Expression of Ca2؉ -inhibitable types V and VI adenylyl cyclases was studied by reverse transcription-polymerase chain reaction in rat renal glomeruli and nephron segments isolated by microdissection. Quantitation of each mRNA was achieved using a mutant cRNA which differed from the wild type by substituting two bases to create a new restriction site in the corresponding cDNA. Type VI mRNA was present all along the nephron but was more abundant in distal than in proximal segments. The expression of type V mRNA was restricted to the glomerulus and to the initial portions of the collecting duct. Expression of the Ca 2؉ -insensitive type IV mRNA studied on the same samples was evidenced only in the glomerulus. The functional relevance of the expression of Ca 2؉ -inhibitable isoforms was studied by measuring cAMP content in the microdissected outer medullary collecting duct which expressed both type V mRNA (2367 ؎ 178 molecules/mm tubular length; n ‫؍‬ 8) and type VI mRNA (5658 ؎ 543 molecules/mm, n ‫؍‬ 8). Agents known to increase intracellular Ca 2؉ in this segment induced a Ca 2؉ -dependent inhibition on either arginine vasopressin-or glucagon-stimulated cAMP level. The characteristics of these inhibitions suggest a functional and differential expression of types V and VI adenylyl cyclases in two different cell types of the rat outer medullary collecting duct.In the past few years, the control of cAMP content in mammalian cells has become more intricate by the description of several types of adenylyl cyclase with different regulatory properties (1-3). Among the eight isoforms of adenylyl cyclase cloned up to date, the type V and the type VI are characterized by an activity negatively regulated by sub-micromolar concentrations of Ca 2ϩ . This property, established in vitro on membrane preparations (4 -7), has been observed also on the cAMP content measured on cultured cells from different tissues that express Ca 2ϩ -inhibitable AC 1 isoforms (4, 8 -13). These results demonstrate therefore that type V and type VI adenylyl cyclases can be inhibited in intact cells in response to a rise in [Ca 2ϩ ] i . Northern blot analyses have demonstrated that types V and VI AC mRNAs are expressed in the rat kidney (8,14). The renal tissue is, however, structurally highly heterogeneous and includes, in addition to the nephron epithelial cells, other cell types such as interstitial and vascular cells (15). The main functions of the kidney are achieved by the glomerulus and the different segments of the nephron, and many of these physiological processes, including the maintenance of Ca 2ϩ homeostasis (16), are regulated by the cAMP and/or the phospholipase C pathway. In addition, recent data demonstrated the expression of an extracellular Ca 2ϩ receptor in the rat kidney that might participate in Ca 2ϩ -sensitive regulations in some segments of the nephron (17). In this context, the presence of Ca 2ϩ -inhibitable AC mRNAs in the rat kidney (8,14) suggests that these isoforms might contribute to the regulation of physiological ...

Aims/hypothesis. C-peptide, the cleavage product of proinsulin processing exerts physiological effects including stimulation of Na + ,K + -ATPase in erythrocytes and renal proximal tubules. This study was undertaken to assess the physiological effects of connecting peptide on Na + ,K + -ATPase activity in the medullary thick ascending limb of Henle's loop. Methods. Na + ,K + -ATPase activity was measured as the ouabain-sensitive generation of 32 Pi from γ[ 32 P]-ATP and 86 Rb uptake on isolated rat medullary thick ascending limbs. The cell-surface expression of Na + ,K + -ATPase was evaluated by Western blotting of biotinylated proteins, and its phosphorylation amount was measured by autoradiography. The membrane-associated fraction of protein kinase C isoforms was evaluated by Western blotting. Results. Rat connecting peptide concentration-dependently stimulated Na + ,K + -ATPase activity with a threshold at 10 −9 mol/l and a maximal effect at 10 −7 mol/l. C-peptide (10 −7 mol/l) already stimulates Na + ,K + -ATPase activity after 5 min with a plateau from 15 to 60 min. C-peptide (10 −7 mol/l) stimulated Na + ,K + -ATPase activity and 86 Rb uptake to the same extent, but did not alter Na + ,K + -ATPase cell surface expression. The stimulation of Na + ,K + -ATPase activity was associated with an increase in Na + ,K + -ATPase α-subunit phosphorylation and both effects were abolished by a specific protein kinase C inhibitor. Furthermore, connecting peptide induced selective membrane translocation of PKC-α. Conclusion/interpretation. This study provides evidence that in rat medullary thick ascending limb, C-peptide stimulates Na + ,K + -ATPase activity within a physiological concentration range. This effect is due to an increase in Na + ,K + -ATPase turnover rate that is most likely mediated by protein kinase C-α phosphorylation of the Na + ,K + -ATPase α-subunit, suggesting that C-peptide could control Na + reabsorption during non-fasting periods. [Diabetologia (2003) 46:124-131]