Danielle Chabardès

PTH sensitive adenyl cyclase activity in different segments of the rabbit nephron

SummaryPTH sensitive adenylate cyclase activity was measured in 9 different segments of the nephron, isolated by microdissection from collagenase-treated rabbit kidney slices.The enzyme of the following segments was stimulated by PTH, 1 U/ml: PCT. (proximal convoluted tubule); PR (pars recta); CAL (cortical portion of the thick ascending limb); DCT (distal convoluted tubule); BCT (first, branched portion of the collecting tubule); the segments which did not respond to PTH were: TDL (thin descending limb); MAL (medullary portion of the thick ascending limb); CCT (cortical portion of the collecting tubule distally adjacent to BCT); MCT (collecting tubule from the outer medulla).PTH sensitive adenylate cyclase per mm tubule in PR was half that measured in PCT.Half maximal stimulation corresponded to 50–100 mU/ml PTH (1–2×10−8M) in both PCT and PR, and to about 350 mU/ml in CAL. PTH (1 U/ml) stimulation factors ranged from 5 to 60 depending on the structures.It is concluded that in addition to PCT and PR, CAL and BCT might be target structures involved in the physiological actions of PTH on the kidney.

Vasopressin dependent adenylate cyclase in single segments of rabbit kidney tubule

SummaryAVP dependent adenylate cyclase activity was measured in single pieces of 8 different tubular segments isolated from collagenase treated rabbit kidneys.High responses were observed in all the tested portions of the collecting tubule, that is its cortical branched part (BCT), its cortical straight part (CCT) and its outer medullary part (MCT). Dose response curves indicated in CCT: 2 fold threshold stimulation at 10−11 M AVP, 27 fold stimulation at 10−6 M AVP, half maximal stimulation at about 10−9 M AVP.Both the medullary (MAL) and, to a lesser exten, the cortical (CAL) portions of the thick ascending limb were also observed to contain AVP sensitive adenylate cyclase (for MAL: 2 fold threshold stimulation at 10−9 M AVP, 9 fold stimulation at 10−7 M AVP, half maximal stimulation at 5×10−9 M AVP).In contrast, nearly no responsiveness to AVP was observed in the proximal convoluted tubule, in the thin descending limb of the loop and in the distal convoluted tubule (DCT).The limited response obtained in DCT (which is a structure generally considered as a target site for AVP) as well as the clearcut effect elicited by AVP in MAL (the functioning of which is not known to be controlled by ADH) were unexpected observations; their possible physiological implications will be discussed.

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.

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+.

Catecholamine sensitive adenylate cyclase activity in different segments of the rabbit nephron

SummaryThe sensitivity to catecholamines of the adenylate cyclase (AC) activity contained in single tubule samples was investigated on 10 different well defined segments, isolated by microdissection from collagenase treated rabbit kidneys.No responsiveness to isoproterenol (10−6 M) was observed in the proximal tubule (convoluted and straight portions), the thin descending and thick ascending limbs of the loop of Henle, and the first (“bright”) portion of the distal convoluted tubule (DCTb); in contrast high responses (stimulation factors: 4 to 6 fold) were obtained in the second (“granular”) portion of the distal convoluted tubule (DCTg), as well as in both the “granular” (CCTg) and the “light” (CCTl) portions of the cortical collecting tubule. In absolute value, however, the CCTl response was definitely lower than those measured in DCTg and CCTg, as is its control activity. In the medullary portion of the collecting tubule, the AC response to isoproterenol was rather poor both in absolute and relative terms.Dose-response curves measured on DCTg samples indicated a threshold response with an isoproterenol concentration below 10−8 M; half maximal effect corresponded to about 3×10−8 M. CCTl sensitivity to isoproterenol was of the same order of magnitude.Isoproterenol as well as norepinephrine effects in DCTg and CCTl were completely suppressed by 10−4 M propranolol, indicating that the observed AC stimulation was mediated via receptors of the β type.In β blocked CCTl, 10−6 M norepinephrine did not inhibit vasopressin-induced AC stimulation; in the presence of 10−6 M norepinephrine, 10−4 M phentolamine resulted in no additional AC stimulation in DCTg and CCTl; these data suggest the absence of α receptors inhibiting AC activity in these structures.In DCTg, AC stimulation induced either by 10−6 M isoproterenol or by 1 U/ml PTH were observed to be additive when the two hormones were given together.The presence of catecholamine-dependent AC activity in three distal portions of the rabbit nephron is discussed in relation to its possible physiological implications.

Inhibition of α2-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.

Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments

SummaryA method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 min at 30°C in a medium containing high specific [α-32P]-ATP 3·10−4 M, final volume 2.5 μl.The [32P]-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn;3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10−15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used.Control and fluoride (5 mM) stimulated adenylate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (DCT), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule.Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Fluoride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity.Series of replicates gave a scatter equal to ±20% (S.D. as a per cent of the mean).The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.

Methodology for Enzymatic Studies of Isolated Tubular Segments: Adenylate Cyclase

Adenylate cyclase (AC) is an ubiquitous enzyme present in the plasma membrane of most cell types; it plays a key role in hormonal control of cell function. At the level of the cell membrane, adenylate cyclase is involved in the transduction of the extracellular signal provided by the hormone for the intracellular production of cyclic AMP. In the cell interior, cyclic AMP is responsible for inducing the appropriate biological response, and was accordingly named “the second hormonal messenger.” The specificity of hormone recognition, by the corresponding target cells, results from the presence of stereospecific hormone receptor sites located on the outer border of cell membranes. These receptor sites are coupled to the AC catalytic units facing the cell interior, so that hormone binding to the receptor results in enzyme activation (Figure 1). The nature of the response induced by cAMP depends essentially on the type of the target cell, i.e., on its specific differentiation, and is not directly related to the structure of the stimulating hormone itself.

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

SummaryRenal 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 dependend 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 occuring in the loop during both periods. Ca reabsorption was highly correlated to that of sodium along the proximal tubule and Henles loop. This was not the case for Mg and P. In the 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.

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 μM or 1 μM, 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 apparentKa 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 after 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 competible with a direct action of ANP stimulating cGMP generation in tubules or inhibiting vasopressin-induced cAMP generation in collecting tubules.