Abstract We report the cloning of the mouse surface GPI-anchored Ly-6E.1 protein from a highly metastatic mouse adenocarcinoma cell line CSML-100 by differential display. The expression is specific for the metastatic cell line as the closely related, non-metastatic mouse adenocarcinoma cell line CSML-0 does not express Ly-6E.1. Northern blot analysis reveals expression in a number of mouse tumour cell lines, exclusively metastatic ones. To date, active Ly-6A/E has only been described in lymphoid cells. The correlation between Ly-6E.1 expression, and the ability to metastasize, is discussed. © Federation of European Biochemical Societies.

Abstract The effect of somatostatin and α2-adrenergic agonists on cyclic AMP accumulation was examined in MDCK cells, grown in denned medium. These hormones inhibited vasopressin-induced cyclic AMP formation, without affecting either the basal or the glucagon- and prostaglandin E2-stimulated level. Pretreating the cells with pertussis toxin, or incubating them with MnCl2 at a low concentration reversed the effect of somatostatin and α2-agonists. These results suggest that somatostatin and norepinephrine could selectively modulate the renal effect of vasopressin, via the inhibitory regulatory subunit (Ni) of adenylate cyclase.

Abstract Addition of ATP (but not epinephrine, angiotensin II, vasopressin, or platelet-activating factor) to H-35 hepatoma cells whose cellular lipids have been pre-labelled with [3H]inositol, causes a rapid increase in [3H]inositol trisphosphate. In H-35 cells pre-incubated in the presence of 45Ca2+, ATP causes a similarly rapid release of 45Ca2+. The concentration-effect relationships for inositol trisphosphate formation and Ca2+ efflux are similar to those reported previously for differentiated hepatocytes. These results demonstrate that at least one of the Ca2+-mobilizing receptors normally found on hepatocytes is functionally retained in the H-35 hepatoma cell line and thus could provide a useful model for the study of these receptor mechanisms in liver.

Abstract The Menkes (MNK) protein is a vital component of copper homeostasis in mammalian cells. In this paper we provide the first biochemical evidence that the MNK protein functions as a copper-translocating P-type ATPase in mammalian cells. The enzyme activity in membrane vesicles prepared from Chinese hamster ovary cells overexpressing MNK was ATP-dependent, correlated with the amount of MNK and followed Michaelis-Menten kinetics with respect to copper. The copper transport was observed only under reducing conditions suggesting MNK transports Cu(I). This study opens the way to detailed structure-function studies and assessment of functional MNK derived from patients with Menkes disease.

Abstract The oral antidiabetic sulfonylurea [3H]glibenclamide specifically binds to plasma membranes from a rat β-cell tumor indicating a receptor for sulfonylureas in these membranes. Irradiation of [3H]glibenclamide at 254 or 300 nm in the presence of albumin resulted in covalent labeling of the albumin molecule. Direct photoaffinity labeling of β-cell membranes with [3H]glibenclamide resulted in the covalent modification of two membrane polypeptides with apparent molecular masses 140 and 33 kDa. The extent of labeling of the 140 kDa polypeptide was specifically decreased by sulfonylureas. This suggests that a membrane polypeptide of 140 kDa is a component of the sulfonylurea receptor in the β-cell membrane.

Abstract We examined the effects of short-term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol-induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.

Abstract Little is known about the regulation of signal transducer and activator of transcription (STAT) stability. Here the osmolarity-dependence of STAT3 stability, ubiquitination, Tyr705 phosphorylation, STAT3 transactivation and γ-fibrinogen (γ-FBG) expression was studied in hepatoma cells. Hyper-osmolarity accelerated STAT3 degradation which was prevented by proteasome inhibitors. Hypo-osmolarity stabilized STAT3, most likely due to a decrease in STAT3 ubiquitination. Accordingly, STAT3 Tyr705 phosphorylation, α2-macroglobulin promoter activity and γ-FBG expression were osmosensitive. Modulation of STAT3 stability may contribute to a hydration dependence of acute phase protein expression.

Abstract P84N5 is a death domain containing protein that interacts with the tumor suppressor retinoblastoma protein and induces apoptosis. We cloned and characterized two novel alternatively spliced versions of p84N5. The p84N5 short isoform (p84N5s) lacks the death domain and does not induce apoptosis. We showed that p84N5, but not p84N5s, is cell cycle regulated. We found that p84N5-GFP chimera can rapidly shuttle between the nucleus and the cytoplasm. Taken together, these observations suggest that p84N5 may transmit signals from the nucleus to cytoplasmic effectors.

Abstract Tumor necrosis factor (TNF) plays a central role in the state of insulin resistance leading to type II diabetes. We here describe the crosstalk of TNF with insulin signaling cascades in the mouse muscle cell line pmi28. TNF downregulated insulin induced insulin receptor kinase activity and insulin induced activation of the transcription factor STAT5. Our results provide evidence that the inhibitory crosstalk between TNF and insulin in skeletal muscle cells comprises an interference with the expression of STAT5 regulated genes which may play an important role in the manifestation and/or progression of insulin resistance in muscle cells.

Abstract In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.

