Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells.

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.

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

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.

Oxytocin stimulates migration and invasion in human endothelial cells.

BACKGROUND AND PURPOSE: It has recently been reported that oxytocin is produced by some tumour cell types, and that oxytocin receptors, belonging to the G-protein-coupled receptor (GPCR) family, are expressed in a variety of cell types. Among these, human umbilical vein endothelial cells (HUVECs) respond to oxytocin with an increased proliferation, suggesting a possible role for the hormone in the regulation of angiogenesis. EXPERIMENTAL APPROACH: We employed chemotaxis and chemoinvasion assays to characterize the effect of oxytocin on HUVEC motility, and immunoblot analysis to study its molecular mechanisms of action. KEY RESULTS: We showed that oxytocin stimulates migration and invasion in HUVECs via oxytocin receptor activation. Searching for the molecular mechanism(s) responsible for oxytocin's pro-migratory effect, we identified the Gq coupling of oxytocin receptors and phospholipase C (PLC) as the main effectors of oxytocin's action in HUVECs. We also found that oxytocin stimulates the phosphorylation of endothelial nitric oxide synthase (eNOS) via the phosphatidylinositol-3-kinase (PI-3-K)/AKT pathway, and that the activation of PI-3-K and formation of nitric oxide (NO) are required for the pro-migratory effect of oxytocin. CONCLUSIONS AND IMPLICATIONS: The ability of oxytocin to stimulate HUVEC motility and invasion suggests that the hormone can participate in physiopathological processes where activation of endothelial cells plays an important role, for example, in angiogenesis. Interestingly, both the AKT and eNOS phosphorylation induced by oxytocin receptor activation depended on PLC activity, thus suggesting the existence of a still undefined mechanism connecting PLC to the PI-3-K/AKT pathway, upon oxytocin stimulation.

Nicotine stimulates a serotonergic autocrine loop in human small-cell lung carcinoma.

Small-cell lung carcinoma cells express different plasma membrane nicotinic acetylcholine receptor subtypes. We have now found that interacting with these receptors (-)-nicotine induces a dose-dependent and stereoselective release of [3H]serotonin which is dependent on external calcium and blocked by the specific ganglionic nicotinic antagonist mecamylamine. With the same potency (-)-nicotine stimulates tumor cell proliferation, an effect also blocked by mecamylamine. Serotonin itself stimulates cell proliferation in a dose-dependent manner, an effect blocked by the selective serotonergic receptor antagonists methiotepine and metergoline. These data suggest that nicotine might affect proliferation of small-cell lung carcinoma cells by inducing the release of hormones (such as serotonin) with autocrine capabilities and place both the nicotinic and the serotonergic receptors at key positions in the biological and, possibly, pharmacological approach to this human lung cancer.

Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils.

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.

Transmembrane signalling at the epidermal growth factor receptor.

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.

Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.

Somatostatin inhibits PDGF-stimulated Ras activation in human neuroblastoma cells.

The main physiological role of somatostatin (SST) is the control of hormone secretion. Recently, SST has been shown to exert antiproliferative effects on some human tumors via both direct and indirect mechanisms. We have previously found that in the human neuroblastoma cell line SY5Y the SST analogue lanreotide (BIM 23014) inhibited serum-stimulated cell proliferation and MAP kinase activity. Here, we examine the effect of SST on PDGF-induced Ras activation. We found that SST suppressed PDGF-induced Ras activation in a pertussis toxin (PTx)-independent and peroxovanadate-dependent manner. Ras-specific GTPase activating protein (GAP) activities were not altered by SST treatment. On the contrary, PDGF-induced PDGF receptor phosphorylation was decreased by SST in a PTx-independent, peroxovanadate-dependent manner, likely accounting for the SST-mediated inhibition of PDGF-induced Ras activation.

Epidermal growth factor-induced phosphoinositide hydrolysis. Modulation by protein kinase C.

A short-term treatment with phorbol 12,13-dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)-stimulated phosphoinositide hydrolysis in A431 cells, whereas long-term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF-triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long-term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long-term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.

Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells.

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.

Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells.

