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The human long pentraxin PTX3 has complex regulatory roles at the crossroad of innate immunity, inflammation, and tissue repair. PTX3 can be produced by various cell types, including vascular endothelial cells (ECs), in response to pro-inflammatory cytokines or bacterial molecules. PTX3 has also been involved in the regulation of cardiovascular biology, even if ambiguous results have been so far provided in both preclinical and clinical research. In this study, we compared the proteomic profiles of human ECs (human umbilical vein ECs, HUVECs), focusing on differentially expressed proteins between the control and PTX3-silenced ECs. We identified 19 proteins that were more abundant in the proteome of control ECs and 23 proteins that were more expressed in PTX3-silenced cells. Among the latter, proteins with multifunctional roles in angiogenesis, oxidative stress, and inflammation were found, and were further validated by assessing their mRNAs with RT-qPCR. Nevertheless, the knock down of PTX3 did not affect in vitro angiogenesis. On the contrary, the lack of the protein induced an increase in pro-inflammatory markers and a shift to the more oxidative profile of PTX3-deficient ECs. Altogether, our results support the idea of a protective function for PTX3 in the control of endothelial homeostasis, and more generally, in cardiovascular biology.

Background: Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and "omics" approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms., Methods: We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR., Results: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression., Conclusions: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC 50 values significantly lower than metformin, (range 0.054-0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-β estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS.

Clinical and epidemiological data show that biological sex is one of the major determinants for the development and progression of cardiovascular disease (CVD). Impaired endothelial function, characterized by an imbalance in endothelial Nitric Oxide Synthase (eNOS) activity, precedes and accelerates the development of CVD. However, whether there is any sexual dimorphism in eNOS activity and function in endothelial cells (ECs) is still unknown. Here, by independently studying human male and female ECs, we found that female ECs expressed higher eNOS mRNA and protein levels both in vitro and ex vivo. The increased eNOS expression was associated to higher enzymatic activity and nitric oxide production. Pharmacological and genetic inhibition of eNOS affected migratory properties only in female ECs. In vitro angiogenesis experiments confirmed that sprouting mostly relied on eNOS-dependent migration in female ECs. At variance, capillary outgrowth from male ECs was independent of eNOS activity but required cell proliferation. In this study, we found sex-specific differences in the EC expression, activity, and function of eNOS. This intrinsic sexual dimorphism of ECs should be further evaluated to achieve more effective and precise strategies for the prevention and therapy of diseases associated to an impaired endothelial function such as CVD and pathological angiogenesis.

Aims: In vitro studies on hormone biological activities are commonly performed on cells cultured in nominally hormone-free media consisting of phenol-red-free media supplemented with charcoal-stripped (CS) serum. These media are largely used in almost all cell types, including endothelial cells (ECs)., Methods: Cell number and metabolic activity were measured with standard methods. Angiogenesis was evaluated in a three-dimensional spheroid sprouting assay., Results: When we compared human umbilical vein ECs (HUVECs) cultured in standard conditions (199 medium supplemented with normal serum) with HUVECs grown in the hormone-free medium (phenol-red-free 199 medium supplemented with CS serum), we found that cells stop to grow in the absence of hormones. Notably, neither 17-β2 estradiol nor dihydrotestosterone reversed this inhibition. Moreover, the presence of the CS serum was sufficient to abrogate the ability of HUVECs to sprout in a three-dimensional spheroid assay, thus affecting a functional property of ECs., Conclusions: Our results suggest that one or possibly more substances removed by stripping procedure from serum and different from sex hormones are crucial for the maintenance of in vitro ECs distinctive properties. Therefore, caution should be used when ECs are studied in media containing the CS serum.

Aims: Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis., Main Methods: Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence., Key Findings: We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs., Significance: Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells.

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.

In a previous work, we showed an increased cell motility due to the accumulation and transcriptional activation of the Hypoxia Inducible Factor-1α (HIF-1α) and a reduced mitochondrial energy production in an in vitro model of endothelial dysfunction (ED) represented by human endothelial cells (ECs) chronically deprived of nitric oxide (NO) by L-NAME treatment. In the present study, in the attempt to unravel the pathway(s) linking NO deficiency to HIF-1α accumulation and activation, we focused our attention on Reactive Oxygen Species (ROS). We found that ROS were partially involved in HIF-1α stabilization, but not in the pro-migratory phenotype. Regarding mitochondrial dysfunction, it did not require neither ROS generation nor HIF-1α activity, and was not due to autophagy. Very interestingly, while acute treatment with L-NAME induced a transient increase in ROS formation, chronic NO deprivation by long term L-NAME exposure drastically reduced cellular ROS content giving rise to an antioxidant environment characterized by an increase in superoxide dismutase-2 (SOD-2) expression and activity, and by nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2). These results might have important implications for our understanding of the consequences of NO deprivation in endothelium behavior and in the onset of cardiovascular diseases., (© 2013.)

Glioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated., Results: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N(G)-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O(2) consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells., Conclusion: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction., (© 2011 Cattaneo et al.)

Objective: To evaluate differences in endometrial stromal cell (ESC) migration between patients with and without endometriosis., Design: Differences in ESC migration, cellular morphology, and cytoskeletal-actin dynamics were evaluated in response to platelet-derived growth factor-BB (PDGF-BB) and steroid hormones (17beta-estradiol and progesterone)., Setting: Medical school research laboratory., Patient(s): Endometrial biopsy samples obtained from 43 women: 23 as controls (endometriosis excluded by laparoscopy), 20 with severe or moderate endometriosis (diagnosed by laparoscopy)., Intervention(s): ESCs were treated with and without PDGF-BB, 17beta-estradiol, and progesterone., Main Outcome Measure(s): Cellular migration was evaluated by means of chemotaxis experiments in a Boyden chamber. Cellular morphology and cytoskeletal-actin dynamics were evaluated by immunofluorescence., Result(s): Progesterone stimulated the migratory behavior of ESCs derived from women with endometriosis, while 17beta-estradiol could stimulate motility of ESCs derived from both controls and women with endometriosis, with a greater effect observed in the latter group. No difference in ESC migratory behavior after PDGF-BB treatment was observed between women with and without the disease. Also, PDGF-BB and steroid hormones could modify the organization of actin cytoskeletal structures., Conclusion(s): Ovarian steroids differently affect the migration of ESCs derived from women with and without endometriosis. This effect is likely to involve cytoskeletal reorganization. Nongenomic signaling pathways induced by steroid hormones might have a role in this phenomenon., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.

Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a key regulator of endothelial cell (EC) migration. Whereas the effects of acute NO generation are generally stimulatory, the role of chronic basal NO release has not been explored so far. Here, we addressed this question both in HeLa and in human endothelial cells. In stably transfected HeLa cells, inducibly expressing eNOS, expression of the enzyme per se blunted the phosphorylation of Akt/PKB in response to serum and strongly inhibited chemotaxis, an effect partially blocked by eNOS- and soluble guanylyl cyclase (sGC) inhibitors. Likewise, long-term pre-treatment of non-transfected HeLa cells with nanomolar concentrations of an NO donor inhibited subsequent migration, an effect blocked by sGC inhibition and mimicked by a cGMP analog. Finally, EC migration was stimulated by chronic pre-treatment with an eNOS inhibitor. Thus, in addition to its well-known stimulatory role, eNOS attenuates migration through basal long-term NO release.

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.

Malignant gliomas are highly invasive tumors which are lethal despite aggressive therapy. The motility behavior of two human glioma cell lines i.e. T98G and U87-MG cells was analysed. The glioma cells showed a high degree of basal motility (especially U87-MG cells) that may be related to the considerable local invasiveness of such tumours even in the absence of exogenous factors. The two cell lines responded equally well to platelet-derived growth factor (PDGF) as chemoattractant factor. The phosphatidylinositol 3-kinase (PI3-K) signaling, but not the extracellular signal-related kinase (ERK) signaling, was strongly involved in the PDGF-stimulated glioma cell motility. Somatostatin was capable of inhibiting the migration in both glioma cell lines without affecting crucial targets for motility control like PI3-K and Rac activity. These data suggest that somatostatin, by interfering with a target further downstream to Rac, negatively affects glioma cell motility, and may thus offer a pharmacological approach to controlling the deregulated motility of these aggressive tumoral cells.

