Two for-Met-Leu-Phe-OMe analogues trigger selective neutrophil responses. A differential effect on cytosolic free Ca2+.

For-Thp-Leu-Ain-OMe and for-Met-delta(z)Leu-Phe-OMe are two conformationally restricted fMLP-OMe analogues able to discriminate between different biological responses of human neutrophils. In this paper, we demonstrate that the former peptide, which evokes only chemotaxis, does not alter human neutrophil Ca2+ levels. In contrast, for-Met-delta(z)Leu-Phe-OMe, which induces superoxide anion release and degranulation but not chemotaxis, significantly increases the cation concentration. The chelation of Ca2+ in both extracellular and intracellular media abolishes O2- production triggered by for-Met-delta(z)Leu-Phe-OMe, while the same procedure does not affect neutrophil chemotaxis towards for-Thp-Leu-Ain-OMe. We therefore suggest that chemotaxis, unlike superoxide anion release, is independent of Ca2+ enhancement in human neutrophils.

Studies on fMLP-receptor interaction and signal transduction pathway by means of fMLP-OMe selective analogues.

For-Thp-Leu-Ain-OMe ([Thp(1), Ain(3)] fMLP-OMe) (2), for-Met-delta(z)Leu-Phe-OMe ([delta(z)Leu(2)] fMLP-OMe) (3), for-Thp-Leu-Phe-OMe ([Thp(1)] fMLP-OMe) (4), and for-Met-Leu-Ain-OMe ([Ain(3)] fMLP-OMe) (5) are for-Met-Leu-Phe-OMe (fMLP-OMe) (1) analogues which discriminate between different responses of human neutrophils. Peptides 3 and 5, similar to fMLP-OMe, enhance neutrophil cyclic AMP (cAMP) as well as calcium levels, while analogues 2 and 4, which evoke only chemotaxis, do not alter the concentration of these intracellular messengers. When we tested the peptides' ability to displace [3H]-fMLP from its binding sites, the following order of potency was observed: analogue 1 > 3 > 5 > 2 > 4. A particularly low activity at the receptor level characterized analogues 2 and 4. Their low effectiveness was not improved by the addition of cytochalasin B, by different incubation temperatures, or by the absence of endogenous guanine nucleotides, conditions known to influence fMLP receptor fate and functionality. We speculate that, in certain conditions, the fMLP receptor may undergo conformational changes that impede the binding of pure chemoattractants.

Effect of nitric oxide on arachidonic acid release from human amnion-like WISH cells.

In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [(3)H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; (3)L -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3',5'-cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E(2) (PGE(2)) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [(3)H]arachidonic acid release. Both soluble guanylyl cyclase and PLA(2) inhibitors counteracted SNP response. Exogenous NO increased PGE(2) release, although to a much lesser degree compared with arachidonic acid release. Our results indicate that NO stimulates AA release in WISH cells by activating PLA(2) through a cyclic GMP-dependent mechanism.

Non-selectivity of yohimbine for adrenergic receptors in fish liver.

Most studies on adrenergic receptors (AR) have been performed on mammalian tissues, but the adrenergic ligands routinely utilized seem not always suitable for specific interaction with fish tissues. Here we report that in isolated catfish hepatocytes, yohimbine, usually thought to act as a specific antagonist for AR of the alpha2 subtype, at high concentrations, increases adenylyl cyclase activity and synergistically enhances the forskolin-induced enzyme stimulation. Such effects are counteracted by the beta-AR antagonist propranolol, but not by the alpha-AR antagonist phentolamine. Moreover, yohimbine seems to antagonize both alpha1- and alpha2-adrenergic ligand-binding in catfish liver membrane in a manner somewhat different from the mammalian systems. Together with previous evidence that yohimbine blocks the rise of intracellular calcium induced by epinephrine via alpha1-AR, the present results seem to indicate that this compound is not a suitable tool for studying alpha2-AR in fish liver.

Coexistence of alpha1 and beta adrenergic receptors in the liver of the frog Rana esculenta, the toad Bufo bufo, the lizard Podarcis sicula campestris, and the turtle Pseudemys picta elegans.

