prostaglandin E output. The inhibitory effect of ampicillin is potentiated, in an additive manner, by ceftriaxone, reduced by gentamycin, and eliminated by tetracycline and erythromycin. The finding that diverse classes of antibiotics and their combinations affect amniotic prostaglandin E release should be taken into account in the management of premature labor.

Effect of cyclic AMP level reduction on human neutrophil responses to formylated peptides.

The increase in human neutrophil cyclic adenosine monophosphate (cAMP) levels evoked by formylated peptides is significantly reduced in the presence of MDL 12330A, SQ 22536, GDPssS and clonidine, which inhibit the adenylyl cyclase system by acting at different sites in this enzyme complex. A similar effect is exerted by adenosine deaminase and dipyridamole, which alter the extracellular adenosine concentration. Neutrophil preincubation with adenylyl cyclase inhibitors or dipyridamole reduces chemotaxis and superoxide anion production triggered by peptides; adenosine deaminase, on the contrary, has no effect on neutrophil responses. Our results seem to indicate that: (1) the peptide-induced increase in neutrophil cAMP is due mainly to an action on the adenylyl cyclase system; (2) an enhancement of this cyclic nucleotide, even slight and necessarily transient, is required for chemotaxis and O2 production induced in neutrophils by formylated peptides; and (3) cAMP does not represent the crucial second messenger for adenosine in the modulation of neutrophil responses.

MDL 12330A inhibits the non-neuronal adenylyl cyclase from the freshwater snail Planorbarius corneus, but the neuronal enzyme is activated by this compound.

N-(Cis-2-phenyl-cyclopentyl)azacyclotridecan-2-imine-hydrochloride (MDL 12330A), considered an inhibitor of adenylyl cyclase, has been tested on the enzyme activity of neuronal and non-neuronal tissues from the freshwater snail Planorbarius corneus. The drug dose-dependently activates the basal as well as agonist-stimulated adenylyl cyclase in the ganglionic preparations, while it exerts an inhibitory effect on the enzyme present in the non-nervous tissues examined. SQ 22536 and forskolin, respectively an inhibitor and activator of adenylyl cyclase, behave as generally reported both in central and peripheral tissues of the snail. This is, to our knowledge, the first report of a stimulatory action of MDL 12330A on an adenylyl cyclase system.

Effect of serotonin and neuropeptides on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus.

The effect of serotonin, FMRFamide and the small cardioactive peptide B (SCPB) on adenylate cyclase activity of the central nervous system and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. The amine and the cardioactive peptide stimulated the enzyme, although with different potencies, in all tissues studied and, when tested in combination, an additive activation was obtained. FMRFamide induced differential effects in the various targets: marked stimulation of adenylate cyclase, additive to that provoked by serotonin or SCPB, in salivary glands; inhibition of the enzyme, both alone and in combination with the other neuromediators, in the nervous tissue; whereas no influence was found in adenylate cyclase activity in the buccal mass. In the last of these tissues, the peptide might act through an intracellular second messenger other than cyclic AMP. The responsiveness of adenylate cyclase to these neuromediators in all the central ganglia suggested that they can exert an important role as neurotransmitters and/or neuromodulators in the central nervous system of the snail. Moreover, in the light of the differential sensitivity of adenylate cyclase in the salivary glands and buccal mass, we suggest that serotonin, FMRFamide and SCPB modulate the feeding behaviour of P. corneus in a complex way.

Adenylate cyclase activity from Hirudo medicinalis segmental ganglia: modulation by calcium and calmodulin.

An adenylate cyclase activity stimulated by serotonin and calmodulin is present in the segmental ganglia of the leech Hirudo medicinalis. Removal of the endogenous calcium binding protein does not alter the responsiveness of the enzyme to serotonin. The calmodulin antagonist, trifluoperazine, drastically reduces the amine stimulatory effect on both intact and calmodulin-depleted membranes. We suggest that calmodulin-sensitive and serotonin-stimulated adenylate cyclase are, at least functionally, independent.

The controversial role of cAMP on amnionic prostaglandin release: effect of adenylate cyclase inhibition.

The suggested role of cAMP in the regulation of amnionic prostaglandin release was investigated using two adenylate cyclase inhibitors, MDL 12330A and SQ 22536. These substances exhibited a dose-dependent inhibitory effect on both amnionic enzyme and cAMP levels, but they did not influence prostaglandin E (PGE) release. In addition forskolin and IBMX (3-isobutyl-1-methylxanthine), two drugs known to increase cAMP levels, did not affect PGE output, while dibutyryl cyclic cAMP showed a dose-dependent inhibitory effect. On the basis of our data, the suggested role of amnionic adenylate cyclase in triggering prostaglandin release is not confirmed, and the pathway of phospholipase A2 activation at the onset of labor remains to be elucidated.

Adenylate cyclase activity from Hirudo medicinalis segmental ganglia: modulation by temperature and Mg2+ ions.

