Nuclear co-localization of prolactin and the prolactin receptor in rat Nb2 node lymphoma cells.

Previous studies have indicated that prolactin (PRL) interacts with specific, high affinity, immunoreactive binding sites within isolated rat hepatocyte nuclei. Moreover, endogenous PRL appears to be bound to this site. However, it remained important to demonstrate nuclear PRL receptors and hormonal translocation in an intact cell system. Therefore, we sought nuclear translocation of PRL and its receptor in the nucleus of PRL-dependent Nb2 node lymphoma cells. Utilizing immunofluorescence (IF) microscopy, growth-arrested cells were found to constitutively express the PRL receptor in the nucleus and in the membrane/cytosol compartments. Addition of PRL stimulated rapid hormone internalization followed by translocation to the nucleus within 6-12 hrs. The translocation of PRL was found to be reversible and dependent upon ATP. These results indicate that an early event coupled to the mitogenic action of PRL in Nb2 cells is hormone transport to the nucleus during the G1 and S phases of cell cycle. Once in the nucleus, PRL bound to its receptor may directly influence gene transcription.

Inhibition by genistein of prolactin-induced Nb2 lymphoma cell mitogenesis.

Tyrosine kinase activation in mediating the mitogenic action of prolactin (PRL) has been evaluated. Use was made of genistein, a tyrosine kinase antagonist, and cultured rat Nb2 lymphoma cells, i.e. the lactogen-dependent Nb2-11 line and a lactogen-independent subline, Nb2-SFJCD1. Genistein was found to be a potent growth-inhibitor for both lines, inhibiting 3H-thymidine incorporation in Nb2-11 and Nb2-SFJCD1 cells with IC50s of 4.2 and 6.7 micrograms/ml, respectively. Genistein also inhibited expression and translation of the heat shock protein 70 gene and pp40 protein substrate phosphorylation which, in Nb2-11 cells, followed PRL addition within minutes. Genistein inhibition of DNA synthesis in G1-arrested Nb2-11 cells was most pronounced if the agent was added within 1 h of PRL treatment. The results indicate that, while both Nb2 cell lines have a general growth requirement for tyrosyl phosphorylation, the early, PRL-induced tyrosine kinase activation is a component of the PRL mitogenic signal transduction pathway.

Down-regulation of the rat hepatic sterol 27-hydroxylase gene by bile acids in transfected primary hepatocytes: possible role of hepatic nuclear factor 1alpha.

In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP27) gene is transcriptionally repressed by hydrophobic bile acids. The molecular mechanism(s) of repression of CYP27 by bile acids is unknown. To identify the bile acid responsive element (BARE) and transcription factor(s) that mediate the repression of CYP27 by bile acids, constructs of the CYP27 5'-flanking DNA were linked to either the CAT or luciferase reporter gene and transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the -840/+23, -329/+23, and -195/+23 mCAT constructs. A -76/+23 construct showed no regulation by bile acids. When a DNA fragment (-110/-86) from this region was cloned in front of an SV 40 promoter it showed down-regulation by TDCA. 'Super'-electrophoretic mobility shift assays (EMSA) indicated that both HNF1alpha and C/EBP bind to the -110 to -86 bp DNA fragment. Recombinant rat HNF1alpha and C/EBPalpha competitively bound to this DNA fragment. 'Super'-EMSA showed that TDCA addition to hepatocytes in culture decreased HNF1alpha, but not C/EBP, binding to the -110/-86 bp DNA fragment. A four base pair substitution mutation (-103 to -99) in this sequence eliminated TCA and TDCA regulation of the (-840/+23) construct. The substitution mutation also eliminated (>95%) HNF1alpha, but not C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between -110 and -86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 transcriptional activity by decreasing HNF1alpha binding to the CYP27 promoter.

Titration of some thiazine dyes with TiCl3 at room temperature.

Methylene Blue and some other thiazine dyes are titrated at room temperature visually and potentiometrically in presence of 0.005-0.1N oxalic acid. The effect of oxalic acid on the conditional potentials of the Ti(IV)/Ti(III) couple is presented.

Prospective study of cortical sinovenous thrombosis value of CT scan and imaging.

Forty two consecutive cases of cortical sino venous thrombosis (CSVT) diagnosed by computerised tomographic scan (CT scan) and or magnetic resonance imaging (MRI) constituted the study material. Clinical features are similar despite of varied aetiology. Stroke like presentation was seen in 20 (48 percent) patients, features of raised intracranial pressure were seen in 14 (33 percent) patients and the remaining presented with diffuse encephalopathic features. No definitive aetiological factor could be found in 7 (17 percent) patients. Main CT scan features included empty delta sign (43 percent), cord sign (31 percent) and haemorrhagic or non haemorrhagic infarcts (62 percent), and CT scan was non diagnostic in 6 (14 percent) patients. The MRI imaging features included hyperintense signals in the sinuses more often in sagittal sinus with or without haemorrhagic infarct.

