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The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H+ exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H+ exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for activating Na+/H+ exchange. However, in contrast to observations in HSWP cells, we were able to activate the Na+/H+ exchanger in mouse 3T3 and human WI-38 cells with PMA alone, suggesting that there is some diversity in the mechanism for activation of Na+/H+ exchange in different types of fibroblasts.

The present retrospective study reports the results of a survey conducted on 130 patients given elective abdominal and urinary surgery together with the cultivation of routine intraperitoneal drainage material. The parameters considered were the type of pathology, the condition of the wound, any serohaematic material drained, the presence and duration of postoperative temperature increases and pre and post-operative leukocytosis levels. These parameters were considered in relation to the positive or negative results of the cultural examination. The aim was to demonstrate that routine drainage causes no appreciable infective or mechanical complications.

Immunoconglutinins (Ic) are a group of predominantly IgM antibodies formed towards antigenic determinants exposed in fixed complement components (C3b and C4). Ic production is stimulated by bacterial and viral infections; elevated titers were also found in a number of other diseases involving completement-fixing reactions in vivo. High titers have been associated with the infectious morbidity within a particular population. The object of this investigation was to determine the correlations of autostimulated Ic titre with several parameters in 100 atopic nondesensitized subjects with respiratory allergies and in 140 non atopic, healthy, individuals. Eighty of them had rhinitis and/or conjunctivitis, 9 asthma and 11 rhinitis and asthma. All the atopics were studied also after at least 6 months of specific immunotherapy. None of the subjects received any other therapy at the time of study. Atopic subjects with high titers of Ic are significantly more numerous than controls, without significant correlation with the kind and beginning of atopic syndrome, and with immunotherapy. These results might be attributed to a higher incidence of infections in the atopic population. The Ic might play an important role in the immunoregulation involving complement system.

The EGF receptor, which is homologous to the v-erb-B oncogene product, has intrinsic tyrosine kinase activity, and mediates an increase in polyphosphoinositide turnover and [Ca2+]i. Recently, great progress has been made in understanding the mechanism of signal transduction at this receptor. Jacopo Meldolesi and colleagues discuss how this knowledge may lead to a better understanding of the control of cell proliferation.

The purpose of this study was to compare the new multiple allergosorbent chemiluminescent assay (MAST CLA) system with the RAST and skin prick tests as an adjunct to the diagnosis of inhalant allergies. In this report, MAST CLA and RAST have given similar results, but no technique is as sensitive as skin tests for allergen-specific diagnosis of inhalant allergic disease.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.

Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+]i, measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+]i rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KC1 were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+]i rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+]i rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+]i responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+]i rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine-coated glass slides) by fluorescence techniques (with quin-2 and fura-2). The effect of NGF on [Ca2+]i is delayed (30-40 s of lag phase), slow (t1/2 = 40 s), relatively small (+50-75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extracellular Ca2+ greater than 10 microM) through a pathway different from the voltage-gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action.

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.

alpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system.

It has been reported that rat growth hormone releasing factor (rat GRF-43), similarly to the two human GRFs (GRF-40 and 44) stimulates adenylate cyclase activity in pituitary cells. Controversial findings have been presented by two different groups on the action of GRF on phosphoinositides (PI) metabolism, a phenomenon linked to Ca-- mediated intracellular mechanisms. In the work to be reported, we evaluated the accumulation of inositol phosphates induced by GRF exposure in primary cultures of rat and human pituitary cells. Addition of rat GRF-43 to rat pituitary cells at doses up to 1 microM had no effect on inositol phosphates accumulation, while already at a dose as low as 0.05 nM it increased growth hormone secretion in the incubation medium significantly. In the same cell system, TRH, a known activator of PI breakdown, significantly increased [3H]inositol phosphates. In primary cultures of human somatotrophs from acromegalic subjects as in rats, addition of hpGRF-40 and also of TRH did not elicit any modification in the accumulation of [3H]inositol phosphates. Consistent with in vivo findings, both peptides induced a significant release of GH in the medium. Our results show that the GH releasing effect of GRF does not involve the hydrolysis of phosphatidylinositol in normal rat as well as in tumoral human somatotrophs. In addition it appears that the anomalous response of TRH on adenomatous cells from acromegalic patients is differently mediated in respect to the action of the tripeptide on normal lactotrophs and thyrotrophs.

1 Mazindol is a new anorexigenic agent which possesses a different chemical structure from that of phenylethylamines, but shows a pharmacological profile similar to that of (+)-amphetamine. 2 Mazindol neither altered whole brain monoamine levels (noradrenaline (NA), dopamine, 5-hydroxytryptamine (5-HT)) nor changed NA levels in the hypothalamus or dopamine levels in the caudate nucleus. 3 Mazindol enhanced dopamine turnover rate in the caudate nucleus, as shown by the increased rate of dopamine decline after blockade of catecholamine synthesis by alpha-methyl-p-tyrosine and decreased the conversion index of (3H)-tyrosine into brain NA. 4 Mazindol administration did not modify pargyline-induced decline of 5-hydroxyindoleacetic acid suggesting that 5-HT turnover is not altered by this drug.

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.

The changes in Ca2+ homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca2+ concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca2+-containing medium caused a rapid rise in the cytosolic Ca2+ concentration, that remained elevated for several minutes. In Ca2+-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. The lack of Ca2+ rise seen in attached cells loaded with quin2 and treated with EGF in Ca2+-free medium was probably the result of a Ca2+ buffer artifact. Concomitantly to the Ca2+ signal, EGF induced phosphoinositide hydrolysis, with stimulated accumulation of inositol 1,3,4,trisphosphate and -1,3,4,5-tetrakisphosphate. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca2+ signal elicited by EGF is due to two components: redistribution from an intracellular store (possibly mediated by generation of inositol trisphosphate) and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin (growth factors that cause much greater phosphoinositide hydrolysis and Ca2+ redistribution responses than EGF). It is therefore suggested that the Ca2+ influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.

We examined the effect of chronic opiate treatment on the alpha 2-adrenergic receptor binding of both the agonist [3H]clonidine and the antagonist [3H]yohimbine in the cerebral cortex and kidney of rats. In addition, we demonstrated the presence of an adrenergic receptor of the alpha 2 type on the neurotumor cell line NCB20 and used this cell line as a model for studying the interaction between the opiate and alpha 2-adrenergic systems. In all the three above systems, chronic opiate treatment did not modify the number or the affinity of alpha 2-adrenergic receptors.