C-peptide stimulates Na+,K+-ATPase activity via PKC alpha in rat medullary thick ascending limb.

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 gamma[(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 alpha-subunit phosphorylation and both effects were abolished by a specific protein kinase C inhibitor. Furthermore, connecting peptide induced selective membrane translocation of PKC-alpha. 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-alpha phosphorylation of the Na(+),K(+)-ATPase alpha-subunit, suggesting that C-peptide could control Na(+) reabsorption during non-fasting periods.

Cellular localization of type 5 and type 6 ACs in collecting duct and regulation of cAMP synthesis.

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.

Functional properties of Ca2+-inhibitable type 5 and type 6 adenylyl cyclases and role of Ca2+ increase in the inhibition of intracellular cAMP content.

Among the different adenylyl cyclase (AC) isoforms, type 5 and type 6 constitute a subfamily which has the remarkable property of being inhibited by submicromolar Ca2+ concentrations in addition to Galphai-mediated processes. These independent and cumulative negative regulations are associated to a low basal enzymatic activity which can be strongly activated by Galphas-mediated interactions or forskolin. These properties ensure possible wide changes of cAMP synthesis. Regulation of cAMP synthesis by Ca2+ was studied in cultured or native cells which express naturally type 5 and/or type 6 AC, including well-defined renal epithelial cells. The results underline two characteristics of the inhibition due to agonist-elicited increase of intracellular Ca2+: i) Ca2+ rises achieved through capacitive Ca2+ entry or intracellular Ca2+ release can inhibit AC to a similar extent; and ii) in a same cell type, different agonists inducing similar overall Ca2+ rises elicit a variable inhibition of AC activity. The results suggest that a high efficiency of AC regulation by Ca2+ is linked to a requisite close localization of AC enzyme and Ca2+ rises.

[Regulation of intracellular cAMP content in kidney tubules: role of adenylyl cyclases inhibitable by calcium]. / Régulation du contenu intracellulaire d'AMPc dans le tubule rénal: rôle des adénylyl cyclases inhibables par le calcium.

Adenylyl cyclase (AC) isoforms 5 and 6 can be inhibited by submicromolar concentrations of Ca2+. Quantitative RT-PCR allowed to study the corresponding messengers (mRNA) along the rat nephron. The results demonstrate a significant expression of AC 6 mRNA all along the nephron and of AC 5 mRNA in the glomerulus and the collecting tubule located in the cortex and the outer medulla. Regulation of cAMP synthesis and of intracellular cAMP content in defined renal cell types established the functional expression of AC 5 and AC 6. In particular, adenylyl cyclase activity is strongly stimulated by hormones and can be inhibited by several factors which either increase intracellular Ca2+ concentration or are coupled to G alpha 1. In each renal cell studied, the expression of 5 and 6 isoforms allow to integrate specific, multiple and independent inhibitory pathways which contribute to decrease intracellular cAMP content.

Cell-specific coupling of PGE2 to different transduction pathways in arginine vasopressin- and glucagon-sensitive segments of the rat renal tubule.

1. The aim of the present study was to investigate the transduction pathways elicited by prostaglandin E2 (PGE2) to inhibit hormone-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the outer medullary collecting duct (OMCD) and medullary thick ascending limb (MTAL) microdissected from the rat nephron. 2. In the OMCD, 0.3 microM PGE2 and low concentrations of Ca2+ ionophores (10 nM ionomycin or 50 nM A23187) inhibited by about 50% a same pool of arginine vasopressin (AVP)-stimulated cyclic AMP content through a same process insensitive to Bordetella pertussis toxin (PTX). 3. Sulprostone, an agonist of the EP1/EP3 subtypes of the PGE2 receptor, decreased AVP-dependent cyclic AMP accumulation in OMCD and MTAL samples. The concentration eliciting half-maximal inhibition was of about 50 nM in OMCD and 0.1 nM in MTAL. 4. In MTAL, 1 nM sulprostone and PGE2 inhibited by about 90% a same pool of AVP-dependent cyclic AMP content through a PTX-sensitive, Ca2+ -independent pathway. 5. In the OMCD, PGE2 decreased by about 50% glucagon-dependent cyclic AMP synthesis by a process sensitive to PTX and Ca2+ -independent. Sulprostone 1 nM induced the same level of inhibition. 6. These results demonstrate that PGE2 decrease hormone-dependent cyclic AMP accumulation through a G(alpha)i-mediated inhibition of adenylyl cyclase activity in MTAL cells and glucagon-sensitive cells of the OMCD or through a PTX-insensitive increase of intracellular Ca2+ concentration in AVP-sensitive cells of the OMCD.

Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+.

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.

Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content.