In previous studies we have showed that somatostatin (SST) inhibits cell division, mitogen-activated protein (MAP) kinase and Ras activity in the human neuroblastoma cell line SY5Y. In the present study, we have assessed the role of a series of SST analogs, three of which were selective for SSTR1, SSTR2 or SSTR5, in these cellular events. All the analogs inhibited forskolin-induced cAMP accumulation. Selective stimulation of SSTR1 or SSTR2 but not of SSTR5 inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation. The three analogs inhibited PDGF-stimulated MAP kinase activity, at least at an early time. In contrast, none of the analogs used individually was able to inhibit PDGF-stimulated Ras activity. A combined stimulation of SSTR2 and SSTR5 was necessary to obtain a significant inhibitory effect, suggesting the possibility of receptor heterodimerization. These results indicate that SST inhibition of Ras and MAP kinase activities takes place via different pathways and that SST inhibition of PDGF-induced cell proliferation occurs via a Ras-independent pathway.

Serotonin release and cell proliferation are under the control of alpha-bungarotoxin-sensitive nicotinic receptors in small-cell lung carcinoma cell lines.

Neuronal type nicotinic acetylcholine receptors (nAchRs) have recently been identified in small-cell lung carcinoma. We here show that both nicotine and cytisine stimulate [3H]serotonin release in a dose-dependent manner; this effect is antagonized by alpha-bungarotoxin (alpha Bgtx) and alpha-conotoxin MI (alpha Ctx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both alpha Bgtx and alpha Ctx. By PCR analysis, we demonstrate the presence in SCLC of both the alpha 7 and the beta 2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that alpha Bgtx-sensitive receptors of the alpha 7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.

A somatostatin analogue inhibits MAP kinase activation and cell proliferation in human neuroblastoma and in human small cell lung carcinoma cell lines.

Somatostatin possesses antisecretory and antiproliferative activity on some human tumors. We herein report that, in a human neuroblastoma cell line, the somatostatin analogue BIM 23014 inhibited mitogen-activated protein (MAP) kinase activity stimulated by either insulin-like growth factor-1, whose receptor bears a tyrosine kinase, or carbachol, which acts at a G-protein coupled receptor. In a human small cell lung carcinoma line BIM inhibited serum-stimulated MAP kinase activation. These inhibitory actions occur in a dose range quite similar to that observed for suppression of proliferation induced by the analogue in the same cell lines. The decrease in cAMP elicited by the analogue in the two cell lines is not responsible for its inhibitory action on MAP kinase and cell growth. Moreover, the analogue did not modify intracellular [Ca2+] and pH. An involvement of a phosphatase activity is suggested.

Ca2+ and Ca2+ channel antagonists in the control of human small cell lung carcinoma cell proliferation.

Small cell lung carcinoma cells possess voltage-dependent calcium channels (VDCCs) of the L, omega-conotoxin-sensitive and P-like type. We hypothesized that these VDCCs might regulate the secretion of autocrine growth factors and thus influence the proliferation of these cells. We found that extracellular Ca2+ plays a stimulatory role in the proliferation of the GLC8 cell line. L-type calcium channel blockers of the dihydropyridine, phenylalkylamine and benzothiazepine classes inhibited [3H]thymidine incorporation in these cells, however at concentrations higher than those required to block L-type channel function. Moreover, the growth of murine Swiss 3T3 fibroblasts which do not possess L-type Ca2+ channels, was inhibited by the Ca2+ channel antagonists at the same effective concentrations as in small cell lung carcinoma cells. omega-conotoxin and omega-agatoxin IVA, which block the N- and P-type channel respectively, had no effect on GLC8 cell proliferation. It is concluded that the presence of extracellular Ca2+ is a positive stimulus for small cell lung carcinoma cell growth. However, under our experimental conditions, the calcium channel blockers inhibited DNA synthesis most probably by a mechanism other than VDCC antagonism.

Evidence for receptor subtype cross-talk in the mitogenic action of serotonin on human small-cell lung carcinoma cells.

