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S Greco, C Storelli, and S Marsigliante

In this paper the signal transduction pathways evoked by bradykinin (BK) in MCF-7 breast cancer cells were investigated. BK activation of the B2 receptor provoked: (a) the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2); (b) the translocation from the cytosol to the membrane of the conventional protein kinase C-α (PKC-α) and novel PKC-δ and PKC-ε; (c) the phosphorylation of protein kinase B (PKB/ Akt); (d) the proliferation of MCF-7 cells. The BK-induced ERK1/2 phosphorylation was completely blocked by PD98059 (an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK)) and by LY294002 (an inhibitor of phosphoinositide 3-kinase (PI3K)), and was reduced by GF109203X (an inhibitor of both novel and conventional PKCs); Gö6976, a conventional PKCs inhibitor, did not have any effect. The BK-induced phosphorylation of PKB/Akt was blocked by LY294002 but not by PD98059. Furthermore, LY294002 inhibited the BK-provoked translocation of PKC-δ and PKC-ε suggesting that PI3K may be upstream to PKCs. Finally, the proliferative effects of BK were blocked by PD98059, GF109203X and LY294002. These observations demonstrate that BK acts as a proliferative agent in MCF-7 cells activating intracellular pathways involving novel PKC-δ/-ε, PKB/Akt and ERK1/2.

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S Marsigliante, A Muscella, S Barker, and C Storelli

We have previously shown that angiotensin II (Ang II) has a role at the level of the eel gill chloride cell regulating sodium balance, and therefore osmoregulation; the purpose of the present study was to extend these findings to another important osmoregulatory organ, the kidney. By catalytic histochemistry Na(+)/K(+)ATPase activity was found in both sea water (SW)- and freshwater (FW)-adapted eel kidney, particularly at the level of both proximal and distal tubules. Quantitation of tubular cell Na(+)/K(+)ATPase activity, by imaging, gave values in SW-adapted eels which were double those found in FW-adapted eels (Student's t-test: P<0.0001). This was due to a reduced number of positive tubules present in FW-adapted eels compared with SW-adapted eels. By conventional enzymatic assay, the Na(+)/K(+)ATPase activity in isolated tubular cells from SW-adapted eels showed values 1.85-fold higher those found in FW-adapted eels (Student's ttest: P<0.0001). Perfusion of kidney for 20 min with 100 nM Ang II provoked a significant increase (1.8-fold) in Na(+)/K(+)ATPase activity in FW, due to up-regulation of Na(+)/K(+)ATPase activity in a significantly larger number of tubules (Student's t-test: P<0.0001). The effect of 100 nM Ang II in SW-adapted kidneys was not significant. Stimulation with increasing Ang II concentrations was performed on isolated kidney tubule cells: Ang II provoked a dose-dependent stimulation of the Na(+)/K(+)ATPase activity in FW-adapted eels, reaching a maximum at 100 nM (1.82-fold stimulation), but no significant effect was found in SW-adapted eels (ANOVA: P<0.001 and P>0.05 respectively). Isolated tubule cells stimulated with 100 nM Ang II showed a significant generation of inositol trisphosphate (InsP(3)) and an increment in calcium release from intracellular stores. In conclusion, our results suggest that tubular Na(+)/K(+)ATPase is modulated by environmental salinity, and that Ang II has a role in regulating its activity in FW-adapted eels, probably through an InsP(3)-dependent mechanism.

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A Muscella, S Greco, MG Elia, C Storelli, and S Marsigliante

Here we demonstrated, by RT-PCR analysis, the expression of both angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in a breast cancer epithelial cell line, MCF-7. Ang II was not able to affect the intracellular Ca2+ concentration in Fura-2 loaded cells suggesting that AT1-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na+/K+ATPase in a dose- and time-dependent manner and was mitogenic, with a dose-dependent (1-1000 nM) proliferative effect and a maximal response at 100 nM. Both Na+/K+ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT1, as the effects were completely blocked by DuP 753, a specific AT1 antagonist. CGP 42112, an AT2 antagonist, did not affect Ang II actions. The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na+/K+ATPase in breast cancer epithelial cells, MCF-7, via AT1 activation independently of the Ca(2+) signalling mechanism.

