Adenylate cyclase responsiveness to hormones in various portions of the human nephron.

The action sites for parathyroid hormone (PTH), salmon calcitonin (SCT), and arginine-vasopressin (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 medullay collecting tubule (activity ratios were 7, 14, 15, 3, and 3, respectively). AVP (1 microM) 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 of the 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 as well preserved under the conditions used in this study.

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.

Vasopressin-dependent adenylate cyclase activities in the rat kidney medulla: evidence for two separate sites of action.

This study demostrates the existence of an adenylate cyclase sensitive to vasopressin in the medullary portion of the rat thick ascending limb. Maximal adenylate cyclase stimulations achieved in that segment (31-fold) were higher than those obtained in collecting tubules from the same rats (22-fold). From comparisons of absolute maximal responses it can be calculated that thick ascending limbs account for about 80% of the response to vasopressin of a kidney medulla homogenate. The apparent Km value of adenylate cyclase activation (from 10(-9)-2 x 10(-8) M) in thick ascending limbs was higher in each experiment than that simultaneously measured in the collecting tubules from the same rats (2 x 10(-10)-3 x 10(-9) M). Such a lower sensitivity is probably not due to a greater hormone degradation by the thick ascending limb samples. Experiments using structural analogues of the oxytocin series ([deamino-6-carba]oxytocin and vasotocin) did not give evidence for different vasopressin receptors in the thick ascending limb and the collecting tubule. A step beyond the hormone-receptor interaction, thus, must account for the different patterns of adenylate cyclase response to vasopressin of these two segments.

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.

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.

Inhibition of alpha 2-adrenergic agonists on AVP-induced cAMP accumulation in isolated collecting tubule of the rat kidney.

A microradioimmunoassay for cAMP was developed in order to analyse the effects of alpha-adrenergic agonists on vasopressin (AVP)-induced cAMP cell accumulation in single pieces of microdissected medullary (MCT) and cortical (CCT) rat collecting tubules. Under the experimental conditions chosen (4 min of incubation in the presence of a phosphodiesterase inhibitor), no cAMP could be detected either in the bathing solution or in non-stimulating samples of tubule. In MCT, 10(-6) M AVP stimulated cAMP generation up to 128.3 +/- 9.0 (SEM) fmoles per mm of tubule per 4 min, N = 11. The response was dose-dependent with a KA value below 10(-10) M AVP. The addition of norepinephrine (NE) (10(-5) M in the presence of propranolol) suppressed the larger part of the response to AVP (from 92% with 2 X 10(-11) M AVP to 76% with 10(-6) M AVP); the addition of 10(-7) M NE still reduced by 59% the MCT response to 10(-10) M AVP (26.2 +/- 5.9 vs. 64.0 +/- 6.4 fmoles/mm, N = 3). In CCT, 10(-5) M NE reduced by 84% the cAMP generation induced by 10(-10) M AVP (8.8 +/- 2.0 vs. 54.2 +/- 3.5 fmoles/mm, N = 3). This inhibitory action of NE against the AVP effect in CCT was mimicked by 10(-7) M clonidine; in MCT it was suppressed by phentolamine and yohimbine, but not by prazosin, suggesting that alpha 2-adrenoreceptors are involved. On the other hand, the addition of the alpha-agonists to the incubation solution produced no inhibition of the cAMP cell accumulations induced by glucagon, calcitonin and isoproterenol in CCT, or glucagon in MCT, an observation demonstrating that alpha 2-adrenergic agonists selectively inhibit vasopressin-dependent cAMP generation by these nephron segments.

Multiple hormonal control of adenylate cyclase in distal segments of the rat kidney.