Human endostatin is one of the better characterized endogenous angiogenesis inhibitors, and its ability to modulate vascularization of tumours could be of great therapeutic interest. These properties are not exclusive to the full-length protein, but are shared by some of its synthetic fragments. A number of research groups have partitioned human endostatin in different peptides and have investigated their activity, in order to collect a body of experimental data which could be important in shedding new light on their structure-activity relationships. It was also reported that a small active fragment can become inactive when contained in a larger fragment, revealing an apparent discrepancy in the experimental results. Very few studies have been devoted to the computational analysis of these systems and to the rationalization of their properties using molecular modelling. Through molecular dynamics simulations of human endostatin and of four synthetic fragments, we have been able to rationalize the experimental findings. In particular, we have identified a pattern consisting of six amino acids, namely R-R(G)-A-D-R-A, which appears to be an active epitope if it is properly exposed to the solvent. Interestingly, this pattern can be already present in sequential order in the primary structure, or it can be generated by the spatial approach of two groups of residues, far apart in the primary structure, as an effect of the peptide folding. Comparing the structural features and the time evolution of all the simulated peptides we provide a coherent explanation of their activity or inactivity.

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.

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.

Pharmacological control of the angiogenic process (i.e., the neovascularization necessary for the growth and progression of tumors and metastases) is considered to be one of the most promising approaches to antineoplastic therapy. Endostatin, a 20-kDa protein derived from collagen XVIII, is one of the first recently discovered endogeneous antiangiogenic substances, but its cell targets and mechanism(s) of action are still unknown. We thought it would be interesting to test whether shorter peptides derived from endostatin might preserve its antiangiogenic activity. Four synthetic peptides corresponding to the sequences 6-49 (I), 50-92 (II), 93-133 (III), and 134-178 (IV) of human endostatin were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis. Fragment I (and fragment IV in the tests performed) was found to be fully biologically active in all of the angiogenesis assays, and sometimes showed even greater potency and efficacy than full-length human endostatin itself., (Copyright 2003 Elsevier Science (USA))

1. Prostaglandin E(1) (PGE(1), alprostadil) is used as a vasodilator for the treatment of peripheral vascular diseases. 2. Previous reports suggested a pro-angiogenic effect for PGE(1). 3. We studied the in vitro and in vivo effect of PGE(1), complexed with alpha-cyclodextrin, on the angiogenic process. Contrary to what was expected, we found that, in human umbilical vein endothelial cells (HUVECs), PGE(1) inhibited proliferation, migration and capillary-like structure formation in Matrigel. 4. By RT-PCR studies, the expression of the EP(2) and EP(3) subtypes of the PG receptor was detected in HUVECs. 5. PGE(1) alone stimulated adenylate cyclase activity at micromolar concentrations, while at nanomolar concentrations potentiated the forskolin-induced cAMP accumulation. 6. 8-Bromoadenosine-3':5'-cyclic monophosphate (Br-cAMP) mimicked the inhibitory effect of PGE(1) on endothelial cell growth, motility and tube formation. 7. Sulprostone, an agonist at the EP(3) subtype of PG receptors, mimicked the in vitro anti-angiogenic effects of PGE(1), while butaprost, an EP(2) receptor agonist, had no effect. 8. Finally, in the plug assay model of angiogenesis in mice, PGE(1) showed a strong inhibitory effect on Matrigel neovascularization. 9. Thus, PGE(1) possesses strong anti-angiogenic activity in vitro and in vivo.

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein, a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.

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.

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.

We have previously reported that nicotine stimulates cell proliferation of three small-cell lung carcinoma (SCLC) cell lines by activating nicotinic receptors of the neuronal type. Here we report that, in the GLC-8 SCLC cell line, nicotine stimulates mitogen-activated protein (MAP) kinase activity in a concentration- and time-dependent manner (ED50 = 10 nM). The nicotine effect was antagonized by mecamylamine, an antagonist specific for neuronal nicotinic receptors. The absence of extracellular Ca2+, or pretreatment with pertussis toxin or the tyrosine kinase inhibitor genistein inhibited the action of nicotine on MAP kinase. Moreover, supernatants from nicotine-stimulated cells transferred to cells pretreated with mecamylamine were still capable of activating MAP kinase. On the other hand, the same supernatants transferred to cells pretreated with mecamylamine and pertussis toxin or genistein failed to activate MAP kinase. These findings suggest that nicotine elicits its stimulatory effect on MAP kinase in SCLC cells indirectly by inducing the production and/or release of a factor which then acts via a pertussis toxin- and tyrosine kinase-sensitive route.

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.

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.

