0013-7227/78/0102-1254$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 4 Printed in U.S.A.
Vasopressin-Dependent Adenylate Cyclase Activities in the Rat Kidney Medulla: Evidence for Two Separate Sites of Action* MARTINE IMBERT-TEBOUL,t DANIELLE CHABARDfiS, MADELEINE MONTfiGUT, ANDRE CLIQUE, AND FRANCOIS MOREL Laboratoire de Physiologie Cellulaire, College de France, 75231 Paris, France ABSTRACT. This study demonstrates 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. (Endocrinology 102: 1254, 1978)
F
ROM studies performed on kidney homogenates and membrane fractions it is now clearly established that vasopressin greatly enhances cAMP production in renal medulla, whereas it produces only a small effect in the cortex which does respond to parathyroid hormone (1, 2). On the basis of physiological data available in 1968, Chase and Aurbach considered (1) that the "localization of parathyroid hormone-sensitive adenyl cyclase to renal cortex and vasopressinsensitive adenyl cyclase to renal medulla is consonant with reports that cellular transfer of phosphate (3) and calcium (4) occurs primarily in the proximal portions of the nephron where parathyroid hormone seems to act, and that sodium transport and water permeability are induced by vasopressin primarily in the collecting tubule (5)." It is, thus, now generally admitted that vasopressin-sensitive adenylate cyclase units present in membrane prepara-
tions from kidney medulla correspond to those located along the collecting tubule. Recent work from our laboratory led us, however, to reconsider this interpretation. By using a microtechnique to measure adenylate cyclase (AC) activities on rabbit single tubules (6), we could demonstrate the presence of vasopressin receptors not only in the collecting tubule, but also in the ascending limb of the loop of Henle (7, 8). Such an observation was of some interest, from a biochemical point of view, because it suggested a possible heterogeneity for particulate membrane preparations of rat kidney medulla with regard to vasopressin-sensitive adenylate cyclase. From a physiological point of view, it provided the first evidence for a direct effect of vasopressin on the loop of Henle. The purpose of this paper was to check these results in the rat, a species currently used both in biochemical and physiological studies, to establish whether or not they could Received June 27, 1977. * This work was supported by a grant from the Centre be considered as a general feature and, if conNational de la Recherche Scientifique (LRA 219) and a firmed, to discuss their possible physiological grant from the Institut National pour la Sante et la significance in the light of in vivo data availRecherche Medicale (ATP 44 76 76). able in the rat but not in the rabbit. f To whom requests for reprints should be addressed. 1254 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:59 For personal use only. No other uses without permission. . All rights reserved.
ADH TARGET SITES IN RAT KIDNEY MEDULLA
Materials and Methods Methods Experiments were performed on 19 adult male Wistar rats, 244 ± 40 g (SD), starved 14 h before the experiments but with free access to water. Animals received an iv injection of heparin (Liquemine, Roche, 50 IU) before being bled under pentobarbital anesthesia (Nembutal, 5 mg/100 g). The blood contained in the left kidney was flushed out by in situ perfusion of 5 ml cold "maceration" solution (vide infra) via the abdominal aorta (rate, 0.5 ml/min). The renal vein was then clamped and 5 additional ml were perfused at a higher rate (1.5 ml/min), to swell the kidney. The kidney was then quickly removed, decapsulated, and sliced along the corticomedullary axis. Pieces of tissue were incubated 30-40 min at 35 C in aerated "maceration" solution. They were then washed out in ice-cold "microdissection" solution and transferred into Petri dishes for microdissection. About 100-250 anatomically localized pieces of tubules were isolated in each experiment. Each tubular sample was photographed in order to measure its length and stored for 24 h at 0 C in microdissection medium before enzymatic assay. Such a procedure was adopted after verification that AC responses to vasopressin (10~6 M) achieved in the collecting tubule and the thick ascending limb were not significantly different (P > 0.5 in both cases, according to Student's t test) when measured on the same day as microdissection or after 24-h storage of the samples at 0 C, with the other experimental conditions remaining the same. AC activity contained in a single piece of tubule (0.5-1.0 mm in length) was measured by using a method derived from that described in (9) and (10). Pretreatment