Revealing the large-scale network organization of growth hormone-secreting cells.

Pituitary growth hormone (GH)-secreting cells regulate growth and metabolism in animals and humans. To secrete highly ordered GH pulses (up to 1,000-fold rise in hormone levels in vivo), the pituitary GH cell population needs to mount coordinated responses to GH secretagogues, yet GH cells display an apparently heterogeneous scattered distribution in 2D histological studies. To address this paradox, we analyzed in 3D both positioning and signaling of GH cells using reconstructive, two-photon excitation microscopy to image the entire pituitary in GH-EGFP transgenic mice. Our results unveiled a homologous continuum of GH cells connected by adherens junctions that wired the whole gland and exhibited the three primary features of biological networks: robustness of architecture across lifespan, modularity correlated with pituitary GH contents and body growth, and connectivity with spatially stereotyped motifs of cell synchronization coordinating cell activity. These findings change our view of GH cells, from a collection of dispersed cells to a geometrically connected homotypic network of cells whose local morphology and connectivity can vary, to alter the timing of cellular responses to promote more coordinated pulsatile secretion. This large-scale 3D view of cell functioning provides a powerful approach to identify and understand other networks of endocrine cells that are thought to be scattered in situ. Many dispersed endocrine systems exhibit pulsatile outputs. We suggest that cell positioning and associated cell-cell connection mechanisms will be critical parameters that determine how well such systems can deliver a coordinated secretory pulse of hormone to their target tissues.

Expression of androgen receptor and androgen regulation of NDRG2 in the rat renal collecting duct.

Androgens are known to regulate gene expression in the renal proximal tubule. Whether the distal parts of the nephron, in particular the cortical collecting duct (CCD), where sodium reabsorption is controlled tightly by aldosterone, are also targets for these hormones is unknown. Real-time PCR on rat isolated renal tubules showed that androgen receptor mRNA is not only, as expected, expressed in the proximal tubule, but also in the CCD. We examined the effects of adrenalectomy (ADX) plus castration and in-vivo administration of the active metabolite of testosterone, dihydrotestosterone (DHT), on the intrarenal expression of N-myc downstream regulated gene 2 (NDRG2), an early aldosterone-induced gene located specifically in the CCD. NDRG2 belongs to a newly identified family of differentiation-related genes; although the function of these genes remains elusive, regulation of NDRG1 by androgens has been suggested. Castration plus ADX increased NDRG2 expression (RNase protection assay) significantly in the whole kidney, and a single i.p. injection of DHT caused a significant decrease in NDRG2 expression 4 h afterwards (up to 24 h). Furthermore, real-time PCR on microdissected tubules revealed that the decrease in NDRG2 expression caused by DHT is restricted to the CCD. Thus, aldosterone and androgens have opposite effects on NDRG2 expression in the renal CCD. These results are the first demonstration of androgen-dependent gene regulation in the rat renal CCD.

Gastric parietal cell acid secretion in mice can be regulated independently of H/K ATPase endocytosis.

BACKGROUND & AIMS: Gastric parietal cells secrete acid into the lumen of the stomach. They express a proton pump, the gastric H(+)/K(+) ATPase, the activity of which is tightly regulated. The H(+)/K(+) ATPase traffics between an intracytoplasmic compartment (tubulovesicles) in quiescent parietal cells and the apical plasma membrane in activated cells. These trafficking events are considered to contribute to the control of acid secretion by modulating access to apical K(+) and Cl(-) conductances that are required for transmembrane H(+) ion transport by the H(+)/K(+) ATPase. Here, we have determined whether the control of acid secretion in vivo requires membrane trafficking of the H(+)/K(+) ATPase. METHODS: We developed mice that only express an H(+)/K(+) ATPase beta subunit in which a putative tyrosine-based endocytosis motif in the cytoplasmic tail is mutated. Location of the H(+)/K(+) ATPase and parietal cell ultrastructure and gastric acid secretion were then examined. RESULTS: Parietal cells of these mice lacked a tubulovesicular compartment, and the H(+)/K(+) ATPase was resident exclusively on the apical plasma membrane. Despite the inability of the H(+)/K(+) ATPase to be endocytosed, the gastric acid secretory response to histamine or an antagonist was very similar to that of wild-type mice, indicating that control of H(+)/K(+) ATPase activity can occur independently of intracellular trafficking. CONCLUSIONS: We were able to dissociate the regulation of H(+)/K(+) ATPase activity from intracellular trafficking of the protein. Thus, it is likely that direct regulation of apical K(+) and Cl(-) conductances are sufficient to control gastric acid secretion.

Characterization of rat NDRG2 (N-Myc downstream regulated gene 2), a novel early mineralocorticoid-specific induced gene.

