Combining Cadherin Expression with Molecular Markers Discriminates Invasiveness in Growth Hormone and Prolactin Pituitary Adenomas.

Although growth hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas are considered benign, in many patients, tumour growth and/or invasion constitute a particular challenge. In other tumours, progression relies in part on dysfunction of intercellular adhesion mediated by the large family of cadherins. In the present study, we have explored the contribution of cadherins in GH and PRL adenoma pathogenesis, and evaluated whether this class of adherence molecules was related to tumour invasiveness. We have first established, by quantitative polymerase chain reaction and immunohistochemistry, the expression profile of classical cadherins in the normal human pituitary gland. We show that the cadherin repertoire is restricted and cell-type specific. Somatotrophs and lactotrophs express mainly E-cadherin and cadherin 18, whereas N-cadherin is present in the other endocrine cell types. This repertoire undergoes major differential modification in GH and PRL tumours: E-cadherin is significantly reduced in invasive GH adenomas, and this loss is associated with a cytoplasmic relocalisation of cadherin 18 and catenins. In invasive prolactinomas, E-cadherin distribution is altered and is accompanied by a mislocalisation of cadherin 18, ß-catenin and p120 catenin. Strikingly, de novo expression of N-cadherin is present in a subset of adenomas and cells exhibit a mesenchymal phenotype exclusively in invasive tumours. Binary tree analysis, performed by combining the cadherin repertoire with the expression of a subset of known molecular markers, shows that cadherin/catenin complexes play a significant role in discrimination of tumour invasion.

Coordinate control of Na,K-atpase mRNA expression by aldosterone, vasopressin and cell sodium delivery in the cortical collecting duct.

We have examined the respective influence of aldosterone, vasopressin and cell sodium delivery on Na+,K+-ATPase expression. The level of expression of the mRNA encoding for the alpha1- and beta1-subunits of Na+,K+-ATPase was evaluated in cortical collecting duct (CCD) cells from rats under different aldosterone status, in cells from the rat CCD cell line RCCD1 treated or not with vasopressin and in CCD cells from mice inactivated or not for the a-subunit of the epithelial sodium channel. The amount of mRNA was determined by in situ hybridization. Both aldosterone and vasopressin up-regulate transcripts encoding for the alpha1-subunit of Na+,K+-ATPase while beta1 is unaltered. Interestingly, when cell sodium entry was largely reduced (alphaENaC knock-out mice), the amount of transcripts encoding for the alpha1-subunit of Na+,K+-ATPase was significantly decreased in spite of high plasma aldosterone concentrations. No effect was observed on beta1-subunit. Altogether, these results suggest a coordinated hormonal and ionic control of Na+,K+-ATPase expression by different transcriptional pathways (steroid-receptor, cAMP-dependent and Na+dependent) in CCD cells. These regulations affect only alpha1-subunit of Na,K+-ATPase but not beta1.

Nongastric H+,K+-ATPase: cell biologic and functional properties.

Several members of the H+,K+-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H+,K+-ATPase as well as a number of nongastric H+,K+-ATPase isoforms. Physiological studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. Although much has been learned about the pattern of H+,K+-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties of renal H+,K+-ATPases both in vitro and in situ. Our analysis of ion fluxes driven by a nongastric H+,K+-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H+,K+-ATPase isoforms may make diverse contributions to renal cation transport. We find that the activities of renal H+,K+-ATPases in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H+,K+-ATPase beta-subunit. Transgenic mice expressing a version of this protein in which the signal has been disabled show constitutively active renal K resorption. The identities of the H+,K+-ATPase isoforms that are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established.

A tyrosine-based signal regulates H-K-ATPase-mediated potassium reabsorption in the kidney.