of the tubules and conditions of the assay were the same as those described previously (6, 11). Briefly, all samples were submitted to an osmotic shock and to a freezing step in order to ensure permeability of the tubular cells to nucleotides. Incubation was performed at 30 C in a final volume of 2.5 fd in the presence of [a-32P]ATP, unlabeled ATP (0.25-0.30 HIM), CAMP (1 HIM), Mg ++ (4 DIM), and an ATP-regenerating system. The reaction was stopped after 30 min. cAMP was separated from other 32P-labeled compounds by double filtration on Dowex and aluminium oxide columns, according to Salomon et al. (12). AC activities were expressed as femtomoles (10~15 mol) of cAMP formed per 30-min incubation period/mm tubular length. We previously determined in the rabbit that, under the conditions used,
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the amount of cAMP formed was proportional to the incubation time (up to 60 min) and to the length of tubule contained in the samples (6). Composition of the media The "microdissection" solution was a modified Hanks' medium as described previously (6). The "maceration" solution was of the same composition as the microdissection solution except that its calcium concentration was 2 mM and it contained bovine serum albumin (BSA) at a final concentration of 0.1% (wt/vol), collagenase at 0.07-0.1% (wt/vol) according to the activity of the batch, and hyaluronidase at 0.1% (wt/vol). Compounds Collagenase from Clostridium histolyticum was purchased from Worthington Biochemical Corporation; hyaluronidase (bovine testes), B grade, was from Calbiochem. Pure arginine-vasopressin (AVP) (13) and [ 1,6 - a - deaminocystathionine ] oxytocin ([deamino-6-carba]oxytocin) were kindly donated by Dr. T. Barth. Pure synthetic vasotocin was kindly donated by Dr. P. Cohen. Other compounds were the same as those specified by Bockaert et al. (10). Radiochemicals [a-32P]ATP (5-20 Ci/mmol) was purchased from New England Nuclear; [3H]cAMP (ammonium salt, 21 Ci/mmol) was obtained from the Commissariat a l'Energie Atomique (Saclay, France).
Results The presence of target sites of vasopressin was investigated in the medullary portion of thick ascending limbs and collecting tubules. Under the experimental conditions used, tubular segments located in the outer medulla could be easily identified and microdissected. Thick ascending limbs were characterized by their small diameter (about 22 /mi, as determined from photographs), their sharp outlines, and their junctions with thin ascending limbs at the transition between outer and inner medulla. Collecting tubules were characterized by their larger diameter (about 37 jtim), their occasional branchings, and their transparent aspect.
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IMBERT-TEBOUL ET AL.
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TABLE 1. Effect of AVP on AC activities in its medullary target sites in the rat nephron
Segment
cAMP formed (fmol/mm/30 min) mean ± SE Control
AVP (10- 6
M)
15.1 ± 1.8 465.3 ± 42.8 MAL MCT 680.6 ± 55.8 31.6 ± 4.4 AC activities (femtomole cAMP formed/30-min incubation time/mm tubular length) were measured in the absence (control) or presence of AVP in MAL and MCT. Mean values reported here were calculated from data obtained in 19 rats. In each rat, AC activities were calculated from three to five replicate samples.
MAL Basal
1) 2) 3)
AVP (10~6 M)
1) 421.8 2) 337.4 3) 353.6
14.9 11.7 9.6
MCT
2.8 (4) 3.0 (4) 2.5 (4)
37.1 36.0 30.7
96.5 (5) 49.2 (4) 104.7 (4)
745.7 834.3 756.3
-H -H -H
All data presented in this paper were obtained from AC activity measurements performed on MAL and MCT samples isolated from the same rats. Mean AC activities measured in the absence (control) and presence of AVP (10~6 M) are reported in Table 1. Results show that both segments contain an AVP-sensitive AC. The amount of cAMP formed under AVP stimulation (stimulated minus control) was lower in MAL than in MCT (P < 0.001). However, in relative terms, it corresponded to a greater AC stimulation (31 and 22 times in MAL and MCT, respectively). This observation was reproducible in spite of large variations observed from one experiment to another in basal activities (MAL, 6-27 fmol/mm/30 min; MCT, 12-40), as well as in maximal activation by AVP (see the range of absolute values in Table 3). Such variations, which were also observed with kidney homogenates (2,14), are unlikely to result only from individual differences between animals. As shown in Table 2, when AC activities were measured in the same experiment in three different rats, the range of variation for basal and AVP-stimulated values was much more limited for both MAL and MCT.