The basal, serotonin- and fluoride-stimulated activities of homogenate preparations from Hirudo medicinalis segmental ganglia increase with incubation temperature, reaching a maximum at 35 degrees C. Both basal and fluoride-stimulated activities yield linear Arrhenius plots, whereas serotonin-enhanced activity shows a break at 25 degrees C. Increasing Mg2+ concentrations stimulate the adenylate cyclase both in the absence and in the presence of serotonin. The affinity of the enzymatic system for the cation is unaffected by the amine.

Two new formylated peptides able to activate chemotaxis and respiratory burst selectively as tools for studying human neutrophil responses.

Two new For-Met-Leu-Phe-OH (FMLP) methyl ester analogues, For-Thp-Leu-Ain-OMe [Thp1, Ain3] and For-Met-delta zLeu-Phe-OMe [delta zLeu2], able to activate selectively chemotaxis and superoxide anion (O2-) release, respectively modulate intracellular cyclic AMP (cAMP) levels in different ways. FMLP and [delta zLeu2] enhance human neutrophil cAMP levels per se, and this effect is potentiated by Ro 201724, a non-xanthinic phosphodiesterase (PDE) inhibitor, whereas it is counteracted by 3-isobutyl-1-methyl-xanthine (IBMX), a blocker of both phosphodiesterase and adenosine receptors. In contrast, [Thp1, Ain3] is ineffective. However, no formylated peptides influence cAMP phosphodiesterase activity. Neutrophil preincubation with Ro 201724 or IBMX drastically reduces chemotaxis and superoxide anion (O2-) production triggered by peptides. Our results suggest that: (1) peptide-induced cAMP increase is probably indirect, and due mainly to the action on adenosine-sensitive adenylate cyclase; (2) formylated peptide, endowed solely with chemotactic activity is unable to increase neutrophil cAMP concentration; (3) cAMP elevation may represent a feed-back mechanism to inhibit the physiological responses induced by formylated peptides.

Epidermal growth factor stimulation of lecithin release by human amnion.

Lecithin release from human amnion disks was assayed in basal conditions as well as under epidermal growth factor administration. The tissue responded to the peptide by increasing the phospholipid release. A significant effect was observed only after 30 min of treatment. A possible role of epidermal growth factor-induced amniotic lecithin release in fetal lung maturation as well as in the mechanism of delivery is discussed.

Modulation of amnionic adenylate cyclase and cAMP phosphodiesterase by prostaglandins E1 and F2 alpha.

The action of prostaglandins E1 and F2 alpha on adenylate cyclase and cAMP phosphodiesterase was assayed in cell membrane fractions of amnion obtained before and after labor from term physiological pregnancies. Both prostaglandins stimulate the activity of adenylate cyclase in a dose-dependent manner. After spontaneous delivery both prostaglandins stimulate the activity of phosphodiesterase at lower doses and inhibit it at higher ones while the stimulatory effect of low doses is missing before labor. Such a behavior could represent a mechanism for the preservation of the cAMP-mediated processes throughout the entire event of delivery.

Effects of the small cardioactive peptide B (SCPB) on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus.

The effect of the small cardioactive peptide B (SCPB) on adenylate cyclase activity of the central nervous system (CNS) and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. This peptide stimulates enzyme activity in a dose-dependent manner in all the tested ganglia, in the salivary glands, the buccal mass and the oesophagus. The amount required for half-maximal stimulation is approx 0.1 microM. Moreover, the response of adenylate cyclase to SCPB progressively increases with time, without desensitizing. On the other hand, the peptide is ineffective on heart adenylate cyclase. The responsiveness of adenylate cyclase activity to the peptide and the wide distribution of SCPB-related immunoreactivity in all the tested ganglia suggest that this substance may exert an important role as neurotransmitter as well as neuromodulator in the CNS of the snail. Since SCPB is consistently present in the buccal and cerebral ganglia, associated nerves, salivary glands, buccal mass and oesophagus, and in the light of the appreciable effects exerted by SCPB on adenylate cyclase activity in the examined peripheral tissues, we suggest that the peptide is strongly involved in the control of feeding behaviour of P. corneus.

Adenylate cyclase from Hirudo medicinalis segmental ganglia: modulation by physiological and non-physiological agents.

1. In Hirudo medicinalis segmental ganglia GTP is essential for the full expression of the stimulatory action of serotonin on the adenylate cyclase activity. The amine, in turn, increases the overall affinity of the enzymatic system for GTP. 2. GTP gamma S and Gpp(NH)p, non-hydrolysable analogues of GTP, dose-dependently enhance the basal enzyme activity, but impair the stimulatory effect of serotonin. 3. Fluoride ions biphasically modulate the leech adenylate cyclase both in the absence and in the presence of GTP. The ion effect is also influenced by non-physiological guanine nucleotides.

Betamethasone-induced lecithin release in vitro from the fetal membranes.