Experimental studies on the changes of adrenergic receptors in rat liver with acute injury and in endotoxemia.

Previous studies suggested that hepatic alpha and beta adrenergic receptors is stable and it is in dynamic equilibrium under normal conditions. However, at a pathological condition these receptors may be changed. We studied the changes of adrenergic receptors in the rat liver with acute injury and in endotoxemia by light microscopic autoradiography. Our experimental results reveal that in the liver of rats with acute injury and in endotoxemia there were changes in varying degrees of alpha and beta adrenoceptors. Moreover, the changes were not uniform in various structures of the rat liver. Glucagon can keep adrenergic receptors stable on hepatic cells and muscular cells of blood vessels in the rat liver. Our experiment provides evidence for clinical use of adrenoceptor antagonists in the treatment of acute and chronic liver disease.

Zinc accumulation in fiddler crabs Uca annulipes latreille and Uca triangularis (Milne Edwards).

The effect of exposure time and concentration on the tissue-specific accumulation of Zn in two fiddler crabs, Uca annulipes and Uca triangularis, obtained from polluted (Visakhapatnam Harbor) and unpolluted (Bhimilipatnam) areas was studied. Hepatopancreas registered major accumulation of Zn when the crabs were exposed to sublethal concentrations (6.38 to 15.39 ppm) for 30 days. However, when the crabs were exposed to different concentrations of Zn (LCO, LC25, LC50, LC75) ranging from 12.93 to 99.38 ppm, higher accumulation was recorded in gills than in hepatopancreas. There was a progressive increase in the percentage body burden of Zn in hepatopancreas, but that in gills showed a decreasing trend. The standardized body burdens for both species of crabs were calculated to evaluate their sensitivity to exposure time as well as to concentration. The fractional retention coefficients for exposure time estimated for exposed crabs indicate that U. triangularis from Bhimilipatnam, having the highest value, is sensitive and U. annulipes from Visakhapatnam Harbor, showing the lowest value, is resistant.

Rapid activation of mitogen-activated protein kinase and p21ras by prolactin and interleukin 2 in rat Nb2 node lymphoma cells.

Several serine/threonine and tyrosine kinase signal transduction pathways have been recently linked to prolactin (PRL) action in lymphoid cells. Utilizing the lactogen-dependent, rat pre-T lymphoma cell line, Nb2-11, and the autonomous subline, Nb2-SFJCD1, studies were conducted to determine whether PRL- or interleukin-2 (IL-2)-stimulated Nb2 cell proliferation is coupled to the activation of p21ras and mitogen-activated protein (MAP) kinase. Stimulation of Nb2-11 cells, growth-arrested in the early G1 phase of the cell cycle, with PRL or IL-2 rapidly (5-10 min) provoked GTP binding to Ras, enhanced tyrosyl phosphorylation of MAP kinase, significantly increased its enzymatic activity, and caused its nuclear translocation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase C, similarly activated Ras and MAP kinase but failed to cause its nuclear translocation. Tyrosine kinase antagonism with genistein inhibited PRL-stimulated Ras and MAP kinase activation. In other experiments, Ras and MAP kinase were each found to be constitutively active in the Nb2-SFJCD1 line. The addition of PRL to these cultures enhanced the activity of these signaling proteins. Finally, the effects of PRL, IL-2, TPA, and phosphatase inhibition on Nb2-11 cell population density and [3H]thymidine uptake were compared. The addition of PRL, IL-2, and TPA significantly stimulated[3H] thymidine incorporation, while only the polypeptide growth factors augmented cell density. Phosphatase inhibition had no effect on either parameter. These results indicate that Nb2 cell proliferation is associated with the early activation of Ras and MAP kinase. Moreover, tyrosyl phosphorylation upstream of Ras activation appears to be required for its subsequent stimulation of mediators, which activate MAP kinase. Protein kinase C activation may be coupled to MAP kinase activation but is not sufficient for Nb2 cell proliferation.

The nuclear prolactin receptor: a 62-kDa chromatin-associated protein in rat Nb2 lymphoma cells.