Using the single tubule adenylate cyclase microassay, we investigated in vitro in three different segments of the rat nephron whether the effects of various hormones are additive when these hormones are tested in combination. In the cortical portion of the thick ascending limb (CAL), no additivity of the effects of glucagon, calcitonin, and PTH was observed. In the medullary portion of the thick ascending limb (MAL), the effects of vasopressin and glucagon were only partly additive, and the effects of vasopressin and calcitonin were fully additive. In the cortical collecting tubule (CCT), the effects of calcitonin and vasopressin were nonadditive in the kidneys in which vasopressin alone induced a high cyclase stimulation, whereas they were fully additive when vasopressin induced a low cyclase stimulation. The data suggest that in each segment, the hormones tested stimulated the same cells: no additivity was observed when cyclase Vmax acted as the limiting factor of the response; partial or full additivity was observed when the number of hormone receptors acted as the limiting factor of the response. As a consequence, calcitonin, glucagon, and PTH should induce the same effects in CAL; vasopressin, glucagon, and calcitonin, the same effects in MAL; and vasopressin and calcitonin, the same effects in CCT.

Functional segmentation of the rabbit distal tubule by microdetermination of hormone-dependent adenylate cyclase activity.

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.

[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.

Distribution of hormone-dependent adenylate cyclase in the nephron and its physiological significance.

In addition to the well established action of PTH in proximal tubules and of AVP in collecting tubules, polypeptide hormones were recently shown to regulate transport properties in other tubular portions. Although still scarce, such physiological studies using isolated perfused tubules demonstrated hormonal effects in those nephron segments observed to contain responsive adenylate cyclase and not in the others. Moreover, the same effects were elicited by applying exogenous cAMP or cAMP derivatives. There is, therefore, good evidence that hormone-dependent adenylate cyclase is involved in the cell mechanisms through which many hormones regulate tubular functions. The effects obtained varied depending on the segment of tubule used. It is not yet established whether the nature of the hormonal effect induced via cAMP is entirely specified by the responding cell types or is also specified by the hormone itself. Further studies are needed to clarify this important problem, as well as many other as yet unsolved questions. There is obviously much more to learn about the hormonal regulation of tubular cell functions by using appropriate biochemical and physiological micromethods.

[Vasopressin action sites along the nephron (author's transl)]. / Les sites d'action de la vasopresine dans le rein.

Sites of action of vasopressin along the nephron were investigated by using a microassay for adenylate cyclase for single pieces of tubule microdissected from collagenase-treated kidneys. In the rabbit, not only the medullary and cortical portions of collecting tubules, but also the thin and thick segments of the ascending limb of Henle's loop were observed to contain adenylate cyclase highly responsive to arginine-vasopressin. In contrast, the other segments of the nephron-including the descending limb of the loop, the distal convolution, and the connecting tubule -- were unresponsive to vasopressin. Qualitative and quantitative species difference were noted between rabbit, rat, mouse and man, regarding vasopressin responsiveness in distal tubules and ascending limbs. As an example, adenylate cyclase in thick ascending limb is not sensitive to vasopressin in man whereas, in rat, it is as responsive as the collecting tubule. The physiological relevance of these results is discussed.

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).

Influence of extracellular fluid volume expansion on magnesium, calcium and phosphate handling along the rat nephron.

Renal tubular handling of P, Ca, Mg and Na was studied in the rat both before and during mild hypertonic NaCl loading (ECVE), using micropuncture and clearance techniques and electron microprobe analysis. Micropuncture was performed at the late proximal and early distal tubule sites. ECVE significantly increased the urinary output of all four elements. In the case of Mg, the increase was relatively small and depended on slight but statistically unsignificant inhibition of reabsorption all along the entire length of the nephron. For Ca, it depended on the inhibition of proximal reabsorption, partially compensated by increased reabsorption along the loop. For P, it depended on proximal inhibition, no important net phosphate movement occurring in the loop during both periods. Ca reabsorption was highly correlated to that of sodium along the proximal tubule and Henel's loop, Ca and Mg reabsorption were closely related to the load delivered at the beginning of the structure. These observations are compatible with the view that tubular reabsorption of Ca and Mg is concentration rather than Tm limited, and that reabsorption of Ca, unlike that of Mg, is linked to the movements of sodium. Following ECVE, the difference between early distal and urinary deliveries increased significantly for Ca and P, but not for Mg. For phosphate, this difference accounted for by 45% of the delivery at the early distal tubule site, at variance with microinjection data obtained in the rat under similar salt loading conditions, which indicated that 17% only of the phosphate distal delivery were reabsorbed along the terminal segments. This discrepancy is discussed in terms of nephron functional heterogeneity.

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)

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)

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.