Isoforms of the H-K-ATPase participate in active K resorption in the renal collecting tubule. The cytoplasmic tail of the beta-subunit of the gastric H-K-ATPase includes a 4 amino acid motif which is highly homologous to tyrosine-based endocytosis signals. We have generated transgenic mice expressing an H-K-ATPase beta-subunit in which the tyrosine residue in this sequence has been mutated to alanine. Mice expressing the mutated protein manifest constitutive hypersecretion of gastric acid, demonstrating that the beta-subunit tyrosine-based motif is required for the regulated endocytosis of the H-K pump and hence the cessation of gastric acid output. To test the possibility that the tyrosine-based sequence in the tail of the H-K-ATPase beta-subunit plays a role in regulating the function of renal H-K-ATPases, we examined renal K clearance in normal and in transgenic mice. Blood pressure, urine volume, glomerular filtration rate (GFR), plasma Na, and Na excretion are similar in control and transgenic mice. However, plasma K concentrations are significantly higher in transgenic mice (4.76 +/- 0.13 meq/l in transgenic and 4. 12 +/- 0.04 meq/l in control; n = 9, P < 0.05) and K excretion is lower in the transgenic animals (fractional excretion of K was 26.2 +/- 3.62% in transgenic and 50.1 +/- 4.78% in control; n = 9, P < 0. 01). These data suggest that the tyrosine-based signal in the cytoplasmic tail of the H-K-ATPase beta-subunit functions in the kidney as it does in the stomach to internalize H-K pump and thus inactivate pump function. Its elimination may result in the constitutive presence of the pump at the cell surface and lead to excessive urinary K reabsorption.

Sorting of P-type ATPases in polarized epithelial cells.

The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.

Sorting and trafficking of ion transport proteins in polarized epithelial cells.

Transport proteins are targeted to specific plasmalemmal domains in polarized epithelial cells. The molecular signals that govern these sorting events are just beginning to be elucidated. In many cases, the cell surface delivery of transport proteins is subjected to tight regulation. Several different mechanisms appear to participate in these trafficking processes.

A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion.

Gastric acid secretion is mediated by the H/K-ATPase of parietal cells. Activation of acid secretion involves insertion of H/K-ATPase into the parietal cell plasmalemma, while its cessation is associated with reinternalization of the H/K-ATPase into an intracellular storage compartment. The cytoplasmic tail of the H/K-ATPase beta subunit includes a four residue sequence homologous to tyrosine-based endocytosis signals. We generated transgenic mice expressing H/K-ATPase beta subunit in which this motif's tyrosine residue is mutated to alanine. Gastric glands from animals expressing mutant beta subunit constitutively secrete acid and continuously express H/K-ATPase at their cell surfaces. Thus, the beta subunit's tyrosine-based signal is required for the internalization of H/K-ATPase and for the termination of acid secretion. As a consequence of chronic hyperacidity, the mice develop gastric ulcers and a hypertrophic gastropathy resembling Menetrier's disease.

Role of protein phosphatase in the regulation of Na+-K+-ATPase by vasopressin in the cortical collecting duct.

In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral Na+-K+-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown. The aim of this study was to investigate whether this AVP-induced increase in basolateral Na+-K+-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP) inhibitors was examined on both the specific 3H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent 86Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na+-K+-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A without effect on PP1. These data suggest that PP2A is implied in the regulation of Na+-K+-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na+-K+-ATPases is mediated by a PP2A-dependent dephosphorylation process.

Cellular localization and regulation of CHIF in kidney and colon.