Previously we demonstrated that lactogen-dependent Nb2 cells express a nuclear prolactin (PRL) receptor. Thus, the nuclear receptor expressed in PRL-dependent Nb2-11 and -independent Nb2-SFJCD1 cells was characterized. Initially, the potential proteolytic processing of internalized 125I-rPRL was investigated. Radiolabeled hormone eluted from a Sephadex G-100 column with a retention time identical to that found for stock hormone, indicating that nuclear PRL was intact. Experiments to investigate the nuclear distribution of the hormone demonstrated that 90% of 125I-rPRL bound to chromatin; the remaining was distributed between "sap-protein" and nucleoplasmic fractions. Chromatin-bound PRL was resistant to high salt and detergent extraction indicating a tight association. Immunoprecipitation and immunoblot analysis revealed the PRL receptor to be 62 kDa in each cell line. Affinity crosslinking experiments and immunoprecipitation demonstrated that 125I-rPRL complexed with a protein(s) of similar M(r) in intact cells. 125I-rPRL binding was saturable and of high affinity (Kds of 180 and 170 pM, for Nb2-11 and Nb2-SFJCD1 lines, respectively). PRL binding was competitively inhibited by ovine and bovine PRLs and hGH, but not by rat GH, and by monoclonal antibodies (McAbs) which recognize the lactogen binding site. These results demonstrate that: (1) 125I-rPRL translocates intact to the Nb2 cell nucleus and tightly associates with chromatin; (2) the chromatin receptor specifically binds 125I-rPRL with high affinity; and (3) the chromatin receptor is essentially identical to its membrane counterpart with respect to mass, binding characteristics, and McAb recognition.

Nuclear translocation of prolactin: collaboration of tyrosine kinase and protein kinase C activation in rat Nb2 node lymphoma cells.

Recent evidence has suggested that prolactin (PRL), internalized by lactogen-dependent Nb2 lymphoma cells, is actively translocated to the nucleus where it binds to PRL receptors. Moreover, the mitogenic action of PRL in these cells has been separately linked to protein tyrosyl phosphorylation and activation of protein kinase C (PKC). Therefore, the coupling of PRL internalization and nuclear translocation to the activation of these signal transduction pathways was investigated. Results from control experiments indicated that 30% of internalized and 5% total cell-associated 125I-rat PRL could be recovered within nuclei obtained from Nb2 cells previously incubated with the radiolabel for 3 h at 37 degrees C. Furthermore, internalized PRL was found to be intact and not associated with any carrier proteins. Addition of tyrosine kinase (TK) antagonists, genistein or tyrphostin, significantly reduced cell surface binding, internalization, and nuclear translocation of 125I-rat PRL. In contrast, neither the level of cell-associated nor internalized hormone differed between cells treated with the PKC antagonists, staurosporine or calphostin C, and control cultures. Instead, PKC inhibition significantly reduced nuclear PRL translocation. The inhibitory effects of the TK and PKC antagonists on PRL internalization and nuclear translocation in intact Nb2 cells were verified by immunofluorescence microscopy in parallel experiments. In other experiments, each of the kinase inhibitors blocked PRL-stimulated Nb2 cell proliferation in a concentration-dependent manner. It is concluded that activated TK and PKC collaborate in the process of PRL internalization and translocation to the nucleus. TK activation may participate in PRL receptor binding or hormone internalization while activation of PKC appears to be required for its nuclear targeting. Since TK and PKC activation are required for lactogen-stimulated Nb2 cell proliferation, we suggest that a component of the mitogenic pathway in these cells is a direct nuclear interaction of PRL.

Prolactin-induced phosphorylation and nuclear translocation of MAP kinase in Nb2 lymphoma cells.

The coupling of prolactin (PRL) receptor ligation to activation of mitogen-activated protein (MAP) kinase was sought in rat Nb2 lymphoma cells, a pre-T lymphocyte line dependent upon lactogens for proliferation. Addition of PRL (20 ng/ml) to Nb2 cells, growth arrested in the early G1 phase of cell cycle, stimulated rapid tyrosyl phosphorylation of MAP kinase (min). Phosphorylated MAP kinase subsequently translocated to the nucleus, with kinetics essentially identical to those demonstrated for nuclear accumulation of PRL. The rapidity of MAP kinase activation suggests an intermediary role for this enzyme in PRL receptor signalling. Moreover, nuclear translocation of MAP kinase provides an interactive mechanism by which PRL, together with its nuclear receptor, may regulate transcription requisite for mitogenesis.

Bile salt-induced apoptosis of hepatocytes involves activation of protein kinase C.

