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 ...

This study was designed to correlate morphological alterations induced in rat collecting tubule by potassium depletion with changes in the activity of enzymatic markers of the cell basolateral membrane. Results show the following responses. 1) Potassium depletion induced a huge and progressive hypertrophy of the outer medullary collecting tubule (MCT). Hypertrophy was paralleled by enhancements of vasopressin- and forskolin-dependent adenylate cyclase (AC) activities. Glucagon-sensitive AC was also increased, but with a different kinetics, whereas isoproterenol-dependent AC was only modestly stimulated. 2) In cortical (CCT) and papillary collecting tubules, AC response to hormones did not change. The concentrating defect of K-deprived rats, therefore, does not appear to result from an intrinsically defective adenylate cyclase system in any portion of the collecting tubule. Decreased AC response of the medullary thick ascending limb to vasopressin and glucagon, observed after 3-5 wk of K depletion, might account, at least in part, for reduced hypertonicity of medullary tissue. 3) Na+-K+-ATPase activity fell in CCT, probably in relation to decreased K secretion. Conversely, in MCT, Na+-K+-ATPase rose much more than tubular volume. The physiological significance of this latter observation remains to be established.

The effect of prostaglandins and alpha-adrenergic agonists on arginine vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) production was investigated in microdissected rat and rabbit cortical collecting tubules (CCT) incubated in vitro. In rabbit CCT, addition to all media of a prostaglandin synthesis inhibitor increased this production; exogenous prostaglandin E2 (PGE2) induced a reproducible dose-dependent inhibition of cAMP accumulation. Maximal inhibition (mean: 57.5%) was observed with 0.3 microM PGE2, and threshold inhibition was observed with concentrations ranging from 3 to 10 nM PGE2. Inhibition of cAMP levels in rabbit CCT was also obtained with 0.3 microM PGF2 alpha (mean inhibition: 44.3%) but not with alpha-adrenergic agonists studied under the same conditions. The opposite was observed in rat CCT studied in parallel: the alpha-agonists inhibited cAMP production by up to 80%, but PGE2 had no effect.

The possible regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by arachidonic acid (AA) was studied in segments, microdissected from the rat kidney, which are sensitive to arginine vasopressin (AVP). In the presence of 5 microM indomethacin, the addition of 5 microM AA did not impair AVP-dependent cAMP accumulation (measured during 4 min at 35 degrees C) in the cortical or outer medullary collecting tubule, but decreased this response in the thick ascending limb with an inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhibited by 34.4 +/- 9.6% (SEM) and 65.8 +/- 5.4% with 1 microM and 5 microM AA, respectively, N = 5 experiments. AVP-, glucagon- and calcitonin-sensitive cAMP levels in MTAL were inhibited by 5 microM AA to a similar extent. AA-induced inhibition was unaffected by the presence of inhibitors of AA metabolism: (1) either 10 microM indomethacin or 50 microM ibuprofen added to all media; (2) a 10-min pre-incubation and a 4-min incubation of MTAL samples with 10 microM eicosa-5,8,11,14-tetrayonic acid, (3) a 1-h preincubation with either 30 microM SKF-525A, 20 microM ketoconazole, or 20 microM nordihydroguariaretic acid. In contrast to AA, 11 other saturated or unsaturated fatty acids had no inhibitory effect on the AVP-dependent cAMP level. In fura-2-loaded MTAL samples, AA induced a slow increase of the intracellular calcium concentration ([Ca2+]i) which reached 21.0 +/- 3.8 nM and 92.9 +/- 21.4 nM over basal values (n = 11) at 2 min and 4 min, respectively, after the beginning of the superfusion of 5 microM AA. AA-induced inhibition of AVP-dependent cAMP accumulation was due neither to the increase in [Ca2+]i elicited by AA, nor to an activation of protein kinase C because this inhibition: (1) was not blocked when MTAL samples were incubated either in zero Ca2+ medium, or in the presence of 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA) to chelate [Ca2+]i, and (2) it was not reproduced by a pre-treatment of MTAL segments with a phorbol ester. Pre-incubation of MTAL (6 h at 35 degrees C) with 500 ng/ml pertussis toxin (PTX) prevented AA-induced inhibition: in the presence of PTX inhibition was 24.7 +/- 6.6% vs 10 nM AVP, as compared to 81.6 +/- 4.0% in control groups, i.e in the absence of PTX, N = 6.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasopressin (AVP) plays a key role in maximal urine concentration by stimulating NaCi reabsorption in the medullary thick ascending limbs of Henle (MAL) and by increasing water permeability in the medullary collecting tubules (MCT). These effects of AVP in MAL and MCT are mediated by cAMP.