Channel inducing factor (CHIF) is a novel cDNA recently cloned from a rat distal colon cDNA library of dexamethasone-treated animals. While its expression in Xenopus oocytes evokes a potassium channel activity similar to that induced by Isk (minK), its cellular role is not clear. CHIF exhibits significant homologies with proteins that are putatively regulatory (phospholemman, gamma-subunit of Na(+)-K(+)-ATPase, Mat-8) while it differs from the small-conductance potassium channel Isk. We have studied the tissue specificity of CHIF expression in rat by in situ hybridization. CHIF is selectively present in the distal parts of the nephron (medullary and papillary collecting ducts and end portions of cortical collecting tubule) and in the epithelial cells of the distal colon. No expression of CHIF was found in renal proximal tubule, loop of Henle and distal tubule, proximal colon, small intestine, lung, choroid plexus, salivary glands, or brain. To gain some insight into CHIF function, we have investigated, using in situ hybridization and ribonuclease protection assay, whether CHIF mRNA expression could be altered in some situations. In the distal colon, corticosteroid hormones, sodium restriction, low-potassium diet, and metabolic acidosis significantly increased CHIF mRNA expression. In the kidney, metabolic acidosis was the only condition that showed an increase in CHIF mRNA expression. Some of these treatments also altered the expression of the colonic H(+)-K(+)-ATPase mRNA. In summary, CHIF mRNA is selectively expressed in the medullary collecting duct of the kidney and in the epithelium of the distal colon; its expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion, and metabolic acidosis. The precise cell-specific functions of CHIF remain to be established.

Differential regulation of putative K(+)-ATPase by low-K+ diet and corticosteroids in rat distal colon and kidney.

K+ homeostasis depends on K+ absorption in digestive and renal epithelia. Recently, a cDNA encoding for a putative K(+)-adenosinetriphosphatase (ATPase) alpha-subunit has been characterized. We studied its expression by ribonuclease protection assay and in situ hybridization in the distal colon and the kidney of rats in various physiological states. In the distal colon of control rats, high expression of the colonic putative K(+)-ATPase mRNA was restricted to the surface epithelial cells. A low-K+ diet did not modify this expression, adrenalectomy decreased it, and aldosterone or dexamethasone treatment for 2 days restored normal levels. In the kidney of control rats, levels of K(+)-ATPase mRNA were very low. A low-K+ diet revealed a clear mRNA expression, which is consistent with a recent report [J.A. Kraut, F. Starr, G. Sachs, and M. Reuben. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F581-F587, 1995]. This expression was restricted to the outer medullary collecting duct, presumably in principal cells. Changes in corticosteroid status did not influence the renal expression. Our results, together with previous studies on K+ absorption and K(+)-ATPase activity, suggest that more than a single molecular form of K(+)-ATPase is likely to be responsible for the regulation of K+ absorption in the colon and distal nephron.

Synergistic action of vasopressin and aldosterone on basolateral Na(+)-K(+)-ATPase in the cortical collecting duct.

The respective effects of aldosterone and arginine vasopressin (AVP) were examined on the number of active Na(+)-K(+)-ATPase and their pumping activity in nonperfused microdissected mouse cortical collecting tubules (CCD) by measuring specific 3H-ouabain binding and ouabain-sensitive 86Rb uptake. In adrenalectomized (ADX) animals, incubation of CCD with AVP (10(-8) M for 5 min) had no effect on the number of pumps. In contrast, in ADX animals replete with aldosterone, AVP induced a approximately equal to 40% increase in the number of pumps. This was accompanied by a approximately equal to 60-65% increase in ouabain-sensitive Rb uptake. AVP effect was dose-dependent (10(-10)-10(-8) M) and was reproduced by dDAVP, forskolin and 8-Br cAMP, indicating a V2 pathway. It was inhibited by amiloride 10(-5) M, and did not occur in CCD incubated in hyperosmotic solution, suggesting that the signal was transmitted via apical sodium entry and cell swelling. Finally, the AVP-dependent increase in the number of pumps was rapid (within 5 min) and transient (< 25 min). These results demonstrate that, in the CCD, aldosterone and AVP act synergistically to increase not only the apical sodium entry but also the basolateral Na(+)-K(+)-ATPase transport capacity: AVP allows a rapid recruitment and/or activation of an aldosterone-dependent pool of latent Na(+)-K(+)-ATPase.

Role of cell volume variations in Na(+)-K(+)-ATPase recruitment and/or activation in cortical collecting duct.

