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In PC12 cells, cultured in the presence of nerve growth factor to increase their complement of muscarinic receptors, treatment with carbachol induces muscarinic receptor-dependent rises in free cytosolic Ca2+ as well as hydrolysis of membrane phosphoinositides. Experiments were carried out to clarify the relationship between these two receptor-triggered events. In particular, since inositol-1,4,5-trisphosphate (the hydrophilic metabolite produced by the hydrolysis of phosphatidylinositol-4,5-bisphosphate) is believed to mediate intracellularly the release of Ca2+ from nonmitochondrial store(s), it was important to establish whether it can be generated at resting cytoplasmic concentration of Ca2+ (approximately 0.1 microM). Cells incubated in Ca2+-free medium were depleted of their cytoplasmic Ca2+ stores by pretreatment with ionomycin. When these cells were then treated with carbachol, their cytosolic concentration of Ca2+ remained at the resting level, whereas inositol-1,4,5-trisphosphate generation was still markedly stimulated. Our results demonstrate that an increase in the concentration of cytosolic Ca2+ is not a necessary intermediate between receptor activation and phosphoinositide hydrolysis, and therefore support the second-messenger role of inositol-1,4,5-trisphosphate.

Understanding the mechanisms that control cell growth is a central problem in cell biology today. During the last few years, it has become increasingly clear that one or more defects in the physiological mechanisms that regulate cell proliferation can be the cause of tumor cell growth. The study of normal cell division is therefore important not only per se, but especially in relation to its pathological implications.

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.

Most excitable cells respond to depolarization with a rapid increase of their Ca2+ permeability, due to the opening of voltage-gated Ca2+ channels (Ca2+ VOCS) in the plasma membrane (Hagiwara and Byerly 1981; Reuter 1984). Opening of these channels causes the cytosolic concentration of Ca2+, [Ca2+]i, to rise quickly, beginning in the cytosolic region(s) immediately adjacent to the plasma membrane. The [Ca2+]i rise is opposed by processes that expell Ca2+ from the cell (active pumping by a Ca2+ ATPase and Na+/Ca2+ exchange) or cause its segregation within intracellular, membrane-bounded campartments (a high affinity microsomal store as well as the mitochondria). Up to now, studies on Ca2+ VOCs were carried out primarily by electro-physiology (classical techniques as well as patch clamping) and by Ca2+ influx experiments. By the use of these techniques, a number of important achievements were obtained (Hagiwara and Byerly 1981; Reuter 1984), including the recent demonstration of the heterogeneous nature of Ca2+ VOCs. Following the simple nomenclature proposed by Nowicky et al. (1985), the various types of Ca2+ VOCs so far identified will be referred to here as the T (transient, or low-threshold) VOCs, probably involved in pacemaking; the L (long-lasting, or high-threshold), the classical Ca2+ VOCs that are affected by dehydropyridine drugs; and the N (neither transient, nor long-lasting) VOCs, present in neurons, that might be preferentially located in the presynaptic membrane (Nowicky et al. 1985).

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells Chromaffin cellainvestigated (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. AbstractA 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.

Summary The present study shows that the intraventricular injection of muscimol (250 ng/ventricle), a potent GABA receptor agonist, strongly antagonized morphine and β-endorphin analgesia. Furthermore, isoguvacine, a compound structurally related to muscimol, and nipecotic acid and guvacine, inhibitors of GABA uptake, administered intraventricularly markedly attenuated the analgesic effect of morphine. These results further substantiate that the GABAergic system is involved in morphine analgesia. A possible mechanism of action is discussed.

PC12 is a line of neurosecretory cells originally developed by Greene and Tischler (1976) from a rat pheochromocytoma. This line has been extensively used in a number of laboratories around the world. Morphologically, growing PC12 cells resemble chromaffin cells of the adrenal medulla, although their secretion granules are smaller and less numerous (Greene and Tischler, 1976, 1982; Tischler and Greene, 1978; Watanabe et al., 1983). Because of this and other differences with respect to chromaffin cells, PC12 are now considered to be undifferentiated sympatoblasts. Their major catecholamine is dopamine, accumulated within secretory granules together with noradrenaline (Greene and Tischler, 1976, 1982; Rebois et al., 1980). Other neurotransmitters are synthesized in PC12 cells. Among these, acetylcholine is stored in organelles (not yet identified unambiguously), which are heavier than regular synaptic vesicles and lighter than the bulk of dopamine containing granules (Schubert et al., 1977). Peptides (enkephalins; neurotensin; Tischler et al., 1983; Panerai and Meldolesi, in preparation), as well as proteins typical of chromaffin and other secretory organelles (chromogranin A; secretogranin; Lee and Huttner, 1983) are present in PC12 cells, but their possible colocalization with dopamine has not been established with certainty yet.

