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 ...

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The cellular distribution of Ca(2+)-inhibitable adenylyl cyclase (AC) type 5 and type 6 mRNAs in rat outer medullary collecting duct (OMCD) was performed by in situ hybridization. Kidney sections were also stained with specific antibodies against either collecting duct intercalated cells or principal cells. The localization of type 5 AC in H(+)-ATPase-, but not aquaporin-3-, positive cells demonstrated that type 5 AC mRNA is expressed only in intercalated cells. In contrast, type 6 AC mRNA was observed in both intercalated and principal cells. In microdissected OMCDs, the simultaneous superfusion of carbachol and PGE(2) elicited an additive increase in the intracellular Ca(2+) concentration, suggesting that the Ca(2+)-dependent regulation of these agents occurs in different cell types. Glucagon-dependent cAMP synthesis was inhibited by both a pertussis toxin-sensitive PGE(2) pathway (63.7 +/- 4.6% inhibition, n = 5) and a Ca(2+)-dependent carbachol pathway (48.6 +/- 3.3%, n = 5). The simultaneous addition of both agents induced a cumulative inhibition of glucagon-dependent cAMP synthesis (78.2 +/- 3.3%, n = 5). The results demonstrate a distinct cellular localization of type 5 and type 6 AC mRNAs in OMCD and the functional expression of these Ca(2+)-inhibitable enzymes in intercalated cells.

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Herbal and traditional medicines are being widely used in practice in many countries for their benefits of treating different ailments. A large number of plants in Morocco were used in folk medicine to treat immune-related disorders. The objective of this study is to evaluate the immunomodulatory activity of protein extracts (PEs) of 14 Moroccan medicinal plants. This activity was tested on the proliferation of immune cells. The prepared total and PEs of the plant samples were tested using MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay on the splenocytes with or without stimulation by concanavalin-A (Con-A), a mitogenic agent used as positive control. The results of this study indicated different activity spectra. Three groups of activities were observed. The first group represented by Citrullus colocynthis, Urtica dioica, Elettaria cardamomum, Capparis spinosa and Piper cubeba showed a significant immunosuppressive activity. The second group that showed a significant immunostimulatory activity was represented by Aristolochia longa, Datura stramonium, Marrubium vulgare, Sinapis nigra, Delphynium staphysagria, Lepidium sativum, Ammi visnaga and Tetraclinis articulata. The rest of the plant extracts did not alter the proliferation induced by Con-A. This result was more important for the PE than for the total extract. In conclusion, this study revealed an interesting immunomodulating action of certain PEs, which could explain their traditional use. The results of this study may also have implications in therapeutic treatment of infections, such as prophylactic and adjuvant with cancer chemotherapy.