The aim of this study was to examine whether cell volume variations could play a role in the previously reported Na(+)-K(+)-ATPase pump recruitment and/or activation induced by an increase in intracellular Na concentration (Nai) in cortical collecting ducts (CCD). Isolated CCD from kidneys of aldosterone-repleted mice were incubated in hyper-, hypo-, or isosmotic solutions with and without Na to modify Nai and cell volume independently. Nai, cell volume, and the number of basolateral pumps were measured using 22Na, image analysis, and specific [3H]ouabain binding, respectively. Ouabain-sensitive 86Rb uptake was also measured. In CCD with high Nai, pump recruitment and/or activation was observed only when an increase in tubular volume was associated with Na load. Pump recruitment and/or activation was also induced by cell swelling in the absence of Na load. Recruited and/or activated pumps display an affinity for ouabain and a specific activity (ouabain-sensitive Rb uptake per pump unit) similar to basal pumps. We conclude that 1) cell swelling is implied in the process of Nai-dependent pump recruitment and/or activation, 2) cell swelling can promote pump recruitment and/or activation independently of Na load, 3) basal and recruited and/or activated pumps probably correspond to the same Na(+)-K(+)-ATPase isoform.

Effects of acidosis and alkalosis on mechanical properties of hypertrophied rat heart fiber bundles.

Effects of alkalosis (pH 7.4) or acidosis (pH 6.8) on the intrinsic mechanical properties of control and pressure-overloaded rat hearts were studied in Triton X-100-treated left ventricular fiber bundles. In control bundles, Ca sensitivity [pCa required for one-half maximal response (pCa50)] was 5.520 +/- 0.012 at pH 7.1. Alkalosis increased it by 0.357 +/- 0.018 pCa unit, whereas acidosis decreased it by 0.365 +/- 0.014 pCa unit with no change in Hill coefficient. Maximal tension was decreased by acidic pH and increased by alkaline pH. Stiffness was measured by the response to quick length changes. Acidosis decreased maximal stiffness but increased the stiffness-to-force ratio, whereas alkalosis increased maximal stiffness but had no effect on stiffness-to-force ratio, suggesting that acidosis decreased the force generated per cross bridge. Alkalosis increased the time constant of tension recovery following a quick stretch from 10.6 +/- 0.66 to 17.45 +/- 1.83 ms, suggesting a decreased cross-bridge cycling rate. Pressure overload induced by thoracic aortic stenosis for 4-6 wk led to a 200% cardiac hypertrophy associated with a shift from fast to slow ventricular myosin. pCa50 of hypertrophied bundles was not different from control (5.541 +/- 0.012). Ca sensitivity was increased by 0.383 +/- 0.008 in alkaline medium and decreased by 0.325 +/- 0.009 in acidic medium. Stiffness-to-force ratio was decreased in acidic pH, and the time constant of tension recovery was increased from 31.0 +/- 0.4 to 34.9 +/- 0.25 ms by alkalosis. In hypertrophied bundles, maximal tension was decreased by acidic pH but not changed by alkalosis. These results show that in the small pH range of our study 1) pH changes have symmetrical effects on Ca sensitivity in both control and hypertrophied bundles, 2) a decrease or an increase in H+ concentration does not have symmetrical effects on the mechanics of the cross bridges, and 3) changes in the phenotype of contractile proteins induced by aortic stenosis do not influence Ca sensitivity, only moderately influence the response to pH changes, and mainly affect the cross-bridge cycling rate.

A putative H(+)-K(+)-ATPase is selectively expressed in surface epithelial cells of rat distal colon.

Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.

Adrenalectomy reduces alpha 1 and not beta 1 Na(+)-K(+)-ATPase mRNA expression in rat distal nephron.

