Analysis of proton (H+) transport by insideout vesicles derived from highly purified chicken osteoclast (OC) membranes has revealed the presence of a newly discovered type of vacuolar H+ ATPase (V-ATPase). Unlike vesicles derived from any other cell type or organelle, H+ transport in OC-derived vesicles is sensitive to V-ATPase inhibitors (Nethylmaleinuide and Bafilomycin Al) and vanadate (IC5, 100 uzM), an inhibitor previously found to affect only P-type ATPases. The OC H+ ATPase contains several V-like subunits (115, 39, and 16 kDa) but subunits A and B of the catalytic domain of the enzyme differ from that of other V-ATPases. In OCs, subunit A has a mass of 63 kDa instead of the 67-70 kDa expressed in monocytes, macrophages, and kidney microsomes, which contain a vanadate-insensitive H+ ATPase. Moreover, two types of57-to 60-kDa B subunits are also found: one is expressed predominantly in OCs and the other is expressed in kidney microsomes. The OC H+ pump may therefore constitute a class ofH+ ATPase with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme. This H+ ATPase is involved in resorption of bone and may be expressed in a cell-specific manner, thereby opening possibilities for therapeutic intervention.

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Calcitonin is a calcium regulating peptide hormone with binding sites in kidney and bone as well as in the central nervous system. The mechanisms of signal transduction by calcitonin receptors were studied in a pig kidney cell line where the hormone was found to regulate sodium pumps. Calcitonin receptors activated the cyclic adenosine monophosphate (cAMP) or the protein kinase C (PKC) pathways. The two transduction pathways required guanosine triphosphate (GTP)-binding proteins (G proteins) (the choleratoxin sensitive Gs and the pertussis toxin sensitive Gi, respectively) and led to opposite biological responses. Moreover, selective activation of one or the other pathway was cell cycle-dependent. Therefore, calcitonin may induce different biological responses in target cells depending on their positions in the cell cycle. Such a modulation of ligand-induced responses could be of importance in rapidly growing cell populations such as during embryogenesis, growth, and tumor formation.

To investigate the microRNA (miRNA) profile in patients with different stages of pseudoexfoliation (PXF). Methods: Peripheral blood of patients with PXF (naïve to medical therapy and with no systemic disease/drugs) with ocular hypertension (OHT) and pseudoexfoliation glaucoma (PXG) was evaluated in triplicate for miRNA profiling using polymerase chain reaction (PCR) arrays. Those identified in the discovery stage were validated with evaluation of serum transforming growth factor-β1 (TGF-β1) levels by ELISA. The downstream targets of TGF-β1 and unfolded protein response (UPR) were analyzed using reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). Predicted targets of the identified miRNA and KEGG pathway analysis were done using miRbase and DIANA tools mirPathv3.1. Results: We found hsa-miR-122-5p, hsa-miR-124-3p and hsa-miR-424-5p to be upregulated in PXG targeting 3 specific pathways namely TGF-β1, fibrosis/ECM and proteoglycan metabolism with common effectors like SMAD/3/2. The unfolded protein response (UPR) genes were significantly downregulated in all stages of PXF suggesting this as the key mechanism for protein aggregates in PXF syndrome. Serum TGF-β1 was significantly upregulated as disease progressed to later stages in PXG. This elevation in advanced stages was associated with significantly differential expression of downstream pathways and fibrotic genes in OHT compared to PXG predominantly through the SMAD3, a canonical pathway marker. Conclusion: Circulatory miRNA differentially regulating TGF-β1 and downstream targets including UPR genes may be the key mechanisms for glaucoma onset in PXF.

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Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,KATPase in the proximal kidney tubule cell line LLC-PKj in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.Calcitonin (CT), a 32-amino acid hormone with profound hypocalcemic effects due to its action in bone and the kidney (1, 2), also increases urinary excretion of Na (3). The mechanisms underlying this effect of CT on the kidney tubule, however, have not been elucidated. In the kidney, Na reabsorption results mostly from activity of the basolateral Na,K-ATPase and the apical Na/H exchanger (NHE) in the proximal tubule and/or Na channels in the distal tubule (4, 5). We have reported (6) that CT regulates Na,K-ATPase in a proximal kidney tubule cell line (LLC-PK1) (7), with the effect of the hormone depending on the position of the cells in the cell cycle; during G2, CT increases cAMP via the stimulatory guanine nucleotide-binding protein (G, protein) (8)(9)(10). The second NHE isoform is found in the apical membrane domain of renal, intestinal, and gall bladder epithelia (10). This apical NHE is less sensitive to inhibition by amiloride and is primarily involved in Na+ reabsorption and proton secretion. A third isoform has recently been reported in the intestine (11) but it is not yet well characterized.CT activates several signal transduction pathways (6, 12-14), some differentially along the kidney tubule (15, 16), which could affect the activity of the NHEs (17). The major objective of this study was therefore to determine whether CT regulates the NHEs in LLC-PK1 cells, whether such effects varied with the phase of the cell cycle, and how the observed responses, if any, could be related to the changes in Na,K-ATPase activity and the natriuretic effect of the hormone.MATERIALS AND METHODS Materials. Salmon calcitonin was from Rorer Central Research (King of Prussia, PA). Ethylisopropyl amiloride (EIPA) was obtained from Merck Sharp and Dohme. Ouabain and all other chemicals were from Sigma. [3H]Ouabain (specific activity, 20 Ci/mmol; 1 Ci = 37 GBq), [3H]thymidine (specific activity, 15 Ci/mmol), and 22NaCl (specific activity, 100-1000 mCi per mg of sodium) were from Amersham.Cells. LLC-PK1 ...