Abstract We report the cloning of the mouse surface GPI-anchored Ly-6E.1 protein from a highly metastatic mouse adenocarcinoma cell line CSML-100 by differential display. The expression is specific for the metastatic cell line as the closely related, non-metastatic mouse adenocarcinoma cell line CSML-0 does not express Ly-6E.1. Northern blot analysis reveals expression in a number of mouse tumour cell lines, exclusively metastatic ones. To date, active Ly-6A/E has only been described in lymphoid cells. The correlation between Ly-6E.1 expression, and the ability to metastasize, is discussed. © Federation of European Biochemical Societies.

Abstract The effect of somatostatin and α2-adrenergic agonists on cyclic AMP accumulation was examined in MDCK cells, grown in denned medium. These hormones inhibited vasopressin-induced cyclic AMP formation, without affecting either the basal or the glucagon- and prostaglandin E2-stimulated level. Pretreating the cells with pertussis toxin, or incubating them with MnCl2 at a low concentration reversed the effect of somatostatin and α2-agonists. These results suggest that somatostatin and norepinephrine could selectively modulate the renal effect of vasopressin, via the inhibitory regulatory subunit (Ni) of adenylate cyclase.

Abstract Addition of ATP (but not epinephrine, angiotensin II, vasopressin, or platelet-activating factor) to H-35 hepatoma cells whose cellular lipids have been pre-labelled with [3H]inositol, causes a rapid increase in [3H]inositol trisphosphate. In H-35 cells pre-incubated in the presence of 45Ca2+, ATP causes a similarly rapid release of 45Ca2+. The concentration-effect relationships for inositol trisphosphate formation and Ca2+ efflux are similar to those reported previously for differentiated hepatocytes. These results demonstrate that at least one of the Ca2+-mobilizing receptors normally found on hepatocytes is functionally retained in the H-35 hepatoma cell line and thus could provide a useful model for the study of these receptor mechanisms in liver.

Abstract The Menkes (MNK) protein is a vital component of copper homeostasis in mammalian cells. In this paper we provide the first biochemical evidence that the MNK protein functions as a copper-translocating P-type ATPase in mammalian cells. The enzyme activity in membrane vesicles prepared from Chinese hamster ovary cells overexpressing MNK was ATP-dependent, correlated with the amount of MNK and followed Michaelis-Menten kinetics with respect to copper. The copper transport was observed only under reducing conditions suggesting MNK transports Cu(I). This study opens the way to detailed structure-function studies and assessment of functional MNK derived from patients with Menkes disease.

Abstract The oral antidiabetic sulfonylurea [3H]glibenclamide specifically binds to plasma membranes from a rat β-cell tumor indicating a receptor for sulfonylureas in these membranes. Irradiation of [3H]glibenclamide at 254 or 300 nm in the presence of albumin resulted in covalent labeling of the albumin molecule. Direct photoaffinity labeling of β-cell membranes with [3H]glibenclamide resulted in the covalent modification of two membrane polypeptides with apparent molecular masses 140 and 33 kDa. The extent of labeling of the 140 kDa polypeptide was specifically decreased by sulfonylureas. This suggests that a membrane polypeptide of 140 kDa is a component of the sulfonylurea receptor in the β-cell membrane.

Abstract We examined the effects of short-term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol-induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.

Abstract Little is known about the regulation of signal transducer and activator of transcription (STAT) stability. Here the osmolarity-dependence of STAT3 stability, ubiquitination, Tyr705 phosphorylation, STAT3 transactivation and γ-fibrinogen (γ-FBG) expression was studied in hepatoma cells. Hyper-osmolarity accelerated STAT3 degradation which was prevented by proteasome inhibitors. Hypo-osmolarity stabilized STAT3, most likely due to a decrease in STAT3 ubiquitination. Accordingly, STAT3 Tyr705 phosphorylation, α2-macroglobulin promoter activity and γ-FBG expression were osmosensitive. Modulation of STAT3 stability may contribute to a hydration dependence of acute phase protein expression.

Abstract P84N5 is a death domain containing protein that interacts with the tumor suppressor retinoblastoma protein and induces apoptosis. We cloned and characterized two novel alternatively spliced versions of p84N5. The p84N5 short isoform (p84N5s) lacks the death domain and does not induce apoptosis. We showed that p84N5, but not p84N5s, is cell cycle regulated. We found that p84N5-GFP chimera can rapidly shuttle between the nucleus and the cytoplasm. Taken together, these observations suggest that p84N5 may transmit signals from the nucleus to cytoplasmic effectors.

Abstract Tumor necrosis factor (TNF) plays a central role in the state of insulin resistance leading to type II diabetes. We here describe the crosstalk of TNF with insulin signaling cascades in the mouse muscle cell line pmi28. TNF downregulated insulin induced insulin receptor kinase activity and insulin induced activation of the transcription factor STAT5. Our results provide evidence that the inhibitory crosstalk between TNF and insulin in skeletal muscle cells comprises an interference with the expression of STAT5 regulated genes which may play an important role in the manifestation and/or progression of insulin resistance in muscle cells.

Abstract In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.