P2Y receptors present in the native and isolated rat glomerulus.

Extracellular ATP can mobilize intracellular calcium in rat glomeruli by interacting with P2Y receptors. However, the identity of the receptor subtypes involved is not known. In the present study, we have used RT-PCR to identify mRNAs for specific P2Y receptor subtypes expressed in the rat glomerulus: mRNA for P2Y1, P2Y2, P2Y4 and P2Y6 receptors was detected. Functional expression of P2Y1 and P2Y2/P2Y4, but not P2Y6, receptors in intact glomeruli was confirmed by measuring the relative stimulation of the inositol phosphate pathway induced by selective agonists of a particular receptor subtype. Finally, we have used available polyclonal antibodies to confirm the expression of P2Y1 and P2Y2 in the glomerulus, in mesangial cells and glomerular epithelial cells (podocytes), respectively; but we could not demonstrate P2Y4 or P2Y6 receptor expression by this means. In a separate series of experiments, we have examined the possibility that intra-renal sympathetic nerve terminals are a source of extracellular ATP and that this would be supported, though not excluded, by supersensitivity to ATP following denervation. Nucleotide-induced stimulation of the inositol phosphate pathway was measured in both control rats and rats that had been sympathectomized by intraperitoneal injection of 6-hydroxydopamine. The response to norepinephrine was measured as a positive control. In the sympathectomized rats, the effect of norepinephrine was significantly enhanced, whereas ATP-induced inositol phosphate production was unaffected, being similar in both groups of animals.

Axial distribution and characterization of basolateral P2Y receptors along the rat renal tubule.

BACKGROUND: Several groups have identified P2Y receptors in the basolateral membrane of the rat nephron. These studies have not covered all segments of the nephron and have relied solely on the relative potency of receptor agonists for classification. METHODS: We measured purine and pyrimidine-induced changes in intracellular free calcium concentration ([Ca(2+)](i)) in anatomically defined segments of the rat nephron. To complement these functional studies, we have used reverse transcription-polymerase chain reaction methodology to identify specific P2Y receptor transcripts in these segments. RESULTS: Adenosine 5'-triphosphate (ATP) mobilized [Ca(2+)](i) in all nephron segments, except for the thick ascending limb of Henle, which was poorly responsive. Adenosine (100 micromol/L) was without effect, confirming that the effect of ATP was mediated by P2 receptors. In the proximal convoluted tubule (PCT) and outer medullary collecting duct (OMCD), there was evidence for two receptor subtypes with characteristics of P2Y(1)- and either P2Y(2)- or P2Y(4)-like receptors. A novel finding in the thin limbs was the presence of a receptor with properties of both P2Y(2) and P2Y(4) receptor subtypes. To aid classification, we identified P2Y receptor mRNA in rat nephron segments. In the PCT and OMCD and thin ascending limb of Henle, we found expression of P2Y(1), P2Y(2), and P2Y(4) receptors. In the descending limb of Henle, P2Y(1) and P2Y(2) mRNA was found, but P2Y(4) was not expressed. CONCLUSION: These data suggest that extracellular ATP can influence tubular cell function in all segments of the rat nephron, through P2Y receptors via multiple (and coexpressed) P2Y receptor subtypes.

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.

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+.

Relationship between extra- and intracellular calcium in distal segments of the renal tubule. Role of the Ca2+ receptor RaKCaR.

The effect of extracellular calcium ([Ca2+]e) on cytosolic calcium ([Ca2+]i) was investigated in thick ascending limbs and collecting ducts from the rat kidney, using the fluorescent dye fura-2. In cortical collecting ducts, basolateral but not apical changes in [Ca2+]e were associated with parallel changes in [Ca2+]i. Basal [Ca2+]i was hardly modified by nifedipine and verapamil but was decreased by 60% by basolateral La3+. Increasing peritubular [Ca2+]e triggered Ca2+ release from intracellular stores. This effect was not reproduced by agonists of the renal Ca2+-receptor RaKCaR, e.g., Ba2+, Mg2+, Gd3+, and neomycin, but was reproduced by Ni2+. Ni2+-induced mobilization of intracellular Ca2+ was larger in the inner medullary collecting duct, a segment which poorly responds to increasing [Ca2+]e. In the cortical thick ascending limb, removing basolateral Ca2+ hardly altered [Ca2+]i but increasing [Ca2+]e or adding Ba2+, Mg2+, Gd3+ and neomycin released intracellular calcium. These data demonstrate that (1) basolateral influx of calcium occurs in cortical collecting ducts, under basal conditions; (2) this influx occurs through nonvoltage gated channels, permeable to Ba2+, insensitive to verapamil and nifedipine, and blocked by La3+; (3) increasing [Ca2+]e stimulates the influx and triggers intracellular calcium release, independently of the phospholipase C-coupled receptor RaKCaR; (4) RaKCaR is functionally expressed in thick ascending limbs; (5) another membrane receptor, sensitive to Ni2+ but not to Ca2+ is present in the collecting duct.