AC activity (fmol/mm/30 min) ± SD (n)
Condition
-H -H -H
Evidence for an AC sensitive to vasopressin in the thick ascending limb
TABLE 2. Variations observed from animal to animal in AC responses to AVP
•H -H -H
Samples of thick ascending limb (MAL) were isolated from the inner strip of outer medulla just after their junctions with thin ascending limbs. Medullary samples of collecting tubule (MCT) were isolated at the same level.
Endo • 1978 Vol 102 • No 4
3.1 (4) 9.6 (4) 8.7 (4)
± 220.3 (3) ± 167.7 (5) ± 125.2 (4)
The amount of cAMP formed in MAL and MCT was measured under basal and AVP-stimulated conditions in the same experiment in three different rats (numbered 1, 2, and 3). For all three rats, AC activities were measured under standard conditions.
The proportionality between cAMP production and the length of the segments used was analyzed in the experiment depicted by Fig. 1. MAL and MCT segments (from 0.3-3.5 mm/sample) were isolated from the same kidney and exposed to moderate concentrations of AVP (lO"10 and 10"9 M for MCT and MAL, respectively) in order to determine whether or not there was detectable hormone degradation during the time course of the 30-min incubation period. It is shown that the amount of cAMP formed increased proportionally to the length of the tubule in MAL and MCT, which suggests that under our experimental conditions none of these segments caused much degradation of the vasopressin (and ATP) present in the medium. Linearity also shows that it is valid to express AC activities/U length in the case of MAL and MCT samples. It was calculated from this experiment that 159 ± 34 and 205 ± 42 (SD) fmol cAMP were formed per 30-min incubation time, per millimeter tubular length in MCT (n = 22) and MAL (n = 24), respectively, SD values (±20% of the mean in both cases) give a rough estimate of the reproducibility of our micromethod for measuring AC activities. Such a value is in good agreement with that previously calculated from a large number of data (11). It should be noted that part of the scatter must be attributed here to functional heterogeneity and not to technical factors, because by using this technique, AC determinations are necessarily performed on separate tubular
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ADH TARGET SITES IN RAT KIDNEY MEDULLA
-800
MCT
MAL
-800
o
-400
0/ /
o
-400
•-' / O
2
3 0 Tubular length (mm)
1
segments originating from different nephrons and not on replicate samples from a homogeneous preparation.
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FIG. 1. AVP-stimulated cAMP production as a function of tubule length. The left-hand panel corresponds to MCT stimulated by AVP (1(T10 M); the right-hand panel corresponds to MAL stimulated by AVP (1(T9 M). The amount of cAMP formed/30-min incubation time was closely correlated to the length of the sample in both MAL (y = 198x + 14; n = 24; r = 0.95) and MCT (y = 186X - 54; n - 24; r = 0.93). AC activities were measured as described under Materials and Methods. Each point corresponds to a single AC activity measurement.
AC stimulation (per cent of the max.)
100-
Comparison of effects of AVP on the collecting tubule and the thick ascending limb Dose-response curves were performed between 10" n and 10~6 M AVP in six rats. One of these experiments is illustrated by Fig. 2. With respect to MCT, a significant (P < 0.001) 3-fold AC stimulation was observed in the presence of AVP at 10"n M, whereas maximal activation occurred at 10~8 M. Half-maximal stimulation corresponded to 2 X 10~10 M AVP. In MAL, AC activation was nearly maximal at 10"8 M, as in MCT, but the threshold AC response occurred only at 10"10 M (3-fold stimulation, P < 0.001) and the apparent Km was about 9 X 10"10 M, suggesting a lower sensitivity to AVP in MAL than in MCT. As shown in Table 3, similar results were obtained from the five other experiments. As for basal activities, large variations in maximal AC responses and in apparent Km were observed from experiment to experiment. It should be noted, however, that in all rats the apparent Km of AC activation by AVP was higher in MAL than in MCT. Fig. 3 compares the magnitude of responses induced by the same concentration of AVP in MAL and MCT samples isolated from the same rats, in nine different experiments. All individual experimental points fall above the line of identity, reflecting the fact that mean
-9 AVP
(log M)
-7
FIG. 2. AVP dose-response curves in MAL and MCT. MAL (O) and MCT (•) from the same rat were incubated as described under Materials and Methods, in the presence of increasing concentrations of AVP. Maximal AC activations achieved in each of these segments were taken as 100% activation (for absolute values see Table 3, exp 4). For each concentration tested, AC stimulations were plotted on the ordinate as a percentage of the maximal effect. Each point is the mean of three to five replicate AC activity measurements. Apparent Km values were 2 x 10"'° and 9 X 10"10 M for MCT and MAL, respectively.