In mammals and birds the characteristics of alpha1 adrenergic receptors and their biological role in liver metabolism have been clearly described, although the predominance of receptor subtypes varies with species. In contrast, the actual presence of hepatic alpha1 adrenergic receptors in fish, amphibians, and reptiles has been questioned. Only recently has their existence been demonstrated in some fish species and also in the wood frog Rana sylvatica. The present study assessed the presence of alpha1 adrenergic binding sites on hepatic membranes of frogs, toads, lizards, and turtles using the specific alpha1 adrenergic receptor antagonist [3H]prazosin; for comparison, the binding of the specific beta adrenergic receptor antagonist [3H]CGP-12177A was evaluated in the same preparations. alpha1 Adrenergic receptors are indeed present in the liver of the ectotherms examined. Specific binding is saturable, reversible, and linear as a function of tissue concentration. The binding data indicated the presence of two classes of binding sites displaying high and low affinities with Kds in the nanomolar and micromolar ranges, respectively. The present study provides the first evidence for the presence of alpha1 adrenergic receptors in the liver of toad, lizard, and turtle while confirming their existence in another species of frog, Rana esculenta.

Adenylyl cyclase activity and glucose release from the liver of the European eel, Anguilla anguilla.

The properties of adenylyl cyclase (AC) in liver membranes of the European eel (Anguilla anguilla) and the involvement of cAMP in glucose release from isolated hepatocytes in response to catecholamines were studied. Basal enzyme activity seemed essentially unaffected by GTP, while a biphasic response to increasing nucleotide concentrations was obtained in the presence of epinephrine. Eel liver AC was dose-dependently stimulated by guanosine 5'-O-(3-thiotriphosphate) and inhibited by guanosine 5'-O-(2-thiodiphosphate). AC activity, intracellular cAMP levels, and glucose release from isolated hepatocytes were significantly enhanced by NaF, forskolin, epinephrine, and phenylephrine. The rise in cAMP production stimulated by catecholamines was counteracted by propranolol, but not by phentolamine. Catecholamine-induced glucose output was instead partially antagonized by both phentolamine and propranolol. Complete inhibition was obtained only by the simultaneous presence of the two adrenergic antagonists. Glucose release from the cells was induced by dibutyryl cAMP and by the calcium ionophore ionomycin. In summary, these data provide the first characterization of eel liver AC system and suggest a direct role for cAMP in the catecholamine-dependent glucose output. Furthermore, the involvement of calcium ions in this cellular response is hypothesized.

Inhibition of neutrophil responses by cyclosporin A. An insight into molecular mechanisms.

OBJECTIVE: Cyclosporin A (CsA) is an effective agent in rheumatoid arthritis (RA), slowing joint damage progression. Its therapeutic effect on T lymphocytes has been studied extensively, but there is little information available about neutrophils, the cells responsible for a substantial proportion of inflammation. A study was performed to investigate the in vitro effects of CsA on neutrophil functions triggered by several agonists and determine whether the drug could counteract the binding of formyl-methionyl-leucyl-phenylalanine (fMLP) to its receptor and/or modulate changes in the intracellular Ca(2+) concentration ([Ca(2+)]i). METHODS: CsA was added to neutrophils 5-50 min before the incubation steps for neutrophil function assays (chemotaxis, superoxide anion production, lysozyme release), calcium measurements and receptor binding experiments. RESULTS: CsA appeared to be particularly effective in lowering chemotaxis, superoxide anion production and lysozyme release induced by different agonists. However, it did not significantly affect either basal or agonist-stimulated neutrophil [Ca(2+)]i and the interaction between fMLP and its receptor. CONCLUSIONS: Because of its in vitro inhibition of neutrophil functions, CsA appears to have considerable potential as an anti-inflammatory drug. Moreover, as it is also a potent immunosuppressive agent, it may reduce the progression of joint damage in RA. More work remains to be done to clarify the molecular mechanism of CsA action on neutrophils.