Cholinergic agonists increase phosphoinositide metabolism and cell calcium in isolated rat renal proximal tubule.

The intracellular signaling involved in cholinergic modulation of renal proximal tubule functions has not been addressed. We report that acetylcholine and carbachol increase the production of inositol phosphates and the intracellular calcium concentration in rat proximal tubule. Muscarinic M3 receptors are involved, given the inhibitory effects of selective antagonists (4-diphenylacetoxy-N-methylpiperidine > > pirenzepine > methoctramine). The muscarinic response is absent in the early part of proximal straight tubule. The response is smaller and more variable in proximal convoluted tubule segments selected at random from cortical tubule suspensions than in the early part of proximal convoluted tubule. This contrasts with the norepinephrine response, which has almost the same magnitude all along the proximal tubule. We conclude that cholinergic agonists activate muscarinic M3 receptors in proximal tubule and that there is a decreasing response gradient from the early convoluted tubule to the early straight tubule.

Molecular analysis of beta-adrenergic receptor subtypes in rat collecting duct: effects on cell cAMP and Ca2+ levels.

Expression and functional properties of beta-adrenergic receptors (beta-ARs) were studied in rat collecting tubules isolated by microdissection. Reverse transcription-polymerase chain reaction experiments demonstrated that the beta 1- and beta 2-AR mRNAs, but not the beta 3-subtype, are expressed in the cortical collecting duct (CCD). Quantitation of mRNAs, carried out using mutant RNAs as internal standards, further showed that beta 1- and beta 2-ARs transcripts are present at comparable amounts in CCD (3,000-4,000 copies/mm of tubular length), but reach 6-8 times lower levels in the outer medullary collecting duct (OMCD: beta 1, 480 +/- 180; beta 2, 590 +/- 110 copies/mm of tubular length). Functional studies, carried out in CCD, corroborated the expression of these two receptor subtypes. The rank order of potency of beta-agonists for stimulating adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was isoproterenol > norepinephrine = epinephrine, and similar efficiencies were found for a beta 1- and a beta 2-antagonist to inhibit isoproterenol-dependent cAMP formation. Fura 2 fluorescence measurements revealed that isoproterenol (10 microM) induces a biphasic rise of intracellular free Ca2+ concentration ([Ca2+]i), consisting of an initial fast increase (delta [Ca2+]i = 122 nM) followed by a plateau phase (delta [Ca2+]i = 58 nM). In the absence of basolateral Ca2+, the initial peak was still observed, suggesting intracellular Ca2+ release. Norepinephrine and epinephrine, as well as selective beta 1- and beta 2-agonists, also increased [Ca2+]i in CCD. Only slight [Ca2+]i variations were produced by isoproterenol in the OMCD (delta [Ca2+]i = 21 nM) and the cortical thick ascending limb (delta [Ca2+]i = 25 nM). These results show that both beta 1- and beta 2-ARs are expressed in the collecting tubule and that they predominate in the CCD. The two receptor subtypes contribute to cAMP accumulation induced by beta-agonists. They also trigger [Ca2+]i variations, indicating their possible coupling to several transduction pathways in the rat CCD.

[Functional expression of vasopressin receptors V1a and V2 along the mammalian nephron]. / Expression fonctionnelle des récepteurs V1a et V2 de la vasopressine le long du néphron de mammifère.