AC responses achieved in MCT between 10"H and 10~7 M AVP (see inset) were significantly higher than those in MAL (for the level of significance, see legend to Fig. 3). The effect elicited by two structural ana-
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TABLE 3. Km of AC activation by AVP in MAL
and
MCT Exp no. Exp 1 MAL MCT Exp 2 MAL MCT Exp 3 MAL MCT
Maximal effect (fmol/mm/30 min)
Apparent Km (M)
143.3 161.4
3.0 x 10"9 1.0 x 10~9
249.6 365.9
1.7 x 10"8 2.8 x 10~9
462.9 577.1
9.0 X 10"9 1.4 X 10~9
323.8 602.9
9.3 X 10"10 1.8 X 10"10
406.9 651.5
1.0 X 10"9 2.0 X 10"10
842.6 1043.1
3.0 x 10"9 1.0 X 10"9
Exp 4
MAL
MCT Exp 5 MAL MCT Exp 6 MAL
MCT
Endo • 1978 Vol 102 • No 4
IMBERT-TEBOUL ET AL.
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along the collecting tubule and the thick ascending limb. Discussion This study demonstrates the presence of an AVP-sensitive AC in the thick ascending limb in addition to the collecting tubule of the rat kidney medulla. Its sensitivity to low AVP concentrations (10~10 M), as well as the magnitude of its maximal activation by the hormone (465 fmol cAMP formed/mm/30 min, i.e., a 31-fold stimulation factor), suggest that the thick ascending limb is actually a target structure involved in the physiological action of vasopressin in the kidney. Such a conclusion is in agreement with our previous study in the rabbit (7). It should be noted, however, that AVP responses
Paired AVP dose-response curves were determined for MAL and MCT. AVP concentrations between 10"" and 10~6 M were used. The magnitude of the maximal effect was expressed in femtomoles of cAMP formed/30-min incubation time/mm tubular length, in response to AVP (stimulated minus control). Apparent Km values, determined from the curves, correspond to the AVP concentrations inducing the half-maximal effect.
logues of AVP on AC contained in the thick ascending limb and the collecting tubule was also investigated and compared to that induced by AVP in the same experiments. [Deamino-6-carba]oxytocin and vasotocin ([Arg8]oxytocin) were chosen because of their low antidiuretic activity and their high natriuretic properties (15, 16, 17). [Deamino-6carba]oxytocin, which had a lower affinity than lysine-vasopressin for pig kidney plasma membranes (18), was tested between 10~8 and 10~5 M, whereas vasotocin was tested between 10~10 and 10"5 M. In MAL and MCT, AVP and its analogs were found to induce comparable AC stimulations when the vasotocin concentration used was about 10-fold that of AVP and the [deamino-6-carba]oxytocin concentration was 2000-fold that of AVP. When plotted as in Fig. 3, where the hormone concentrations used are not taken into account, the results show that the experimental points corresponding to these two analogs fall within the range of AVP data. On the basis of these experiments, there is, thus, no evidence for a different stereospecificity of receptors located
2 5 5 0 7 5 AC Stimulation in MAL (per cent of the max I
100
FIG. 3. Comparison of effects of AVP and analogues on MAL and MCT. Paired measurements of AC activities were performed, on MAL and MCT using AVP (•, from 10"u-10~6 M, nine rats), vasotocin (D, from 10"10-10"5 M, one rat) and [deamino-6-carba]oxytocin (O, from 10"8-10~5 M, one rat). In both segments, AC activations by AVP (stimulated minus control) were expressed as a percentage of the corresponding maximal response; AC activations by vasotocin and [deamino-6-carba]oxytocin were also expressed as a percentage of the maximal effect elicited by AVP in the same experiment. Each point corresponds to three to five replicate AC activity measurements in each segment. The line represents the line of identity (slope = 1). The inset gives the mean value (± SE) calculated from all rats at each AVP concentration tested (10~12-10~6 M); mean AC activations (% max) achieved in MCT were significantly higher than in MAL in the presence of AVP 10"11 M (P < 0.05, n - 6), 10"10 M (P < 0.01, n = 9), 10~9 M (P < 0.025, n = 9), 10"8 M (P < 0.025, n = 7), and 10"7 M (P < 0.025, n = 8).
