François Morel

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.

Electron Probe Analysis of Tubular Fluid Composition

Electron Probe Analysis (EPA), using a MS 46 microprobe (Cameca, France), has been developped and applied to the determination of Cl, Na, K, Ca, P and Fe concentrations in 48 micropuncture samples col

Phosphate, calcium and magnesium transfers in proximal tubules and loops of henle, as measured by single nephron microperfusion experiments in the rat

SummaryOil blocked superficial proximal tubules were microperfused in situ (30 nl/min) with physiological solutions labelled with45Ca,32Pi and3H-inulin containing either 2 mM (normal Pi) or 0.6 mM (low Pi) as phosphate concentrations. Fluid was successively collected from the first distal, the last proximal and one or two other proximal convolutions of each perfused nephron. 3H,45Ca and32P radioactivities were measured by liquid scintillation with appropriate energy discrimination. Pi, Ca and Mg concentrations were determined by electron probe analysis.Net and unidirectional fluxes were calculated for each tubule from the data provided by paired samples.The results indicate that:1.Proximal calcium net absorption involves large bidirectional fluxes and is not affected by the Pi concentration in the perfusate; calcium absorption along the loop probably includes two different processes, the first, involving bidirectional fluxes, might take place in thepars recta, the second, involving low Ca backflux and active transport against a chemical gradient, might take place along the ascending limb.2.Magnesium net absorption was very low or even absent in the perfused proximal segments; Magnesium net absorption in the loop resulted in an Mg concentration in the early distal fluid lower than that in plasma ultrafiltrate, indicating active transport out of the ascending limb; Mg and Ca net absorptions in the loop were highly correlated.3.Proximal net phosphate absorption was present with normal Pi solutions, but not with low Pi solutions, suggesting that proximal phosphate transport is concentration-limited. Unidirectional lumen-to-plasma Pi flux was almost equal to the net flux with both kinds of solutions, indicating that phosphate reabsorption involves very low permeability of the proximal tubule to phosphate back diffusion. In the loop, net phosphate absorption was very limited (normal Pi) or nil (low Pi), and the absence of any drop in32P specific activity indicates that no bidirectional Pi exchanges took place along the loop of Henle. Mean values for unidirectional and net fluxes of Pi and calcium in the proximal are given in the tables as pmoles per min per mm of tubule, as well as mean values for the net absorption flux of Ca, Mg andPi within the loops (pmoles per min per loop).

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.

Cytosolic free calcium in single microdissected rat cortical collecting tubules

Cytosolic free Ca2+ ([Ca2+]i) was measured in single fragments of rat cortical collecting tubule (CCT) by using fura-2 and a tubule superfusion device. Under basal conditions, i.e. with 1 mM of external Ca2+ ([Ca2+]o), the average steady state [Ca2+]i was 179±16 nM (n=44 tubules). Random alterations of [Ca2+]o between 0 mM and 4 mM led to corresponding variations in steady state [Ca2+]i levels, which were linearly correlated with [Ca2+]o (average slope 93±34 nM [Ca2+]i per 1 mM [Ca2+]o for six tubules). In contrast, [Ca2+]i was little affected by decreasing external Na+ concentration. Cell membrane depolarization with 100 mM of external K+ induced a sustained drop in [Ca2+]i (21% as an average). The data suggest that steady state [Ca2+]i in CCT cells resulted from a non-saturable passive entry of calcium ions across cell membranes balanced with an active extrusion by calcium ATPase (pump and leak mechanism). The passive component cannot be accounted for either by Na+/Ca2+ exchangers nor by voltage-dependent calcium channels; it is best explained by the presence of voltage-independent calcium channels in cell membranes.

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.

Insulin receptors along the rat nephron: [125I] insulin binding in microdissected glomeruli and tubules.

Binding of [125I] Tyr A14 human insulin ([125I] insulin) was measured at 4°C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10−18 mol [125I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5±0.3; proximal convoluted tubule (PCT), 12.6±0.6; pars recta (PR), 4.0±2.3; thin descending limb (TDL), 0.6±0.2; thin ascending limb (TAL), 0.6±0.2; medullary thick ascending limb (MAL), 0.8±0.1; cortical ascending limb (CAL), 2.1±0.1; distal convoluted tubule (DCT), 5.6±1.1; cortical collecting tubule (CCT), 3.2±0.3 and outer medullary collecting tubule (MCT), 2.3±0.1. Specific [125I] insulin binding to glomeruli and tubule segments was time- and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [125I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [125I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [125I] insulin binding sites may correspond to physiological insulin receptors.

Simultaneous Mg, Ca, P, K, Na and Cl analysis in rat tubular fluid

SummaryMg, Ca, P, Na, K and Cl concentrations in non-diuretic rats were measured by electron probe analysis in late proximal and early distal fluid micropuncture samples, as well as in ureteral urine and plasma ultrafiltrate samples. The glomerular indicator used was either3H-inulin or ferrocyanide. The results obtained with inulin show that only 20% of the filtered Mg were reabsorbed along the proximal tubule, whereas 64% were reabsorbed within the loop of Henle. They confirm the existence of a phosphorus reabsorption in the terminal part of the nephron. The tubular handling observed as regards the other ions generally agreed with what is known from the literature. Ferrocyanide, in the concentration used, acted as a non reabsorbable polyanion and reduced Ca reabsorption in the proximal tubule and Mg reabsorption in the loop of Henle; it enhanced K+ tubular excretion in the terminal part of the nephrons; its tubular effects on the other ions were proportionally less pronounced.

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.