In vitro studies on single microdissected segments have been extensively used during the 20 past years to localize V1 and V2 vasopressin receptors within the mammalian kidney, and define their role in the control of water balance. Based on vasopressin-dependent adenylate cyclase activity measurements and quantitative RT-PCR studies, it is now clear that V2 receptors are present along the whole collecting duct from cortex to papilla, and, in most species, in the ascending limb of Henle's loop (thick and thin limb); occasionally in the distal tubule but not in the other segments. The stimulation by cyclic AMP of sodium chloride reabsorption in the thick ascending limb, and of urea reabsorption in the papillary collecting duct indicates that vasopressin--in addition to its well known hydroosmotic effect--also participates in the building up of the corticopapillary gradient of osmotic pressure. As regards the V1a receptor, binding studies as well as quantitative RT-PCR, and measurements of free cytosolic calcium concentration allow us to draw the following conclusions. In the rat, the V1a receptor is absent from the glomerulus, the proximal tubule (convoluted and straight portions), the tick ascending limb of Henle's loop and the terminal portion of the papillary collecting duct. It is present in the thin ascending limb and the cortical and outer medullary portions of the collecting duct. Its presence in the thin descending limb has not, up to now, been explored. By contrast with previous data in the rabbit, the V1a receptor does not alter vasopressin-dependent sodium and water reabsorption in the rat cortical collecting duct. Further studies will be necessary to determine its functional role in that segment, as well as in the thin ascending limb. Finally, vasopressin V2 agonists have been shown to induce intracellular calcium release in the papillary collecting duct, a segment devoid of V1a receptors. This effect--which cannot be ascribed to a cross-reaction with oxytocic receptors--indicates either an unusual coupling of the V2 receptor to phospholipase C or, else, the presence of a new vasopressin receptor.

cAMP-dependent effects of vasopressin and calcitonin on cytosolic calcium in rat CCD.

Fura 2 fluorescence measurements were carried out on microperfused rat cortical collecting ducts (CCD) to investigate the effect of adenosine 3',5'-cyclic monophosphate (cAMP) and adenylate cyclase-stimulating hormones on free cytosolic calcium ([Ca2+]i). Forskolin, 3-isobutyl-1-methylxanthine, and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) all triggered marked and sustained [Ca2+]i variations. Maximal increases elicited by 100 microM CPT-cAMP amounted to 101 +/- 11 nM (mean +/- SE, n = 18). This effect was mostly dependent on the presence of basolateral calcium and totally independent of luminal calcium. It remained unchanged in CCD perfused with sodium-free luminal fluid (82 +/- 10 nM, n = 5), pretreated with 1 mM bath ouabain (113 +/- 20, n = 4), or superfused with sodium-free bath in the presence of ouabain (82 +/- 22, n = 5). The V2 agonist 1-desamino-8-D-arginine vasopressin (DDAVP, 10 nM) increased [Ca2+]i by 57 +/- 5 nM (n = 27), a value 40% lower than that achieved with 10 nM AVP (141 +/- 7, n = 34) but similar to that observed with AVP + a V1a antagonist (57 +/- 6, n = 6). Significant effects could also be obtained with 200 pM DDAVP (31 +/- 6, n = 8) and arginine vasopressin (AVP) (72 +/- 6, n = 16). Rat calcitonin also raised [Ca2+]i by 43 +/- 10 (n = 8) and 66 +/- 8 nM (n = 17) at 1 and 10 nM, respectively, and its effect was not additive to that of CPT-cAMP. Calcitonin and DDAVP effects, like those of CPT-cAMP and forskolin, were nearly abolished in Ca(2+)-free bath, but AVP action on intracellular release persisted. These results show that, in rat CCD, cAMP effects on [Ca2+]i mainly result from basolateral calcium entry. In contrast to rabbit CCD the mechanism is independent on Na reabsorption and basolateral Na+/Ca2+ exchange. Calcitonin and DDAVP effects on [Ca2+]i are probably secondary to increased cAMP production.

Cyclic guanosine monophosphate is the mediator of platelet-activating factor inhibition on transport by the mouse kidney thick ascending limb.