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A Muscella, S Marsigliante, MA Carluccio, GP Vinson, and C Storelli

Cultured human umbilical vein endothelial cells (HUVECs) at passage 4 specifically bound 70 +/- 12 fmol [3,5-3H]Tyr4-Ile5-angiotensin (Ang) II/mg protein, with a Kd of 0.9 +/- 0.36 nM. Binding was eliminated in cells preincubated with a monoclonal antibody (6313/G2) raised against the subtype AT1 of the Ang II receptor. Freshly seeded HUVECs were positive for 6313/G2 antibody by immunocytochemistry, and such immunoreactivity was still retained at passage 4. Incubation of HUVECs for 20 min with different concentrations of Ang II provoked a significant increment in Na+/K+ ATPase activity compared with controls, in a dose- and time-dependent manner. Maximal response was obtained with 1000 pM Ang II after 20 min stimulation and resulted in a 2.2-fold increment in Na+/K+ ATPase activity. This stimulation was abolished when cells were incubated with 1000 pM Ang II in the presence of 1 microM of the specific AT1 subtype inhibitor, DuP753. Moreover, preincubation of HUVECs with 6313/G2 or with 1 mM dithiothreitol also inhibited the stimulatory effect of Ang II. These results suggest that the AT1 receptor subtype mediates the Na+/K+ ATPase response to Ang II in these cells.

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S Marsigliante, A Muscella, S Greco, MG Elia, S Vilella, and C Storelli

In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation.

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S Greco, M G Elia, A Muscella, S Romano, C Storelli, and S Marsigliante

We have previously reported that bradykinin (BK) represents an influential mitogenic agent in normal breast glandular tissue. We here investigated the mitogenic effects and the signalling pathways of BK in primary cultured human epithelial breast cells obtained from a tumour and from the histologically proven non-malignant tissue adjacent to the tumour. BK provoked cell proliferation, increase in cytosolic calcium, activation of protein kinase C (PKC)-α, -β, -δ, -ε and -η and phosphorylation of the extracellular-regulated kinases 1 and 2 (ERK1/2). The following compounds blocked the proliferative effects of BK: Hyp3-BK, a B2 receptor subtype inhibitor; U73122, a phospholipase C-β inhibitor; GF109203X, a protein kinase C (PKC) inhibitor; and PD98059, a mitogen-activated protein kinase kinase inhibitor. Gö6976, a Ca2+-dependent PKC inhibitor, did not have any effect. In conclusion, the mitogenic effects of BK are retained in peritumour and tumour cells; hence, it is likely that BK has an important role in cancer endorsement and progression.

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S Greco, A Muscella, MG Elia, P Salvatore, C Storelli, and S Marsigliante

The effect of angiotensin II (Ang II) on Ca(2+) signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca(2+) probe. Ang II (0.1-1000 nM) induced an intracellular free calcium ([Ca(2+)](i)) transient peak which was unchanged by external Ca(2+ )removal. In Ca(2+)-free medium pretreatment with thapsigargin abolished Ang II-induced Ca(2+ )release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca(2+) response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca(2+)](i) increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca(2+) response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation. Epithelial breast cells expressed PKC-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by Go 6976, a specific inhibitor of PKC-alpha and -beta1, the Ca(2+)-dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca(2+)](i )released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.

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A Muscella, S Greco, MG Elia, E Jimenez, C Storelli, and S Marsigliante

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.

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S Marsigliante, R Acierno, M Maffia, A Muscella, G P Vinson, and C Storelli


The monoclonal antibody 6313/G2 raised against the mammalian type I (AT1) angiotensin II (Ang II) receptor (Ang II-R) also recognises a component in teleost (eel) tissue preparations that binds radiolabelled Ang II, and has an isoelectric point (pI) of 6·5 and molecular mass of 75 kDa. Immunohistochemical analysis using this antibody showed specific binding sites in eel intestine, kidney, gill and liver sections.

The same antibody was used here to evaluate the presence and distribution of Ang II-R in target tissues of the Antarctic teleost icefish (Chionodraco hamatus).

Immunocytochemistry of intestine and gill sections showed that the antibody bound to uniformly distributed intracellular sites and cell surface membranes in absorptive cells in the intestine and chloride and pavement cells in the gills. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. In the kidney, only the tubules in the trunk stained positively while the head (atubular part of the kidney) was negative. In kidney tubules, in contrast with other tissues, the receptor was most concentrated in the cytoplasm underlying the basolateral membranes, with somewhat weaker staining beneath the apical cell membrane. Immunoblotting identified a single component from trunk kidney preparations that focused at pI 5·9 in isoelectric focusing gels and showed a molecular mass of 75 kDa in SDS–polyacrylamide gels.

The data suggest that, as in other teleosts, Ang II has a physiological role in the icefish.

Journal of Endocrinology (1997) 154, 193–200