Marco Buzzi

α-Adrenoceptor-mediated glucose release from perifused catfish hepatocytes

In fish liver catecholamines bind to beta-adrenoceptors (AR) and increase glucose release via cAMP augmentation. Alpha1-AR have recently been shown to mediate IP3 and Ca2+ elevation in catfish and eel hepatocytes, although their coupling to a physiological response has remained doubtful. We have perifused isolated catfish hepatocytes in Bio-Gel P4 columns with epinephrine in the presence of prazosin and/or propranolol, alpha- and beta-AR antagonists, respectively. Ten nM epinephrine stimulated glucose release approximately 3-fold, and this effect was completely antagonized by the simultaneous presence of both alpha- and beta-AR blockers. The two AR antagonists separately inhibited about one-third and two-third of the total stimulation, respectively. Through alpha-AR occupancy, epinephrine provoked a significant increase of glucose release whereas no stimulation was detected in Ca2+-depleted hepatocytes. Glucose release was strongly elevated by both ionomycin and dibutyryl cAMP. These results represent the first direct evidence that alpha-AR transduction pathway is involved in epinephrine-induced glucose release from fish hepatocytes.

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.

Somatostatin-induced control of cytosolic free calcium in pituitary tumour cells

In rat pituitary tumour cells (GC cells), spontaneous oscillations of the intracellular concentration of Ca2+ ([Ca2+]i) induce growth hormone (GH) secretion that is inhibited by octreotide, a somatostatin (SRIF) agonist which binds to SRIF subtype (sst) receptor 2. The effects of its functional activation on the control of [Ca2+]i were investigated using fluorimetric measurements of [Ca2+]i. SRIF decreases the basal [Ca2+]i and the [Ca2+]i rise in response to forskolin (FSK) through the inhibition of L‐type voltage‐dependent Ca2+ channels. Pretreatment with octreotide or with L‐Tyr8Cyanamid 154806, a sst2 receptor antagonist, abolishes the SRIF‐induced inhibition of [Ca2+]i. Octreotide is known to operate through agonist‐induced desensitization, while the antagonist operates through receptor blockade. sst1 and sst2 receptor‐immunoreactivities (‐IRs) are localized to cell membranes. sst2, but not sst1 receptor‐IR, internalizes after cell exposure to octreotide. SRIF‐induced inhibition of basal [Ca2+]i or FSK‐induced Ca2+ entry is blocked by pertussis toxin (PTX). FSK‐induced cyclic AMP accumulation is only partially decreased by SRIF or octreotide, indicating that sst2 receptors are coupled to intracellular pathways other than adenylyl cyclase (AC) inhibition. In the presence of H‐89, an inhibitor of cyclic AMP‐dependent protein kinase (PKA), SRIF‐induced inhibition of basal [Ca2+]i is still present, although reduced in amplitude. SRIF inhibits [Ca2+]i by activating sst2 receptors. Inhibition of AC activity is only partly responsible for this effect, and other transduction pathways may be involved.

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.

Inhibition of amniotic prostaglandin E release by ampicillin

OBJECTIVE The effect of antibiotics in the prevention of preterm labor needs to be further investigated. The aim of this study was to determine the effect of ampicillin on prostaglandin E release from amnion as a possible explanation for its ability to retard preterm labor. STUDY DESIGN The effect of the beta-lactam antibiotic ampicillin on prostaglandin E release from human amnion was tested under basal and stimulated conditions. RESULTS Ampicillin dose dependently inhibits basal prostaglandin E release from amnion in both static and dynamic conditions. In our experiments, 10(-7) mol/L ampicillin (a concentration able to significantly inhibit prostaglandin E output) leaves the microbiologic features of the medium substantially unmodified up to 5 hours of incubation. Moreover, the drug reversibly counteracts the prostaglandin E elevation induced by arachidonic acid or oxytocin. CONCLUSION This finding (i.e., that ampicillin inhibits prostaglandin E release from amnion) may offer an explanation for a beneficial response to ampicillin therapy in the case of preterm labor even in the absence of bacterial infection.

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.

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.

Olfactory transduction mechanisms in sheep

The enzyme adenylyl cyclase from sheep olfactory epithelium is dually regulated by GTP and is highly sensitive to the nucleotide analogues GTPγS and GppNHp, as well as to fluoride ions and forskolin. Many, but not all, odorants tested are able to stimulate adenylyl cyclase in a dose-dependent manner and with different potencies. Such an effect is detectable only in the presence of GTP. The odorants belonging to the putrid class are the least effective in stimulating adenylyl cyclase activity, and only furfuryl mercaptan significantly increases cAMP biosynthesis. Mixtures of two odorants, chosen among those able to activate adenylyl cyclase, induce additive or supra-additive effects, suggesting the presence of many different receptor types. The presence of an alternative olfactory signal transduction process, i.e. the inositol phospholipid second messenger system, has been evaluated. Triethylamine, a putrid odorant completely ineffective on cAMP levels, is able to significantly increase inositol phosphate accumulation, indicating the coexistence of both cAMP- and InsP3-mediated signalling pathways in sheep olfactory epithelium.

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.

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.