Since we have previously shown a direct inhibitory effect of platelet-activating factor (PAF) on Cl reabsorption in the medullary thick ascending limb of Henle's loop (TAL), the aim of this study was to extend this effect to the whole TAL and to further investigate the signaling pathway involved. In microperfused cortical TALs, PAF significantly decreased Cl reabsorption by 50.3 +/- 6.5%. On the one hand, this effect was not modified in the presence of staurosporine and was not mimicked by phorbol ester; chelating cytosolic Ca by BAPTA/AM failed to suppress the inhibitory effect of PAF on Cl reabsorption; moreover, no significant increase in intracellular Ca concentration could be observed in the presence of PAF on isolated tubules. On the other hand, 8-bromo cyclic GMP mimicked the PAF effect on Cl reabsorption and prevented a further effect of this agent; the PAF effect was significantly reduced by H-8, a cyclic GMP-dependent protein kinase inhibitor; in medullary TALs, PAF significantly increased by twofold cyclic GMP content, an effect inhibited by the PAF antagonist BN 50730, whereas PAF did not significantly modify cAMP content in basal or stimulated conditions. Finally, inhibition of nitric oxide production by NAME or NMMA failed to prevent the effect of PAF on Cl reabsorption. It is concluded that the PAF-induced inhibition of Cl reabsorption in the TAL was mediated by cyclic GMP, likely independent of a nitric oxide synthesis.

V2-like vasopressin receptor mobilizes intracellular Ca2+ in rat medullary collecting tubules.

Cytosolic free calcium concentration ([Ca2+]i) was measured in single microdissected rat medullary collecting tubules [outer (OMCD) and inner (IMCD)] to identify receptors involved in vasopressin (AVP)-induced [Ca2+]i increases. In both segments, [Phe2,Orn8]vasotocin ([Phe2,Orn8]VT), a specific V1 agonist, as well as the V2 agonist 1-desamino-8-D-AVP (dDAVP) triggered [Ca2+]i variations. In OMCD, the mean response to 10 nM AVP roughly corresponded to the sum of V1 and V2 agonists effects. In IMCD, dDAVP (10 nM) alone reproduced the calcium response to AVP (delta[Ca2+]i = 243 +/- 34 nM, n = 6, and 248 +/- 27 nM, n = 8, with dDAVP and AVP, respectively). Furthermore, in the same experiments V1 and V2 maximal effects were not additive ([Phe2,Orn8]VT = 154 +/- 21 nM, n = 6; dDAVP + [Phe2,Orn8]VT = 233 +/- 23 nM, n = 9). As AVP, dDAVP released intracellular calcium (delta[Ca2+]i in calcium-free medium = 182 +/- 24 nM, n = 8, vs. 182 +/- 14 nM, n = 6 with 10 nM dDAVP and AVP, respectively). Neither 8-(4-chlorophenyl-thio)-adenosine 3',5'-cyclic monophosphate nor forskolin modified [Ca2+]i. A cross-reaction of dDAVP with an oxytocin (OT) receptor can be excluded since 1) the specific OT agonist [Thr4,Gly7]OT (10 nM) increased only slightly [Ca2+]i (delta-[Ca2+]i = 20 +/- 5 nM, n = 11); 2) the dDAVP response was not altered by the specific OT antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine,4-threonine, 8-ornithine,9-tyrosylamide]vasotocin [d(CH2)5(1),O-Me-Tyr2,Thr4,Tyr-NH2(9)]OVT; 3) it was insensitive to V1 antagonists but was totally blocked by the V1/V2 antagonist [d(CH2)5(1),O-Et-Tyr2,Val4]AVP ([delta[Ca2+]i = 18 +/- 4 nM, n = 6). These results indicate that in IMCD AVP increases [Ca2+]i via both V1 and V2 receptors. [Ca2+]i variations due to V2 receptors involve a mechanism independent of adenylate cyclase and coupled to the same intracellular calcium pool as V1 and V2 receptors.

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)

Isoproterenol increases Ca, Mg, and NaCl reabsorption in mouse thick ascending limb.

The effect of isoproterenol (Iso) on tubular transport in the thick ascending limb of Henle's loop (TAL) was investigated by in vitro microperfusion of MTAL (medullary) and CTAL (cortical) from White Swiss mouse kidney. The pattern of activation of adenylate cyclase along the distal tubule was investigated in this strain: results indicated that Iso stimulated adenylate cyclase fivefold in MTAL and ninefold in CTAL. Data from microperfusion experiments showed that Iso (10(-7) M in the bath) significantly and reversibly increased Ca and Mg reabsorption in CTAL. No net transport of Ca and Mg was observed in MTAL whether Iso was present or not. With regard to Na and Cl, Iso significantly stimulated their reabsorption in both segments and increased the transepithelial voltage in MTAL. Iso abolished K reabsorption in MTAL and induced a net K secretion in CTAL, the latter effect being also observed with 10(-9) M of Iso. When applied on CTAL, propranolol (10(-6) M in the bath) inhibited all these effects. These data indicate that beta-adrenergic agonists are involved in the multihormonal modulation of the TAL function.