We previously reported a significant mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small-cell lung carcinoma cells (SCLC, GLC-8), mediated by both 5-HT1D and 5-HT1A receptors. Here we investigate possible interactions between the two receptor subtypes. Dose-effect curves obtained by simultaneously applying equipotent concentrations of the selective 5-HT1A agonist 8-OH-DPAT and the selective 5-HT1D receptor agonist sumatriptan are shifted to the right, although maximal effects are additive. The nonselective 5-HT antagonist metergoline displays higher potency when both receptor subtypes are activated. The 5-HT1D receptor antagonist GR127935 is markedly more potent against sumatriptan than against the sensitive portion of 5-HT effect. Indeed, both GR127935 and the 5-HT1A antagonist spiperone shift the EC50 for the residual effect of 5-HT from approximately 300 to 120-150 nM, suggesting that blocking one receptor subtype may facilitate activation of the other. Preincubation with either 8-OH-DPAT or sumatriptan suppresses the mitogenic response to the other specific receptor agonist; suppression is complete within 10 min at 37 degrees C, and is not observed when the preincubation is done at 4 degrees C. Measurements of adenylate cyclase activity do not help in interpreting the results. Conversely, measurements of MAP kinase activity reveals biphasic activation with a delayed activation at 1 h, and reproduce the suppression of the effect of the second drug by 15 min preincubation. These findings constitute the first evidence of a reciprocal negative interference between human 5-HT1A and 5-HT1D receptors, and indicate that SCLC GLC-8 cells simultaneously express both receptor subtypes. Mere reciprocal antagonism of the drugs employed cannot account for these data. We suggest that in this cell system cross-talk occurs in the transduction pathways of the two receptor subtypes.

Mitogenic effect of serotonin in human small cell lung carcinoma cells via both 5-HT1A and 5-HT1D receptors.

We have recently shown that the mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small lung carcinoma (SCLC) cells is at least partly due to stimulation of a 5-HT1D receptor type. We now report that the 5-HT1A receptor agonist R(+)-8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT) is also capable of stimulating [3H]thymidine incorporation into SCLC GLC-8 cells, although with lower efficacy than 5-HT. The simultaneous administration of maximal doses of 8-OH-DPAT and the 5-HT1D receptor agonist sumatriptan reproduced the maximal [3H]thymidine incorporation observed with 5-HT alone. The 5-HT1A receptor antagonists spiperone and SDZ 216-525 completely abolished the effect of 8-OH-DPAT (IC50 30 nM for both drugs) behaving as pure antagonists. Accordingly, the two drugs partially inhibited the mitogenic effect of 5-HT. These data indicate that the mitogenic effect of 5-HT in SCLC cells involves both 5-HT1A and 5-HT1D receptor types.

5-HT1D receptor type is involved in stimulation of cell proliferation by serotonin in human small cell lung carcinoma.

Serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter and vasoactive agent, is contained in two small cell lung carcinoma cell lines GLC8 and NCI-N-592 and is released in the culture medium. It also stimulates DNA synthesis in the same cell lines. In GLC8 cells this mitogenic effect is not counteracted by ketanserin, ICS 205-930 and GR 113-808 which are antagonists of the 5-HT2, 5-HT3 and 5-HT4 receptors, respectively. On the contrary, the antagonists metergoline, methysergide, SDZ 21-009 and methiothepin inhibit the 5-HT-stimulated incorporation of [3H]thymidine in GLC8 cells. The 5-HT1D agonist sumatriptan is capable of mimicking 5-HT action on cell proliferation. Both sumatriptan and 5-HT inhibit adenylate cyclase activity at doses which correlate with the mitogenic effect. We conclude that a 5-HT1D receptor type contributes to the mitogenic effect of 5-HT in GLC8 cells. This is the first demonstration of an involvement of the 5-HT1D receptor type in human cell proliferation. The design of specific antagonists for this type of receptor might be useful for the growth control of this very aggressive tumor.

Chronic opiate treatment does not modify alpha 2-adrenergic receptors in rat cerebral cortex, kidney and in the neurotumor cell line NCB20.

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.

alpha-Conotoxin imperialis I inhibits nicotine-evoked hormone release and cell proliferation in human neuroendocrine carcinoma cells.

alpha-Conotoxins are small peptides present in the venom of different species of marine snails of the Conus genus that target nicotinic acetylcholine receptors (nAChRs), with a marked specificity for muscle-type nAChRs. alpha-Conotoxin Imperialis I (alpha-Ctx-Iml), from Conus imperialis, has been recently described as a potent antagonist of mammalian neuronal alpha-bungarotoxin (alpha-Bgtx)-sensitive nAChRs. Human small cell lung carcinoma (SCLC) is a very aggressive tumor composed of neuroendocrine secretory cells. We demonstrated that human SCLC cells express neuronal-type alpha-Bgtx-sensitive nAChRs, and that their activation causes secretion of mitogenic hormones and stimulates cell proliferation, alpha-Ctx ImI inhibits both these nicotinic effects, and could therefore be considered a new important tool for investigating human neuronal-type alpha-Bgtx-sensitive nAChRs.

Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth.

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.