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]

The pure tetrameric form of acetylcholinesterase (EC 3.1.1.7) from the electric eel Electrophorus electricus has been covalently coupled to 2'-O-succinyl-cAMP tyrosine methyl ester and 2'-O-succinyl-cGMP. Both enzymatic conjugates have been used as tracers in a classical heterogeneous competitive enzyme immunoassay allowing the determination of cAMP and cGMP, respectively. The test was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit Immunoglobulin antibody in order to ensure separation between bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by colorimetric assay. When standards or samples were first acetylated by treatment with acetic anhydride, the sensitivity of both assays appeared very good since minimum detectable concentration close to 0.04 pmol/mL (2 fmol/well) could be calculated for each assay. Precision was also very satisfying since the coefficient of variation was less than 5% in the 0.2-10 pmol/mL range. Good correlation was noted between enzymoimmunological and radioimmunological measurements of cAMP performed for different biological samples (urine, serum, or tissue extracts).

A B S T R A C T The action sites for parathyroid hormone (PTH), salmon calcifonin (SCT), and argininevasopressin (AVP) were investigated along the human nephron by measuring adenylate cyclase activity, using a single tubule in vitro microassay. Well-localized segments of tubule were isolated by microdissection from five human kidneys unsuitable for transplantation.PTH (10 IU/ml) increased adenylate cyclase activity in the convoluted and the straight proximal tubule, in the medullary and cortical portions of the thick ascending limb, and in the early portion of the distal convoluted tubule (corresponding stimulated:basal activity ratios were 64, 19, 10, 18, and 22, respectively).SCT (10 ng/ml) increased adenylate cyclase activity in the medullary and cortical portions of the thick ascending limb, in the early portion of the distal convoluted tubule, and, to a lesser extent, in the cortical and the medullary collecting tubule (activity ratios were 7, 14, 15, 3, and 3, respectively).AVP (1 ,tM) stimulated adenylate cyclase activity in the terminal nephron segments only, i.e., the late portion of the distal convoluted tubule, the cortical and medullary portions ofthe collecting tubule (activity ratios 81, 51, and 97, respectively).As measured in one experiment, nearly one-half maximal responses were obtained with 0.1 IU/ml PTH or 0.3 ng/ml SCT in thick ascending limbs and with 1 nM AVP in collecting tubules, suggesting that enzyme sensitivity to hormones was well preserved under the conditions used in this study.

No abstract

Rabbit distal convoluted tubules (DCT) microdissected from collagenase-treated kidneys were observed to contain up to four portions of a different appearance under stereomicroscopic examination: (1) a DCTa portion (generally very short), located right after the macula densa (MD) and resembling the portion of the limb (CAL) located before the MD; (2) a constant, "bright" portion, DCTb; (3) a constant, "granular" DCTg portion which, in most DCT, is connected to a portion of the collecting tubule of a similar "granular" appearance (CCTg); (4) many DCT having contacts with the kidney capsule in the superficial cortex were observed to contain an additional portion of a "light" appearance, DCTl, resembling the portion of the collecting tubule (CCTl) to which these superficial DCT are always branched. The hormone-dependent adenylate cyclase (AC) contained in these different portions was investigated by sectioning microdissected distal structures into successive samples according to the above-mentioned criteria, and by measuring with the help of a previously described micromethod, the enzyme activity contained in each single sample under one of the following conditions: control, parathyroid hormone. (PTH l U/ml), vasopressin, (AVP 10(-6)M), isoproterenol (10(-6)M), fluoride (5 X 10(-3)M). Highly significant and reproducible AC stimulations by these hormones were obtained for the following portions, respectively: DCTa, DCTg and CCTg with PTH; DCTl and CCTl with AVP; DCTg, CCTg and CCTl with isoproterenol. From these data, it is concluded that (a) the distal convoluted tubule can no longer be regarded as a single well-defined functional structure; (b) DCTa is actually a short CAL portion extending beyond MD, (c) DCTg and CCTg are two portions of a same functional segment; (d) similarly, DCTl belongs to the functional segment mainly constituted by CCTl; and, finally, (e) DCTb is the only functional segment which is entirely located in the distal convoluted tubule, i.e., included between the macula densa and the first branching with another tubule.

Insulin (Ins) decreases Na+ delivery in the final urine. To determine whether the loop of Henle participates in this reduction, the effects of Ins were tested on cortical (CTAL) and medullary thick ascending limbs (MTAL) of the mouse nephron, microperfused in vitro. In the MTAL, Ins increased the transepithelial potential difference (Vt) and the Na+ and Cl- net reabsorption fluxes (JNa and JCl, respectively) in a dose-dependent manner, the threshold being below 10(-9) M. At 10(-7) M, Ins reversibly increased JNa and JCl, leaving Mg2+ and Ca2+ fluxes (JMg and JCa, respectively) close to zero. In the CTAL, 10(-7) M Ins reversibly increased Vt, JNa, JCl, JMg, and JCa. In CTAL segments perfused under asymmetrical conditions, with a bath-to-lumen-directed NaCl gradient (lumen 50 mM NaCl, bath 150 mM NaCl), addition of 10(-7) M Ins to the bath resulted in a large increase in JMg and JCa. Thus the responses of CTAL and MTAL to Ins are in all ways similar to those already reported for the adenosine 3',5'-cyclic monophosphate (cAMP)-generating hormones acting on these nephron segments. When 10(-10) M arginine vasopressin (AVP) and 10(-7) M Ins were used in combination, previous addition of one hormone to the bath potentiated the response to the second hormone. In cAMP accumulation experiments, performed in the presence of a phosphodiesterase inhibitor, the amounts of cAMP formed with 10(-7) M Ins and 10(-10) M AVP (which elicit maximal physiological responses in these segments) were in the same range.(ABSTRACT TRUNCATED AT 250 WORDS)

AVP dependent adenylate cyclase activity was measured in single pieces of 8 different tubular segments isolated from collagenase treated rabbit kidneys. High responses were observed in all the tested portions of the collecting tubule, that is its cortical branched part (BCT), its cortical straight part (CCT) and its outer medullary part (MCT). Dose response curves indicated in CCT: 2 fold threshold stimulation at 10(-11) M AVP, 27 fold stimulation at 10(-6) M AVP, half maximal stimulation at about 10(-9) M AVP. Both the medullary (MAL) and, to a lesser extent, the cortical (CAL) portions of the thick ascending limb were also observed to contain AVP sensitive adenylate cyclase (for MAL: 2 fold threshold stimulation at 10(-9) M AVP, 9 fold stimulation at 10(-7) M AVP, half maximal stimulation at 5 X 10(-9) M AVP). In contrast, nearly no responsiveness to AVP was observed in the proximal convoluted tubule, in the thin descending limb of the loop and in the distal convoluted tubule (DCT). The limited response obtained in DCT (which is a structure generally considered as a target site for AVP) as well as the clearcut effect elicited by AVP in MAL (the functioning of which is not known to be controlled by ADH) were expected observations; their possible physiological implications will be discussed.