The early phase of the stimulatory action of aldosterone on sodium reabsorption in tight epithelia involves hormone-regulated genes that remain to be identified. Using a subtractive hybridization technique on isolated renal cortical collecting ducts from rats injected with a physiological dose of aldosterone, we have identified an early response cDNA highly homologous to human and murine NDRG2 (N-Myc downstream regulated gene 2), which consists of four isoforms and belongs to a new family of differentiation-related genes. NDRG2 mRNA was expressed in classical aldosterone target epithelia, and in the kidney, it was specifically located in the collecting duct, the site of aldosterone-regulated sodium absorption. NDRG2 mRNA was increased within 45 min by aldosterone in the kidney and distal colon, whereas it was unaffected in the heart. In the RCCD2 collecting duct cell line, NDRG2 mRNA was enhanced as early as 15 min after aldosterone addition by transcription-dependent effects. NDRG2 was induced by aldosterone concentrations as low as 10(-9) M, and a maximal effect was observed at 10(-8) M. In contrast, the glucocorticoid dexamethasone was ineffective in NDRG2 expression, whereas the glucocorticoid-regulated gene sgk was induced. Taken together, these results indicate that NDRG2 regulation by aldosterone is an early mineralocorticoid-specific effect. Interestingly, NDRG2 is homologous to Drosophila MESK2, a component of the Ras pathway, suggesting that activation of the Ras cascade may play a significant role in mineralocorticoid signaling.

Calcyclin is an early vasopressin-induced gene in the renal collecting duct. Role in the long term regulation of ion transport.

Long-term effects of arginine vasopressin (AVP) in the kidney involve the transcription of unidentified genes. By subtractive hybridization experiments performed on the RCCD(1) cortical collecting duct cell line, we identified calcyclin as an early AVP-induced gene (1 h). Calcyclin is a calcium-binding protein involved in the transduction of intracellular signals. In the kidney, calcyclin was localized at the mRNA level in the glomerulus, all along the collecting duct, and in the epithelium lining the papilla. In RCCD(1) cells and in m-IMCD(3) inner medullary collecting duct cells, calcyclin was evidenced in the cytoplasm. Calcyclin mRNA levels were progressively increased by AVP treatment in RCCD(1) (1.7-fold at 4 h) and m-IMCD(3) (2-fold at 7.5 h) cells. In RCCD(1) cells, calcyclin protein levels were increased by 4 h of AVP treatment. In vivo, treatment of genetically vasopressin-deficient Brattleboro rats with AVP for 4 days induced an increase in both calcyclin and aquaporin-2 mRNA expression. Finally, introduction of anti-calcyclin antibodies into RCCD(1) cells by permeabilizing the plasma membrane prevented the long-term (but not short-term) increase in short-circuit current induced by AVP. Taken together, these results suggest that calcyclin is an early vasopressin-induced gene that participates in the late phase of the hormone response in transepithelial ion transport.

Basolateral translocation by vasopressin of the aldosterone-induced pool of latent Na-K-ATPases is accompanied by alpha1 subunit dephosphorylation: study in a new aldosterone-sensitive rat cortical collecting duct cell line.

The regulation of plasma membrane Na(+)-K(+)-ATPases (NKA) expression by aldosterone and arginin vasopressin (AVP) in the cortical collecting duct (CCD) has been examined in a new rat CCD cell line, designated as RCCD(2). This cell line has maintained many characteristics of the CCD-in particular, the expression of the mineralocorticoid receptor. Mineralocorticoid receptor is expressed at the protein level and binds (3)H-aldosterone (approximately 15 to 20 fmol/mg protein). Short-circuit current (Isc) experiments showed approximately a twofold increase in Isc associated with a decrease in transepithelial resistance when cells were treated with aldosterone concentrations as low as 10(-9) M. This effect on Isc was significant 2 h after aldosterone addition and was still present after 24 h. It was accompanied by an increase in the amount of mRNA encoding for the alpha subunit of the epithelial sodium channel (sixfold) and the alpha1 subunit of NKA (fourfold) after 24 h of hormone treatment. In addition, mRNA expression of the serum- and glucocorticoid-induced kinase (Sgk) was increased by 10(-9) M aldosterone treatment as early as 45 min after hormone addition. As had already been documented in native CCD obtained by microdissection, incubation of RCCD(2) cells for 24 h with aldosterone resulted in the constitution of a latent pool of NKA that could be rapidly recruited by AVP (15 min). NKA biotinylation experiments and preparation of membrane fractions show that this latent pool of NKA is present in the intracellular compartment of the cells and is recruited by AVP in the basolateral membrane through a translocation process. This mechanism is accompanied by dephosphorylation of the alpha(1) catalytic subunit of NKA.

Activation of the amiloride-sensitive epithelial sodium channel by the serine protease mCAP1 expressed in a mouse cortical collecting duct cell line.

This study examines whether serine proteases can activate the amiloride-sensitive sodium channel (ENaC) in mammalian kidney epithelial cells. The transepithelial sodium transport assessed by amiloride-sensitive short-circuit current appears to be sensitive to aprotinin, a protease inhibitor in a mouse cortical collecting duct cell line (mpkCCD(c14)). This result indicated that serine proteases may be implicated in the regulation of ENaC-mediated sodium transport. Using degenerated oligonucleotides to a previously isolated serine protease from Xenopus, xCAP1 (channel activating protease), a novel full-length serine protease (mCAP1), has been isolated and characterized. RNA analysis showed a broad pattern of expression in tissues (kidney, lung, colon, and salivary glands) expressing ENaC. Reverse transcription-PCR experiments also showed that mCAP1 was abundantly expressed in proximal tubule cells and was also expressed in intact and cultured collecting duct cells. Coexpression of the Xenopus, rat, or human alpha-, beta-, and gamma-ENaC subunits in Xenopus oocytes also showed that mCAP1 induces a significant increase in ENaC-mediated current accompanied by a decrease of channel molecules at the cell surface. It is proposed that this novel mouse channel activating protease may act as a regulator of ENaC within the kidney.