Toxic bile salts induce hepatocyte apoptosis, a model relevant to liver injury during cholestasis. However, the signaling mechanisms culminating in bile salt-induced apoptosis remain unclear. Because protein kinase C (PKC) is activated by bile salts in hepatocytes and causes apoptosis in other cells, we tested the hypothesis that bile salt-induced hepatocyte apoptosis is mediated by PKC. The PKC inhibitors chelerythrine and Gö-6976 reduced, whereas a PKC agonist, phorbol 12-myristate 13-acetate (PMA), increased glycochenodeoxycholate (GCDC)-induced hepatocyte apoptosis. Membrane-associated total PKC activity was increased in GCDC-treated hepatocytes. Quantitative immunoblot analysis demonstrated membrane translocation of PKC-alpha, PKC-delta, and PKC-epsilon to hepatocyte membranes after administration of GCDC. Direct activation of PKC-alpha and PKC-delta by GCDC was also demonstrated using recombinant, baculovirus-expressed PKC isoforms in a medium of defined lipid composition. Chelerythrine and Gö-6976 reduced, whereas PMA enhanced, cathepsin B activity during treatment of hepatocytes with GCDC, demonstrating coupling of PKC activity to the protease effector mechanisms of apoptosis. In conclusion, our data suggest for the first time that PKC-dependent signaling pathways play a critical role in bile salt-induced hepatocyte apoptosis.

Hepatocellular protein kinase C activation by bile acids: implications for regulation of cholesterol 7 alpha-hydroxylase.

We have recently shown that taurocholate (TCA) represses the transcriptional activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of the bile acid biosynthetic pathway, through a protein kinase C (PKC)-dependent mechanism in primary cultures of rat hepatocytes. The present studies sought to determine the mechanisms by which bile acids activate hepatic PKC activity and the consequences of this activation on isoform distribution and cholesterol 7 alpha-hydroxylase mRNA levels. TCA (12.5-100 microM for 15 min) increased membrane-associated "classic" isoenzyme cPKC-alpha and "novel" isoenzymes nPKC-delta, and nPKC by two- to sixfold. Membrane-associated PKC progressively increased, and cytosolic PKC decreased, for 1 h after the addition of TCA (50 microM); after 24 h whole cell cPKC-alpha, nPKC-delta, and nPKC were downregulated by 35-55% compared with untreated controls. In a reconstituted assay system, TCA or taurodeoxycholate (10-100 microM) increased calcium-dependent and -independent PKC activity by three- and fourfold, respectively. Taurine-conjugated bile acids stimulated PKC activity in proportion to their hydrophobicity index (r = 0.99). Finally, cholesterol 7 alpha-hydroxylase mRNA was repressed > 75% by phorbol 12-myristate 13-acetate (100 nM for 3 h), a nonselective activator of PKC isoforms. In contrast, selective cPKC-alpha activation with thymeleatoxin (100 nM for 3 h) had no significant effect on cholesterol 7 alpha-hydroxylase mRNA levels. We conclude that bile acids activate hepatocellular PKC, resulting in sequential redistribution and down-regulation of calcium-dependent and -independent isoforms. The calcium-independent PKC isoforms may mediate the repression of cholesterol 7 alpha-hydroxylase mRNA by TCA.

Activation of protein kinase C alpha and delta by bile acids: correlation with bile acid structure and diacylglycerol formation.

The feedback repression of cholesterol 7alpha-hydroxylase transcriptional activity and mRNA levels by taurocholate (TCA) occurs via a protein kinase C (PKC)-dependent signal. To determine whether bile acids could activate PKC indirectly via generation of diacylglycerol (DG), their effects on DG levels in primary cultures of rat hepatocytes were determined using a DG kinase assay. To determine whether bile acids might activate PKC isozymes more directly, their effects on PKC alpha and delta purified from baculovirus expression systems were examined in phosphatidylserine/phosphatidylcholine/Triton X-100 (PS/PC/TX) mixed micelles. Addition of tauroursodeoxycholate (TUDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) (50 microM) to the cells rapidly (15 min) increased DG content in cultured rat hepatocytes to 105%, 155%, and 130%, respectively, as compared to untreated control cultures. Addition of TCA increased PKC alpha specific activity with EC50 of approximately 400 nM; maximal activity was observed with 5 microM. Other taurine-conjugated bile acids (5 microM) increased PKC alpha specific activity (pmol/min/microg protein) in proportion to their relative hydrophobicity: PS/PC/TX 17 +/- 2; + TUDCA 29 +/- 18; + TCA 68 +/-13; + TDCA 166 +/- 21; and, taurochenodeoxycholate 178 +/- 20 (P vs. PS/PC/TX = 0.54, 0.019, 0.002, and 0.001, respectively); unconjugated bile acids gave similar results (r2 for activity vs. hydrophobicity index 0.59). Taurine-conjugated bile acid interaction enthalpies, as determined by dimyristoyl-phosphatidylcholine chromatography, were more highly correlated (r2 = 0.96) with PKC alpha activation than with the hydrophobicity index. TCA also stimulated the activity of purified PKCdelta with EC50 of approximately 150 nM and maximally (2.7-fold) at 1 microM. Free and taurine-conjugated bile acids (1 microM) increased PKCdelta activity according to their hydrophobicity index (r2 = 0.89) and interaction enthalpies (r2 = 0.96).