Lecithin release from fetal membranes and placental tissue was assayed in basal conditions as well as under betamethasone administration. In the absence of the steroid, amnion and chorion released lecithin as a function of incubation time. Both tissues responded to betamethasone by increasing the lecithin release, the amnion exhibited a higher basal level and a greater responsiveness to the hormone than the chorion. Basal lecithin release from placental tissue was unaffected by any steroid concentration.

Receptor-mediated supra-additive activation of guinea pig superior cervical ganglion adenylate cyclase: role of Mn2+ ions and calmodulin.

The effects of Mn2+ and calmodulin were studied on the basal and agonist-modulated adenylate cyclase activity of the guinea pig superior cervical ganglion. The divalent cation strongly stimulates the basal and agonist-modulated enzyme in a concentration-dependent manner. Moreover, in the presence of Mn2+ the inhibitory effects of "high" GTP concentrations and of D-Ala2-Met-enkephalinamide on adenylate cyclase are eliminated, while the stimulation exerted by prostaglandin E2 and the supra-additive activation of the enzyme by the combination of the two drugs are unaffected. In EGTA-washed, calmodulin-depleted membrane preparations, Mn2+ still activates the cyclase but the enkephalin inhibition and the superactivation of the enzyme induced by the combination of opiate and prostaglandin are lost, both in the absence and in the presence of the cation. Reconstituting the depleted membranes with exogenous Ca2+/calmodulin fully restored the enzyme responsivity to the combination and, partially, to the enkephalin. The findings suggest the existence in the guinea pig superior cervical ganglion of both the calmodulin-sensitive and differently regulated calmodulin-insensitive adenylate cyclase.

Supra-additive activation of guinea-pig superior cervical ganglion adenylate cyclase by PGE2 and D-Ala2-Met-enkephalinamide: role of GTP.

The effects of guanine nucleotides were tested on basal and agonist-modulated adenylate cyclase in guinea-pig superior cervical ganglion crude membrane preparations. GTP gamma S and Gpp(NH)p dose-dependently stimulate, while GDP beta S inhibits, both the basal and the prostaglandin E2-stimulated enzyme activity. Low GTP doses, up to 10(-5) M, stimulate, while higher doses inhibit, the ganglionic adenylate cyclase. The GTP-induced diphasic pattern is maintained also in the presence of prostaglandin E2, D-Ala2-Met-enkephalinamide, or a combination of the two drugs. However, the opioid inhibits the enzyme activity, but only at high GTP doses, while the prostaglandin stimulates the enzyme at all GTP concentrations. The effect is potentiated by a combination of prostaglandin and enkephalin. The enhancing effect of the prostaglandin and of the combination with enkephalin is maximally expressed at high, almost physiological, GTP doses.

RMI 12330A, an inhibitor of adenylate cyclase and cyclic AMP-phosphodiesterase activities in the segmental ganglia of the leech Hirudo medicinalis.

The effect of RMI 12330A, an inhibitor of basal as well as serotonin-stimulated adenylate cyclase in the segmental ganglia of the leech Hirudo medicinalis, has been tested on cyclic AMP-phosphodiesterase activity in the same tissue. The drug dose-dependently inhibits high and low affinity enzyme, an effect first evident at an RMI 12330A concentration of 0.5 mM. For both enzyme activities the drug reduces Vmax without substantially influencing Km values. Both basal and serotonin-stimulated adenylate cyclase as well as cyclic AMP-phosphodiesterase activities are inhibited by RMI 12330A in a non-competitive manner.

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 and D-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: role of Mg2+ions.

Agonists modulation of Mg2(+)-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg2+ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with "higher" and "lower" apparent affinity states, are extrapolated. In the presence of D-Ala2-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E2 increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg2(+)-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.

Interactions between prostaglandin E2 and D-ala2-met-enkephalinamide on adenylate cyclase activity in the guinea-pig superior cervical ganglion.

Crude membrane fractions, obtained from superior cervical ganglia of normal and sympathectomized guinea-pigs, have been used to investigate the role of prostaglandin E2 and D-ala2-met-enkephalinamide in the modulation of ganglionic adenylate cyclase as well as their functional interrelationship. In ganglia from normal animals the enzyme activity was stimulated and inhibited, respectively, by the prostaglandin (10(-4)M) and by the opiate pentapeptide (10(-4)M), while little or no effects were observed in denervated preparations. When the two substances were tested in combination, a supra-additive stimulation of adenylate cyclase activity was obtained both in normal and denervated ganglia. In the latter preparation the opiate increased prostaglandin E2 specific binding, suggesting that the mechanism of supra-additivity could involve interactions at receptors level. Furthermore, the supra-additive stimulation of adenylate cyclase activity by the combination of the two drugs was obtained in a narrow range of concentrations since at low prostaglandin E2 doses (10(-7)-10(-6)M) or at very high doses of the opiate (10(-3)M), only the inhibitory effect of D-ala2-met-enkephalinamide was evidenced.