Short (1–10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 miristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts we demonstrate that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA. Feed-back inhibition of surface receptors by protein kinase C-mediated phosphorylation is therefore not general, and cannot be the only process responsible for the attenuation of receptor-mediated responses in eukaryotic cells.

The signals that induce a cell to divide are usually external and in the form of a binding of growth factors. We focussed our attention in defining the sequence of events which occurs after the binding of the mitogens to their surface receptors. One of the early membrane events stimulated by growth factors is a Na+ flux coupled to a H+ efflux that is typically inhibited by amiloride. The importance of this event and of the consequent cytoplasmic alkalinization for the cell proliferation is discussed. Recent data indicate that mitogens increase intracellular Ca++ levels and activate protein kinase C by inducing the hydrolysis of membrane phosphoinositides. A role for Ca++ and protein kinase C in activating Na+/H+ A role for Ca++ and protein kinase C in activating Na+/H+ exchange system is discussed. Finally a model is presented that illustrates the first membrane events stimulated by the growth factors. The model reveals an intimate interconnections between phosphoinositide metabolism, cytosolic Ca++ rise, protein kinase C and cytoplasmic alkalinization.

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.

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 μm 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.

Preincubation of PC12 cells (used both before and after differentiation by NGF) with phorbol myristate acetate (PMA) was without effect on the basal concentration of inositol phosphates (metabolites of phosphoinositide hydrolysis) and of free cytosolic Ca 2+ , but inhibited considerably the increases induced by the cholinergic agonist carbachol via the activation of the muscarinic receptor. Inasmuch as binding was unaffected, this inhibition might occur at the level of receptor coupling to its transduction mechanism(s). Inhibition appeared within 1 min and was maximal after 3 min. The concentrations of PMA needed (10 −9 – 10 −8 M) were in the range believed to cause specifically the activation of protein kinase C. The muscarinic receptor, via the hydrolysis of phosphoinositides and the generation of diacylglycerol, participates in the regulation of the latter enzyme. Our data suggest therefore that the receptor operates under stringent feedback control by the metabolites generated as a consequence of its activation.

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.

Activation of the EGF receptor in A431 cells induces the hydrolysis of phosphoinositides and a transient rise of the cytosolic Ca2+ concentration, [Ca2+], which are completely inhibited by acute pretreatment with activators of protein kinase C, such as phorbol esters. Down regulation of the enzyme (by long-term pretreatment of the cells with phorbol esters) causes the [Ca2+]i response to EGF to increase in magnitude and, especially, to become much more persistent (average t 1 2 of [Ca2+]i decline 9 min with respect to 2.3 min in controls). These results demonstrate that the activation of protein kinase C induced by EGF in intact A431 cells is sufficient to trigger a feed back, autolimitative regulation of the EGF receptor that might play a prominent physiological role in the definition of the mitogenic activity of the growth factor.

Protein kinase C is known to be involved both in initiation and termination of cellular responses due to phosphoinositide breakdown. Here we report that in PC12 cells (a line of neurosecretory cells derived from a rat pheochromocytoma), pretreatment with nanomolar concentrations of phorbol myristate acetate, PMA, which is believed to specifically activate protein kinase C, inhibits the cytosolic-free Ca2+ concentration rise induced by depolarizing agents. In contrast, plasma membrane potential and 45Ca efflux from preloaded cells were unaffected by PMA pretreatment. Inhibition by PMA and diacylglycerol of the cytosolic-free Ca2+ concentration rise induced by depolarization was observed also in another cell line, the insulin secreting line RINm5F. These results raise the possibility that the voltage-gated Ca2+ channel is under inhibitory control by protein kinase C.

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 intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.

Background: Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs). Methods: Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting. Results: Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother’s weight may exert a different influence on male and female babies. Conclusions: The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.

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.

Microinjections of muscimol, a GABA receptor agonist, into the periaqueductal gray matter (PAG) counter-acted the antinociceptive effect of morphine in rats, as measured by the "tail-flick" method. Muscimol's effect was partially reversed by bicuculline.