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 Ca2+-insensitive type IV mRNA studied on the same samples was evidenced only in the glomerulus. The functional relevance of the expression of Ca2+-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 Ca2+ in this segment induced a Ca2+-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.

Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin.

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)

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule.

Previous studies have demonstrated that prostaglandin E2 (PGE2) inhibits arginine vasopressin-(AVP)dependent adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE2 was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30 degrees C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35 degrees C). In contrast to PGE2, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean +/- SEM): 1 nM AVP = 47.1 +/- 6.8 fmol.mm-1 x 4 min-1; AVP+0.3 microM PGE2 = 20.1 +/- 2.7, P < 0.01 versus AVP; AVP + 10 nM PMA = 42.0 +/- 4.7, NS versus AVP; AVP + 50 micrograms/ml OAG = 44.1 +/- 4.8. NS versus AVP, N = 5 experiments. However, 10 nM PMA prevented PGE2-induced inhibition: AVP + PGE2 = 44.2 +/- 3.5% of the response to AVP and 90.3 +/- 3.2% without and with PMA respectively, N = 16. Similar results were obtained with either 50 micrograms/ml OAG or 25 micrograms/ml DOG (AVP + PGE2 + OAG = 92.9 +/- 6.6% of the response to AVP, N = 8; AVP + PGE2 + DOG = 94.1 +/- 5.3%, N = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin stimulates Na+, Cl-, Ca2+, and Mg2+ transports in TAL of mouse nephron: cross-potentiation with AVP.

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)

PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of alpha 2-adrenergic and alpha 1-adenosine agonists, insensitive to pertussis toxin and dependent on extracellular calcium.

The accumulation of cyclic adenosine 3',5'-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A1 agonist of adenosine (-)-N6-(R-phenylisopropyl) adenosine (PIA), an alpha 2-adrenergic agonist clonidine (CLO), and prostaglandin E2 (PGE2). The negative regulation elicited by PGE2 was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 microM PGE2 with either 0.1 microM PIA or 1 microM CLO led to an inhibition of the response to AVP (80.0 +/- 3.5%, SEM, N = 7 and 92.6 +/- 0.8%, N = 5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE2 in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 micrograms/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE2. PGE2-induced inhibition was prevented in a calcium-free medium [0 Ca2+ + 0.1 mM [ethylene-bis (oxyethylene-nitrilo)] tetraacetate (EGTA)]: values were 67.0 +/- 2.1% and 5.8 +/- 8.7% (+/- SEM) in 2 mM Ca2+ and 0 Ca2+ medium, respectively, N = 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of prostaglandin E2 on agonist-stimulated cAMP accumulation in the distal convoluted tubule isolated from the rabbit kidney.

The effects of calcitonin, vasoactive intestinal peptide (VIP), parathyroid hormone (PTH) and isoprenaline on intracellular cAMP accumulation were determined in the distal tubule (DCT) microdissected from collagenase-treated rabbit kidney. In DCTb (the initial "bright" portion) calcitonin (10 ng/ml) elicited a highly reproducible response 203.7 +/- 19.1 fmol cAMP mm-1 4 min-1 (SE,N = 13) whereas VIP-induced cAMP accumulation was less and more variable from one experiment to another (1 microM, 97.2 +/- 17.8 fmol mm-1 4 min-1, SE, N = 12). When used in combination, these two agonists were non-additive, indicating stimulation of a single pool of cAMP in DCTb. In DCTg, ("granular") which consists of at least two cell types, PTH (100 nM) elicited a marked, reproducible accumulation of cAMP (154.3 +/- 27.0 fmol mm-1 4 min-1; SE, N = 5). Isoprenaline (1 microM) and VIP (1 microM) induced much smaller increases in cAMP levels 20.9 +/- 2.7 and 29.4 +/- 4.1 fmol mm-1 4 min-1 (SE, N = 5) respectively, and, when used in combination, were non-additive, demonstrating that VIP and isoprenaline are active on the same cell type. In DCTb, prostaglandin E2 (PGE2) inhibited both calcitonin- and VIP-stimulated cAMP accumulation (calcitonin 57.8 +/- 2.7% inhibition, SE, N = 16; VIP, 80.6 +/- 2.1% inhibition, SE, N = 5). The EC50 values for calcitonin were 1.21 +/- 0.33 ng/ml and 1.83 +/- 0.25 ng/ml (SD, N = 3) in the absence and presence of PGE2 (300 nM) respectively with an IC50 for PGE2 of 26.3 +/- 6.3 nM (SE, N = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic stimulation of phosphoinositide metabolism in isolated rat glomeruli.