Small cell lung cancer (SCLC) cells express cholinergic nicotinic receptors on their membranes, and the stimulatory effect on cell growth of nicotine has been described in cell cultures. We investigated three SCLC cell lines, all showing high levels of fluorescence intensity when labeled with FITC alpha-bungarotoxin in most cells. From one of these cell lines, NCI-N592 a tumor line in athymic nude mice was established. Nude mice were subcutaneously grafted on the same day with tumor fragments and with Alzet osmotic minipumps (200 microliters, 14 days of infusion) infused with serotonin or nicotine to investigate their effects on tumor growth. Two dose levels of each compound were used, namely 20 and 200 micrograms/day. In mice treated with 200 micrograms serotonin, tumors took a shorter time than those of untreated controls to reach 50 mg in volume, meaning that the first steps of tumor growth were faster. In contrast, a delay in tumor appearance was observed in mice treated with low-dose serotonin. No differences were found in tumor growth in the groups of mice treated with nicotine. When the treatment was delivered to already established and vascularized tumors (around 100 mm3), no effect on tumor growth was achieved by serotonin or nicotine. Therefore, in the experimental conditions used in the study, the stimulatory effect of nicotine on an SCLC tumor was not demonstrated.

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.

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.

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.

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.

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.

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.

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein--protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules.

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 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.

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.

Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 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 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.

We investigated the mechanism(s) whereby activation of a growth-factor receptor typically endowed with tyrosine kinase activity, such as the platelet-derived growth factor (PDGF) receptor, triggers phosphoinositide hydrolysis. In Swiss 3T3 cells permeabilized with streptolysin O, an analogue of GTP, guanosine 5'-[gamma-thio]triphosphate, was found to potentiate the coupling of the bombesin receptor to phospholipase C. In contrast, the activation of the enzyme by PDGF occurred in a GTP-independent manner. Moreover, the inactive analogue of GTP, guanosine 5'-[beta-thio]diphosphate, significantly inhibited the bombesin-induced InsP3 generation, whereas it did not decrease the same effect when stimulated by PDGF.

Alpha latrotoxin, purified from the venom of the black widow spider, is a high Mr (130000) protein devoid of detectable enzymatic activity. When applied to vertebrate nerve terminals (of the central as well as peripheral nervous systems) the toxin elicits massive release of neurotransmitters by stimulating the fusion of synaptic vesicles with the presynaptic membrane (exocytosis). Among non-neuronal systems, only the neurosecretory cell line PC12 is sensitive to alpha latrotoxin; all others investigated so far are insensitive. In order to act, alpha latrotoxin requires the presence of divalent cations in the medium. Ca2+ can be substituted by other divalent cations as Sr2+, Ba2+, Mn2+, Mg2+. However, with the last two the catecholamine release response is reduced in PC12 cells and synaptosomes. A specific, high affinity receptor of alpha latrotoxin exists in preparation sensitive to the toxin. This receptor has been purified and found to be a high Mr, integral membrane protein. In the frog neuromuscular junction the receptor is localized exclusively in the presynaptic membrane. Binding of alpha latrotoxin to the receptor in a Ca2+-containing incubation medium induces membrane depolarization (insensitive to tetrodotoxin), stimulation of Ca2+ influx (insensitive to verapamil) with consequent increase in the cytoplasmic free Ca2+ concentration and stimulation of phosphoinositide breakdown. In Ca2+ free medium depolarization is maintained, but free Ca2+ concentration does not rise after toxin application. In conclusion, alpha latrotoxin seems to act through a dual mechanism. The Ca2+-independent part of this mechanism may be mediated by the activation of protein kinase C, triggered by phosphoinositide metabolism. The relevance of these findings for presynaptic physiology is discussed.

Activation of the EGF receptor in A431 cells induces the hydrolysis of phosphoinositides and a transient rise of the cytosolic Ca2+ concentration, [Ca2+]i, 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 t1/2 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.

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 Ca2+, 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.

The mitogens serum, vasopressin and bradykinin stimulate a significant rise in the inositol phosphate content of cultured human fibroblasts within 10 seconds, while serum- and bradykinin-stimulated arachidonic acid release does not occur until after 30 seconds. The release of inositol phosphates is not secondary to a rise in Ca activity since the Ca ionophore ionomycin does not stimulate release of inositol phosphates. Moreover, we show that phospholipase C in human fibroblasts is activated by these mitogens at resting Ca levels since TMB-8, which blocks the mitogen-induced rise in Ca activity, does not affect the serum-stimulated accumulation of inositol phosphates.