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Human endostatin is an endogenous angiogenesis inhibitor, and its ability to modulate tumors vascularization is of great therapeutic interest. The antiangiogenic activity is conserved also in some of its synthetic fragments. Fragment 6-49, in particular, is even more active than full length protein. It covers an endostatin region which shows two phenylalanines unusually exposed on the surface. The effect of the mutation of these residues on fragment 6-49 structure and activity are discussed and compared to those of a similar mutation in full length endostatin. A hypothesis on the fragment active epitope is supported by data from molecular dynamics simulations.

Cell motility and invasion are crucial events for the spread of cancer and, consequently, the metastatic process. Platelet-derived growth factor (PDGF) is not only capable of stimulating the proliferation of SH-SY5Y human neuroblastoma cells, but also their migration and invasion through an extracellular matrix barrier. Experiments using wortmannin and PD98059, specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and of the mitogen-activated protein kinases (ERK 1 and 2) signaling, respectively, show that the activation of both pathways is required for the PDGF-induced cell motility responses. We have previously shown that somatostatin inhibits cell division and ERK 1/2 and Ras activity in SH-SY5Y cells. We report here that it is also capable of potently and effectively inhibiting their PDGF-stimulated migration and invasion. The inhibitory effect of somatostatin is sensitive to pertussis toxin. Although somatostatin does not affect PI3-K, it inhibits ERK 1/2 and the small G-protein Rac activation and ruffle formation induced by PDGF. These results indicate that somatostatin can be considered an anti-migratory and anti-invasive agent that acts by inhibiting ERK 1/2 signaling and the PI3-K pathway via the inhibition of Rac in SHSY5Y 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-HT 1D and 5-HT 1A receptors. Here we investigate possible interactions between the two receptor subtypes. Dose-effect curves obtained by simultaneously applying equipotent concentrations of the selective 5-HT 1A agonist 8-OH-DPAT and the selective 5-HT 1D 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-HT 1D receptor antagonist GR127935 is markedly more potent against sumatriptan than against the sensitive portion of 5-HT effect. Indeed, both GR127935 and the 5-HT 1A antagonist spiperone shift the EC 50 for the residual effect of 5-HT from ≈ 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°C, and is not observed when the preincubation is done at 4°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-HT 1A and 5-HT 1D 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.

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.

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.

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.

The phosphoinositide hydrolysis stimulated by agonist-receptor interaction is an universal mechanism for signal transduction that many different types of cells use to communicate with each other . It is well known that the hydrolysis of a particular phosphoinositide, phosphatidylinositol 4,5-bisphosphate, operated by a specific phospholipase, phospholipase C (PLC), generates at least two second messengers, diacylglycerol which activates proteinkinase C and inositol 1,4,5trisphosphate which mobilizes Cap+ from intracellular stores . Consequently, all the endogenous substances (hormones, neurotransmitters, growth factors) that act via this metabolic reaction activate within the cell all the proteinkinase C and calcium dependent processes . The regulation of PLC activity is therefore crucial for achieving a correct and controlled cell response . It has recently become clear that various types of PLC exist [1] . The enzymes purified from various sources possess molecular weight ranging from 56 to 154 kDa and appear to be both soluble and membrane-associated. Cloning of four of these enzymes (a, /3, y, 6) has revealed a surprisingly low degree of similarity in their primary structure, suggesting different roles and different regulation for each of them . The isoenzymes also display differential tissue locations . All four types have been found to be ubiquitously expressed, but the level of expression is highly variable among tissues and cell lines . For example, the distribution in the various rat brain areas [2] is heterogeneous (PLC-a is highest in the hippocampus, rostral raphe nuclei and cerebellar Purkinje cells ; PLC-f3 is highest in the cerebral cortex, caudate and granule cell layer of the cerebellum ; PLC-)/has a more widespread distribution while PLC-S is low throughout the brain), suggesting the possibility of a preferential association of individual PLC isoenzymes with particular postsynaptic receptors . The mechanism of the receptor-mediated activation of PLC has recently been a matter of intense research . In analogy with the adenylate cyclase system, a G protein (Gp) is considered to be physically and functionally interposed between receptors and PLC [3] . Many studies have shown that in membrane preparations or permeable cells stable analogues of GTP both activate PLC and potentiate the

Serum stimulation of cultured human fibroblasts activates a Na+ influx via an amiloride-sensitive Na+/H+ exchange system. Evidence is presented which indicates that phospholipase activity is an important component of the mechanism by which mitogen receptor occupation leads to activation of Na+/H+ exchange. Serum stimulation of Na+ flux is effectively blocked by inhibitors of phospholipase activity such as mepacrine and U-1002. The Ki values for inhibition of Na+ flux by these agents (10 microM and 18 microM, respectively) are comparable to their Ki values for inhibition of serum-stimulated arachidonic acid release. In contrast, the activation of Na+ influx produced by the divalent cation A23187 is not affected by phospholipase inhibitors indicating that these agents specifically block mitogen activation of Na+ flux rather than nonspecifically disrupting the membrane's ability to perform Na+/H+ exchange. The phospholipase activator melittin stimulates Na+ influx in the absence of mitogens at concentrations that cause a comparable stimulation of arachidonic acid release. The melittin-stimulated Na+ influx is inhibited by amiloride and mepacrine, suggesting that melittin activation of phospholipase activity leads to the activation of the Na+/H+ exchange system. In addition, chronic treatment of cells with dexamethasone, which is known to induce an endogenous phospholipase inhibitor in human fibroblasts, leads to a substantial inhibition of the serum stimulation of both Na+ influx and arachidonic acid release. When taken together, these data support the involvement of phospholipase activity in the serum stimulation of the Na+/H+ exchange system.

The EGF receptor, which is homologous to the ν-erb-B oncogene product, has intrinsic tyrosine kinase activity, and mediates an increase in polyphosphoinositide turnover and [Ca 2+ ] 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 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.

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.

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.

Mazindol, a new anorexigenic agent which possesses a different chemical structure from phenylethylamine derivatives such as amphetamine, causes anorexia along with increases in locomotor activity and body temperature. Mazindol also induces stereotyped behaviour and, if injected into rats with unilateral nigro-striatal lesions, causes turning towards the lesioned side. Mazindol-induced anorexia is antagonized by pretreatment with α-methyl-p-tyrosine or pimozide. Pimozide pretreatment prevents the rotation induced by Mazindol in rats with unilateral nigro-striatal lesions. The involvement of dopamine in the mechanism whereby Mazindol elicits anorexia and turning behaviour is discussed.

Summary In the attempt to further characterize the role of the GABA-ergic system on morphine analgesia, nipecotic acid and guvacine, inhibitors of GABA reuptake, were administered intraventricularly in rats pretreated with morphine. Under these conditions the animals showed a decreased response to the antinociceptive effect of morphine when compared to control rats.