Lucia M. Vicentini

Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts.

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.

Anti-migratory and Anti-invasive Effect of Somatostatin in Human Neuroblastoma Cells INVOLVEMENT OF RAC AND MAP KINASE ACTIVITY

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.

Chronic opiate treatment does not modify α2-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.

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.

Chapter 10 Mechanisms of Growth Factor Stimulation of Na+-H+ Exchange in Cultured Fibroblasts

The data presented in this paper suggest that we have identified several key steps in the sequence of events occurring between binding of growth factors to their surface receptors and the activation of the Na + -H + exchange system. Our current working model is shown in Fig. 4. The initial step after binding of peptide rnitogens appears to be the release of inositol trisphosphate from membrane pools of phosphatidylinositol 4′,5′-bisphosphate (PIP 2 ). This inositol trisphosphate then interacts with an internal Ca2 + storage site to mobilize intracellular Ca 2+ thereby elevating the intracellular Ca 2+ activity. An elevation of Ca 2+ activity then acts through a Ca 2+ -dependent regulatory protein which somehow leads to the activation of the Na + -H + exchanger. The current data are most consistent with calmodulin being the Ca 2+ -dependent regulatory protein involved in the activation process; however, the synergism between A23187 and TPA suggests that protein kinase C may also play some role in this process, although the lack of effect of TPA alone suggests that activation of protein kinase C is not a sufficient stimulus for activation of the Na + -H + exchanger. The current data on phospholipase involvement are most readily explained on the basis of a mitogen activation of phospholipase C activity which acts to release inositol trisphosphate, which in turn mobilizes intracellular Ca 2+ . However, it should be pointed out that in general the compounds (mepacrine and melittin) that we have used to perturb phospholipase activity have been traditionally thought of as interacting with phospholipase A 2 instead of phospholipase C. However, recent results in our laboratory indicate that mepacrine will inhibit the mitogen-induced release of inositol trisphosphate and that melittin will stimulate its release in the absence of mitogens (Jamieson and Villereal, in preparation), suggesting either that these compounds do interact with phospholipase C or that there is some regulation of phospholipase C activity by the breakdown products of phoshpolipase A 2 activity. A report from Majeruss laboratory indicating that mepacrine directly inhibits phospholi pase C activity suggests that effects of mepacrine in the HSWP cell are probably at the level of phospholipase C. In the summary scheme (Fig. 4), the possibility that the release of inositol trisphosphate could be potentiated via the stimulation of a kinase activity which would increase the substrate (PIP2) available to phospholipase C is presented. This possibility is suggested by the observation that certain oncogene products with kinase activity can phosphorylate phosphatidylinositol to the polyphosphorylated forms (Sugimoto et al. , 1984; Macara et al. , 1984).

Fatty acids rather than hormones restore in vitro angiogenesis in human male and female endothelial cells cultured in charcoal-stripped serum

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.

Human umbilical endothelial cells (HUVECs) and sex differences

HUVECs are worldwide used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. The present work characterised the phenotype of HUVECs in terms of morphology, proliferative and migratory capacity and in the gene expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3) to evaluated if they are sexually dimorphic. Moreover, autophagic process was analysed in male and female HUVECs (MHUVECs and FHUVECs), as autophagy is influenced by sex. Umbilical cords were obtained from healthy, normal weight, male and female neonates born to healthy non-obese and non-smoking women. HUVECs morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Real-time PCR was used to evaluate gene expression for oestrogen and androgen receptors and for NOS3, while the expression of the primary molecules involved in autophagic process [(Akt, the mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] and NOS3 were analysed by western blotting. FHUVECs showed significantly higher proliferation and migration rate, and NOS3 mRNA and protein expression than MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating a higher autophagy in male cells as also indicated by ultrastructural analysis showing a buildup of autophagic vacuoles at different stages in MHUVECs. The expression of oestrogen and androgen receptor genes, the protein expression of Akt, mTOR, and cellular size and shape were not influenced by sex. Male and female neonates did not differ in body weight, but the weight of male babies was positively associated with the weight of the mother, suggesting that the weight of the mother may exert a different influence on male and female babies. Our findings indicate that sex differences exist from prenatal life and are parameter- specific, suggesting that a better quality of the research on the endothelium in vitro can be obtained by analyzing HUVECs of both sexes as well as its translational value. Moreover, the sex differences observed in HUVECs could help the diseases of adulthood because endothelial dysfunction has a key role in cardiovascular diseases, diabetes mellitus, neurodegeneration and immune diseases.