M. Montegut

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.

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.

The isolated frog skin epithelium: Presence of α and β adrenergic receptors regulating active sodium transport and water permeability

SummaryThe effects of stimulation of α and β adrenergic receptors on short circuit current (S.C.C.), Na+ and Cl− fluxes and osmotic water permeability were studied on isolated frog skin epithelial layers separated from the dermis.Low norepinephrine doses (final concentrations in the incubation medium ranging from 5×10−9 to 10−8 M) produced increased water permeability and S.C.C. The latter was entirely accounted for by an increase in the active Na+ influx. Na+ outflux and Cl− fluxes were not modified. Both these effects disappeared after treatment with the β blocking agent, Propranolol. Higher norepinephrine doses (final concentrations: 10−7 to 10−6 M) produced: 1. an increase in water permeability lower than that produced by low doses, the highest doses failing to increase water permeability, and 2. a triphasic change in S.C.C.: after an initial increase, S.C.C. dropped to its resting value and then rose again to a sustained value. Na+ and Cl− flux measurements showed that the variation in S.C.C. reflected variations in active Na+ transport. When the same high norepinephrine doses were applied after treatment with the α blocking agent Phentolamine, the effects observed were identical to those obtained with low doses.On β blocked preparations, large doses of norepinephrine inhibited the water permeability and sodium transport increases induced by theophylline or oxytocin but did not modify those induced by 3′5′-cyclic AMP. The inhibition was suppressed after blocking α receptors.From the foregoing, it was concluded that both α and β adrenergic receptors are present in frog skin epithelial cells and are involved in the regulation of water and sodium permeability.It is suggested that the inhibitory effect of α stimulation resulted from the inhibition of cyclic-AMP generating system, the activity of which is under the positive control effect of oxytocin and β stimulation.

The isolated frog skin epithelium: Permeability characteristics and responsiveness to oxytocin, cyclic AMP and theophylline

SummaryThe combined treatment of the frog skin with collagenase and hydrostatic pressure enables complete separation of the epithelial layer from the supporting dermis. The separation entirely preserves the epitheliums passive permeability and active sodium transport capacity. The short circuit current, d.c. resistance, unidirectional fluxes of Na+ and Cl− ions, and osmotic water permeability were found identical on series of isolated epitheliums and intact skins obtained from the same groups of animals. Furthermore, the isolated epithelium is fully responsive to oxytocin, cyclic AMP and theophylline—three agents known to enhance active sodium transport and water permeability of the intact skin.

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.

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 microdissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC resposes 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 glucagonsensitive 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.

Impaired Response to Vasopressin of Adenylate Cyclase of the Thick Ascending Limb of Henle’s Loop in Brattleboro Rats with Diabetes insipidus

Brattleboro rats with diabetes insipidus (DI) exhibit an impaired urinary concentrating ability in response to exogenous vasopressin (AVP) and a decreased AVP-sensitive adenylate cyclase (AC) in homog