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ADH TARGET SITES IN RAT KIDNEY MEDULLA achieved in the rat thick ascending limb are, both in absolute and relative values, higher and of a more constant magnitude than those { generaUy achieved in the rabbit (only 90 fmol cAMP formed/mm/30 min, which corres p o n d s to a mean 9-fold stimulation in the /presence of AVP 10~6 M, as described earlier Because the thick ascending limb and the collecting tubule have different diameters, comparison of AC activities/mm tubular length does not give accurate information on their respective ability to generate cAMP in response to vasopressin. From determinations of the outer diameters of these segments (MAL, 22 /xm; MCT, 37 /mi), it can be roughly calculated, however, that there is about 1.7 times more tubular surface/mm tubular length in the collecting tubule than in the thick ascending limb. As compared to the ratio for mean absolute AC responses/mm tubular length (MCT:MAL, 1.5), this value suggests that the amount of cAMP formed/U membrane surface must be rather comparable in the thick ascending limb and the collecting tubule. From our observations, there are about five thick ascending limbs/collecting tubule in the outer medulla of the rat; it can thus be calculated that thick ascending limbs would account for about 80% of the effect of vasopressin on a membrane preparation from outer medulla. This is certainly an overestimation in the case of a whole medulla homogenate, because thick limbs are absent from the inner medulla. Nevertheless, the bulk of plasma membranes present in a whole medulla particulate fraction is obviously provided by outer medulla, so that thick ascending limbs must, in any case, play a greater part than collecting tubules in the overall AC response of an homogenate to vasopressin. Such a conclusion is in agreement with the fact that the apparent Km of AC activation by AVP, measured on kidney medulla homogenates (from 2 X 10~9-2 X 10"8 M; (1, 2, 19-22) is very close to that measured on the thick ascending limb (from 10~9-2 X 10~8 M) and higher than that measured in the collecting tubule (2 X 10~10-3 X 10"9 M) in our experiments.
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The shift observed in MAL and MCT doseresponse curves (Fig. 2) is unlikely to be the result of a greater hormone degradation by MAL than by MCT samples. If such an inactivation process occurred, the data of Fig. 1 should indicate a drop in the response of the thick ascending limb to AVP with increasing amounts of tissue in the sample. On the contrary, cAMP production as a function of tubular length increased linearly in both segments, even in the presence of relatively low AVP concentrations in the medium. Neither can it be explained by the existence of molecular receptors of a different stereospecificity towards AVP along the thick ascending limb and the collecting tubule because, in our experiments, these two segments had apparently the same ability to discriminate between AVP and structural analogues such as vasotocin and [deamino-6-carba]oxytocin (Fig. 3). In addition, previous kinetic studies of [ 3 H]vasopressin binding to plasma membranes from kidney medulla did not provide any evidence for heterogeneity of the receptor population (22, 23). Consequently, a step beyond the hormone-receptor interaction must be involved to account for the difference in the shape of MAL and MCT dose-response curves; for example, the number of hormone-receptor sites per cyclase unit may differ in the two structures or a difference in coupling efficiency may exist in MCT and MAL. The data do not allow us to discriminate between these two hypotheses. At any rate, the difference observed in vitro for the threshold of AC activation suggests that the in vivo concentrations of circulating vasopressin necessary to induce the final biological response could be higher for the thick ascending limb than for the collecting tubule. If results obtained in the rabbit can be extrapolated to the rat, the fact that phosphodiesterase activity was reported to be higher in the thick ascending limb than in the collecting tubule (24) would tend to reinforce this conclusion. On the basis of physiological data, it is now well established that the thick ascending limb actively reabsorbs sodium chloride and has a low water permeability (25, 26); both of these properties account for the delivery of a hy-
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IMBERT-TEBOUL ET AL.