The abundance of mRNA of alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-isoforms of Na(+)-K(+)-ATPase was examined in several renal structures of normal and adrenalectomized (ADX) rats. In situ hybridization with 35S-labeled cRNA probes was performed on kidney sections from adult rats. The number of silver grains per unit surface area was quantified over cells of the glomerulus, proximal convoluted tubules (PCT), early distal tubules (EDT), and cortical collecting ducts (CCD). In normal rat kidney, alpha 1- and beta 1-mRNA was detected in PCT, EDT, and CCD, with the following range of magnitude: EDT > CCD > PCT > glomerulus. The amount of alpha 1- and beta 1-mRNA was equivalent. A large abundance of these two mRNA species was also found in the medullary thick ascending limb of the loop of Henle. Expression of alpha 2, alpha 3, and beta 2 was very low and evenly distributed over any cell type. In ADX, a significant decrease in alpha 1-mRNA (30%) was observed in EDT and CCD, with no change in PCT. beta 1-mRNA abundance was unaffected by adrenalectomy. These results indicate that 1) in the rat kidney alpha 1- and beta 1-mRNA are coexpressed at a similar level that varies along the renal tubule according to the cell type, 2) minute expression of alpha 2-, alpha 3-, and beta 2-mRNA is present in the kidney, and 3) corticosteroid depletion reduces the expression of alpha 1- and not beta 1-mRNA in the corticosteroid-sensitive tubular cells.

Multiple patterns of 11 beta-hydroxysteroid dehydrogenase catalytic activity along the mammalian nephron.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to be a protective enzyme of the mineralocorticoid receptor (MR). We have previously demonstrated (Bonvalet et al, J Clin Invest 86:832-837, 1990) that 11 beta-OHSD is colocalized with MR along the rabbit nephron. In the present study, we examined whether 11 beta-OHSD is similarly located along the nephron of other mammals. Various tubular segments were microdissected from the mouse, rat, and rabbit nephron, and incubated for two hours at 37 degrees C in the presence of 11 nM [3H]-corticosterone (B). Thereafter, the respective amounts of B and [3H]-11dehydrocorticosterone (A) in the incubation solution were measured by HPLC. In the rabbit, the mouse and the rat, about 520 pmol/10 mm of B were transformed into A in tubular segments possessing MR, that is, the distal parts of the nephron (distal and collecting tubule). Differences appeared in the aldosterone-insensitive proximal tubule; in both the initial and final parts of this segment, 11 beta-OHSD activity was low (26 pmol/10 mm) in the rabbit and the mouse, and relatively high in the rat (328 pmol/10 mm). In the cortical part of the loop of Henle, where the presence of MR is still under discussion, 11 beta-OHSD activity was low in the mouse (70 pmol/10 mm), high in the rat (533 pmol/10 mm) and intermediate in the rabbit (227 pmol/10 mm). The comparison of these results with previous data obtained with immunohistochemical methods suggests that the proximal and distal nephron might express different isoforms of 11 beta-OHSD.

Time course of sodium-induced Na(+)-K(+)-ATPase recruitment in rabbit cortical collecting tubule.

In cortical collecting tubules (CCD) of aldosterone-repleted rabbit kidney, an increase in intracellular sodium concentration (Nai) induces the recruitment and/or activation of latent Na(+)-K(+)-ATPase pumps (Blot-Chabaud et al., J. Biol. Chem. 265: 11676-11681, 1990). The present study was addressed to determine the time course of this Nai-dependent pump recruitment and to examine some of the factors possibly involved in this phenomenon. CCD from adrenalectomized rabbits complemented with aldosterone and dexamethasone were incubated at 4 degrees C either in a K(+)-free saline solution (Na(+)-loaded CCD) or in a sucrose solution (control CCD) and then rewarmed for various time periods to allow pump recruitment to occur. The number of pumps in the membrane was determined by specific [3H]ouabain binding; Nai was measured using 22Na. A rise in Nai induced a threefold increase in the number of basolateral pumps, which was fully achieved within 1-2 min. This pump recruitment was reversible within 15 min after restoration of low Nai. It was unaffected by inhibitors of cytoskeleton and Ca2+ ionophore A 23187. The blocker of the Na(+)-H+ antiporter, amiloride, did not prevent it. The protein kinase C activator, phorbol 12-myristate 13-acetate, did not induce it in the absence of Na+. We conclude that Nai is a major determinant of pump recruitment and/or activation, which occurs over a very short period of time. It may constitute a rapid adaptative response to an increase in the cell Na+ load.