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.

Alterations of enzymatic activities along rat collecting tubule in potassium depletion.

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.

Sites of prostaglandin E2 (PGE2) synthesis along the rabbit nephron.

The purpose of this study was to establish whether the nephron segments recognized as PGE2 target sites in the rabbit, i.e. the proximal tubule, the thick ascending limb and the collecting tubule, are also sites of PGE2 production. We therefore developed a microimmunoassay sensitive enough to allow the measurement of PGE2 on microdissected tubular segments about 1 mm in length. Under the conditions used (30 min incubation at 20 degrees C), a basal rate of PGE2 production was measured in the cortical (CCT) and medullary portions of the collecting tubule, as could be expected. In the presence of 10(-4) M sodium arachidonate, it was shown that: (1) The thin descending limb (TDL) is also an active site of PGE2 formation. When expressed per mm tubule length the amounts formed were lower in TDL than in CCT (14.1 +/- 2.7 SE pg/mm, n = 5, vs. 93.5 +/- 10.7, n = 8). They were quite comparable, however, when expressed per microgram total proteins (0.70 ng in TDL vs 0.6 in CCT). (2) A slight PGE2 production was noted in the connecting tubule but it was likely due to contamination by adjacent CCT cells. (3) In the other nephron segments, only negligible amounts of PGE2 were formed, which are probably of no physiological significance.

Influence of chronic ADH treatment on adenylate cyclase and ATPase activity in distal nephron segments of diabetes insipidus Brattleboro rats.

The medullary thick ascending limb (MAL), but not the medullary collecting tubule (MCT), has been shown to have an impaired adenylate cyclase (AC) responsiveness to ADH and a selective hypoplasia in Brattleboro diabetes insipidus (DI) rats. Since chronic ADH administration has been found to increase epithelium volume and basolateral membrane surface area in MAL but not in MCT, we investigated whether chronic ADH infusion would affect the hormone-sensitive AC and the Na-K-ATPase activity--two markers of the basolateral membrane--in single micro-dissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC responses to in vitro AVP and glucagon and Na-K-ATPase activity increased to the same extent as did epithelium volume (60-80%); 2. changes in the other segments were independent of any morphological alteration. In the cortical thick ascending limb, AVP and glucagon-sensitive AC decreased by 30-40% whereas Na-K-ATPase activity did not change. In the collecting tubule, AC response to in vitro AVP was not altered by ADH-treatment but glucagon-sensitive AC dropped by 50% and Na-K-ATPase activity doubled, independently of any variation in plasma aldosterone and glucagon levels. These results show that, in the MAL, the ADH-induced variations in enzyme activity are a reflection of the enlargement of the basolateral membrane surface area. Further studies are needed to clarify the origin of enzymatic alterations in the other segments.

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.

Ontogenesis of hormone-dependent adenylate cyclase in isolated rat nephron segments.

Ontogenesis of hormone-dependent adenylate cyclase (AC) was investigated in rat kidney by single tubule microassay between two days postnatal and adulthood. This approach allowed us to analyze the kinetics of vasopressin-sensitive AC maturation in its tubular target sites, namely the thick ascending limb and collecting tubule. It was also possible to compare in a single segment--the thick ascending limb--the kinetics of AC ontogenesis for three hormones-- vasopressin, calcitonin, and parathyroid hormone. The results show that 1) 2 days after birth AC is still poorly responsive to vasopressin, especially in the thick ascending limb. By contrast, this segment exhibits marked AC responses to calcitonin and parathyroid hormone. 2) For a given hormone, the kinetics of AC ontogenesis depends on the segment in which the receptor-enzyme complex is located. 3) For a given segment, the pattern of AC maturation is specific for each hormone. These data indicate that the process of tubular AC maturation cannot be accounted for simply by an increase in basolateral membrane area or by the synthesis of new catalytic units. More specific mechanisms must be involved that regulate independently the synthesis of each kind of hormone receptor and/or its coupling to cyclase.