poosmotic fluid to the distal tubule in the presence as well as absence of vasopressin (27). This renders it most unlikely that the final effect of vasopressin on this segment is the regulation of its permeability to water, as is the case in the collecting tubule (see, for example, 28). It has been reported by Atherton et al. (29) that the in vivo perfusion of low ADH concentrations in rats undergoing water diuresis induces an anti-diuretic effect without much change in the medullary content of sodium, whereas the perfusion of higher hormone concentrations permits the sequestration of sodium in the interstitium. To interpret these data, these authors proposed the existence of two types of vasopressin target sites in kidney medulla: 1) high affinity sites, located along the collecting tubule and involved in the control of water permeability; and 2) low affinity sites responsible for the accumulation of sodium ions in interstitial tissue, either by a decrease of the blood flow entering the medulla (vasa recta hypothesis) or, as previously suggested by Morel (30), by an increase of sodium chloride reabsorption in the thick ascending limb of the loop. The presence in the rat thick ascending limb of an AC with a lower sensitivity to vasopressin than in the adjacent collecting tubule fits perfectly with this latter hypothesis (without excluding, of course, a possible concomitant action of the hormone via vasa recta). It should be noted, however, that an increased interstitial accumulation of sodium chloride in response to vasopressin, although confirmed by some authors (31-34), has been negated by others (35, 36) and thus remains to be further substantiated. Such an indirect observation is, in any case, insufficient to allow a definite conclusion as to the nature of vasopressin action on the thick ascending limb. Micropuncture experiments were performed by two different groups in order to answer this question, but, unfortunately, the results obtained in the rat (37) and the dog (38) were at variance and did not lead to a clearcut response. Thus, it is clear that more direct techniques have to be used to establish the nature of vasopressin action on the thick ascending limb
Endo • 1978 Vol 102 • No 4
of Henle's loop. The technique for in vitro microperfusion of rabbit isolated tubules developed by Burg et al. (39) is, a priori, adequate although limited by the fact that, in the rabbit, responses to vasopressin in the thick ascending limb are generally low. In this respect, the recent report that the microperfusion method can also be applied to the rat (40) appears quite promising. References 1. Chase, L. R., and G. D. Aurbach, Renal adenyl cyclase: anatomically separate sites for parathyroid hormone and vasopressin, Science 159: 545, 1968. 2. Rajerison, R., J. Marchetti, C. Roy, J. Bockaert, and S. Jard, The vasopressin sensitive adenylate cyclase of the rat kidney; effect of adrenalectomy and cortico steroids on hormonal receptor-enzyme coupling, J Biol Chem 249: 6390, 1974. 3. Pitts, R. F., R. S. Gurd, R. H. Kessler, and K. Hierholzer, Localization of acidification of urine, potassium and ammonia secretion and phosphate reabsorption in the nephron of the dog, Am J Physiol 194: 125, 1958. 4. Duarte, C. G., and J. F. Watson, Calcium reabsorption in proximal tubule of the dog nephron, Am J Physiol 212: 1355, 1967. 5. Berliner, R. W., and C. M. Bennett, Concentration of urine in the mammalian kidney, Am J Med 42: 777, 1967.