[Vasopressin-stimulated adenyl cyclase activity in the ascending portions of rabbit kidney nephron loops]. / Présence d'une adenyl-cyclase stimulée par la vasopressine dans la branche ascendante des anses des néphrons du rein de Lapin

The adenylate-cyclase activity contained in the successive segments of the loop of Henle was measured using single tubular pieces isolated by microdissection. Vasopressin (10 (-6) M) had no significant effect on either the pars recta or the thin descending limb; whereas, it stimulated the enzyme contained in the thin ascending limb (8.9 fold as a mean value) and that contained in the medullary portion of the large ascending limb (14.7 fold).

Hormone-sensitive adenylate cyclase along the nephron of genetically hypophosphatemic mice.

The response of the adenylate cyclase (AC) activity to PTH and calcitonin was measured along the nephron of normal (N) and mutant hypophosphatemic (Hyp) mice of the C 57 BL/6J strain, using in vitro single tubule AC microassay. In each experiment, a Hyp mouse was paired to a N mouse from the same litter. In the presence of PTH (10 U/ml), AC activities (femtomoles cAMP per millimeter of tubule per 30-min incubation) were reduced in the proximal convoluted tubule of Hyp mice as compared to N mice in all experiments (448 +/- (SEM) 46 vs. 831 +/- 79, N = 4, P less than 0.01). Some decrease in AC response to PTH also was noted in the cortical portion of the thick ascending limb of the loop of Henle (476 +/- 70 in Hyp mice vs. 719 +/- 83 in N mice, N = 4, P = NS). The Hyp and N AC responses to PTH were similar in the "bright" and "granular" portions of the distal convoluted tubule (1524 +/- 177 in Hyp mice and 1538 +/- 228 in N mice, N = 4). The other segments tested were not responsive to PTH (except the pars recta of the proximal tubule). In the presence of salmon calcitonin (10 ng/ml), a striking 5- to 12-fold increase in AC activity of the "bright" and "granular" portions of the distal convoluted tubule was observed in each Hyp mouse as compared to its paired N control (2434 +/- 618 vs. 399 +/- 56, N = 6, P less than 0.01). The AC response to calcitonin was also increased, though to a lesser extnet (Hyp/N = 1.8) in the "light" portion of the distal tubule (590 +/- 60 in Hyp and 352 +/- 36 in N mice, P less than 0.01). Other segments of the mouse nephron were also observed to contain calcitonin-sensitive AC, but the responses were of limited magnitude only and were not statistically different in Hyp and N mice. Dose-response curves showed that the decrease of the response to PTH in the proximal tubule as well as the increase of the response to calcitonin in the distal tubule were present in Hyp mice for the whole range of hormone concentrations tested. In both structures, the apparent Km for the cyclase activation by the hormone was similar in the Hyp and its paired N mouse.

Atrial natriuretic peptide effects on cGMP and cAMP contents in microdissected glomeruli and segments of the rat and rabbit nephrons.

A microradioimmunoassay has been developed in order to measure the changes in cGMP cell content induced in vitro by atrial natriuretic peptides (ANP) in either glomeruli or defined portions of tubules microdissected from collagenase treated rat and rabbit kidneys. When tested at 0.1 microM or 1 microM, all ANP analogues used produced in rat glomeruli a 20-25 fold increase in cGMP accumulation compared to basal values. Threshold responses were obtained with about 1 nM ANP and apparent Ka values ranged between 5 and 50 nM. Atriopeptin III led to similar results in glomeruli isolated from rabbit. Under the same experimental conditions, no cGMP could be detected in any ANP-treated nephron segment from the rat kidney (namely, from the proximal convoluted tubule up to the outer medullary collecting tubule) nor in cortical collecting tubules isolated from the rabbit kidney. Moreover, ANP did not alter the forskolin-induced increase in cAMP content in glomeruli or collecting tubules, nor the AVP-induced increase in cAMP content in collecting tubules. Our data confirm the marked effect of ANP on cGMP generation by isolated glomeruli from rat and rabbit; however, they are not compatible with a direct action of ANP stimulating cGMP generation in tubules or inhibiting vasopressin-induced cAMP generation in collecting tubules.

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)