Cholinergic effects on kidney function have been observed in some mammals but the intrarenal localization and the cellular mechanisms of these effects are poorly defined to date. The aim of this work was to study the effects of carbachol on phosphoinositide metabolism in freshly isolated rat glomeruli labeled with myo-[3H]inositol. Carbachol rapidly and markedly stimulates phosphoinositide metabolism with a 50% effective concentration of 3 microM. The enormous magnitude of the response is enlightened by the use of 10 mM lithium, which provokes in the presence of the agonist a large accumulation of inositol phosphates and a corresponding depletion of cellular free inositol. The response is inhibited by 85% by pirenzepine, is pertussis toxin insensitive, and shows no desensitization at maximum dose of carbachol up to 40 min of stimulation.

Establishment of renal cell lines derived from S2 segments of the proximal tubule.

The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology. These cell lines were maintained in culture for at least 24 passages, then cells were frozen. One of the cell lines, the RKPC-2, was selected and further characterized. RKPC-2 cells formed domes on impermeable supports, indicating fluid and solute transport. RKPC-2 cells formed continuous monolayers of low transepithelial resistance on collagen-coated filters. They were able to accumulate tetraethylammonium, an organic cation; however, no significant transcellular transport could be measured. We conclude that this cell line which shows characteristics of epithelial cells has maintained certain properties of intact proximal tubules, in particular the capacity to accumulate organic cations.

Two mechanisms of inhibition by prostaglandin E2 of hormone-dependent cell cAMP in the rat collecting tubule.

The effect of exogenous prostaglandin E2 (PGE2) on hormone-dependent adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated by microradioimmunoassay in collecting tubules microdissected from the cortex (CCT) or outer medulla (MCT) of the rat kidney. Two phosphodiesterase inhibitors were used: either a xanthine derivative (isobutyl-methylxanthine (IBMX, 1 mM] active on all forms of phosphodiesterase or Ro 20-1724 (50 microM) active on the phosphodiesterase type III. A prostaglandin synthesis inhibitor was added to all media. In the presence of IBMX, 0.3 microM PGE2 inhibited by 39.1% the response induced in the CCT by the beta-adrenergic agonist isoproterenol (1 microM). Under the same experimental conditions, arginine vasopressin (AVP)-stimulated cAMP accumulation in CCT or MCT was not affected by PGE2. In the presence of Ro 20-1724, 0.3 microM PGE2 did not modify the response to 1 nM AVP in CCT but inhibited this response in MCT samples (mean inhibition: 52.7%). The inhibition by PGE2 was dose dependent with a maximum at 0.3 microM, observed for all concentrations of AVP tested (from 50 pM to 1 nM) and did not affect the concentration of AVP inducing half-maximal cAMP accumulation. In a second experimental series performed in the presence of adenosine deaminase, an A1-adenosine agonist [theta)-N6-(R-phenylisopropyl)adenosine (PIA, 0.1 microM] also decreased the response to 1 nM AVP in the MCT. The addition of an A1-adenosine antagonist relieved the effect of PIA but did not modify the inhibition observed with PGE2. Thus PGE2 decreased the synthesis of cAMP in beta-adrenergic sensitive cells in rat CCT and might affect the catabolism of AVP-dependent cAMP level rather than its synthesis in rat MCT.

Developmental pattern of cyclic guanosine monophosphate production stimulated by atrial natriuretic peptide in glomeruli microdissected from kidneys of young rats.

Cyclic guanosine monophosphate (GMP) productions by alpha rat atrial natriuretic peptide 1-28 (alpha-rANP), carbamylcholine or sodium nitroprusside were assessed in isolated glomeruli microdissected from collagenase-treated kidneys of 2- to 34-day-old and adult rats. In both young and adult animals, alpha-rANP-stimulated cyclic GMP generation was proportional to the number of glomeruli and was enhanced in a dose-dependent and saturable fashion with increasing alpha-rANP concentrations. The apparent activation constant values were 6.4 nM for 5-day-old and 9.7 nM for adult rats. Maximal doses of either alpha-rANP or rANP 5-28 elicited similar responses in young and adult animals. Clear differences appeared between the developmental patterns of cyclic GMP productions stimulated by either alpha-rANP, carbamylcholine or sodium nitroprusside. The response to alpha-rANP was very large in the youngest rats tested, declined sharply during the suckling period and represented about 1.6 times the adult control level in 34-day-old rats. In contrast, the response to carbamylcholine was low after birth and rose progressively with age up to the adult level at the end of the weaning period, and the response to nitroprusside seemed to be independent of the animal's age.

Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase.

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).

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron.

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 microM GTP, 1 microM VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Half-maximal stimulation of AC by VIP was observed at 26 +/- 10 nM (n = 3) in OMCTi and at 19 nM (n = 2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.

Effect of PGE2 and alpha-adrenergic agonists on AVP-dependent cAMP levels in rabbit and rat CCT.

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.