6. Imbert, M., D. Chabardes, M. Montegut, A. Clique, and F. Morel, Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments, Pfluegers Arch 354: 213, 1975. 7. Imbert, M., D. Chabardes, M. Montegut, A. Clique, and F. Morel, Vasopressin-dependent adenylate cyclase in single segments of rabbit kidney tubule, Pfluegers Arch 357: 173, 1975. 8. Imbert, M., D. Chabardes, M. Montegut, A. Clique, and F. Morel, Presence d'une adenyl cyclase stimulee par la vasopressine dans la branche ascendante des anses des nephrons du rein de lapin, C R Acad Sci (Paris) 280: 2129,1975. 9. Krishna, G. B., B. Weiss, and B. B. Brodie, A simple sensitive method for the assay of adenyl cyclase, J Pharmacol Exp Ther 163: 379, 1968. 10. Bockaert, J., C. Roy, and S. Jard, Oxytocin-sensitive adenylate cyclase in frog bladder epithelial cells. Role of calcium, nucleotides and other factors in hormonal stimulation, J Biol Chem 247: 7073,1972. 11. Morel, F., D. Chabardes, and M. Imbert-Teboul, Methodology for enzymatic studies of isolated tubular segments: adenylate cyclase, In Martinez-Maldonado, M. (ed.), Methods in Pharmacology, vol 4B, Renal Pharmacology, Plenum Press, New York, chap. 7, in press. 12. Salomon, Y., C. Londos, and M. Rodbell, A highly
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ADH TARGET SITES IN RAT KIDNEY MEDULLA
13.
14.
15.
16.
17. 18.
19.
20.
21. 22.
23.
24.
25.
26.
sensitive adenylate cyclase assay, Anal Biochem 58: 541, 1974. Pruzik, Z., E. Sedlakova, and T. Barth, Isolation of [Arg-8]vasopressin from the neurophysin complex by free-flow continuous electrophoresis, Hoppe Seylers Z Physiol Chem 353: 1837, 1972. Rajerison, R. M., D. Butlen, and S. Jard, Effects of in vivo treatment with vasopressin and analogues on renal adenylate cyclase responsiveness to vasopressin stimulation in vitro, Endocrinology 101: 1, 1977. Barth, T., I. Krejci, B. Kupkova, and K. Jost, Pharmacology of cyclic analogues of deamino-oxytocin not containing a disulphide bond (carba analogues), Eur J Pharmcol 24: 183, 1973. Sawyer, W. H., W. Y. Chan, and H. B. van Dyke, Antidiuretic responses to neurohypophysial hormones and some of their synthetic analogues in dogs and rats, Endocrinology 71: 536, 1962. Chan, W. Y., and W. H. Sawyer, Saluretic actions of neurohypophysial peptides in conscious dogs, Am J Physiol 201: 799, 1961. Roy, C, T. Barth, and S. Jard, Vasopressin-sensitive kidney adenylate cyclase. Structural requirements for attachment to the receptor and enzyme activation: studies with oxytocin analogues, J Biol Chem 250: 3157, 1975. Beck, N. P., T. Kaneko, U. Zor, J. B. Field, and B. B. Davis, Effects of vasopressin and prostaglandin Ei on the adenyl cyclase-cyclic 3'-5'-adenosine monophosphate system of the renal medulla of the rat, J Clin Invest 50: 2461, 1971. Dousa, T., 0. Hechter, I. L. Schwartz, and R. Walter, Neurohypophyseal hormone-responsive adenylate cyclase from mammalian kidney, Proc Natl Acad Sci 68: 1693, 1971. Neer, E. J., The vasopressin-sensitive adenylate cyclase of the rat renal medulla, J Biol Chem 248: 4775, 1973. Rajerison, R. M., D. Butlen, and S. Jard, Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation, Mol Cell Endocrinol 4: 271, 1976. Bockaert, J., C. Roy, R. Rajerison, and S. Jard, Specific binding of [3H]lysine-vasopressin to pig kidney plasma membranes: relationship of receptor occupancy to adenylate cyclase activation, J Biol Chem 248: 5922, 1973. Shanta, T. R., W. D. Woods, M. B. Waitzman, and G. H. Bourne, Histochemical method for localization of 3':5'-nucleotide phosphodiesterase, Histochemie 7: 177, 1966. Burg, M. B., and N. Green, Function of the thick ascending limb of Henle's loop, Am J Physiol 224: 659, 1973. Rocha, A. S., and J. P. Kokko, Sodium chloride and
27. 28. 29.
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water transport in the medullary thick ascending limb of Henle: evidence for active chloride transport, J Clin Invest 52: 612, 1973. Wirz, H., Der osmotische Druck in den corticalen Tubuli der Ratten Niere, Helv Physiol Pharmacol Ada 14: 353, 1956. Grantham, J. J., and M. B. Burg, Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules, Am J Physiol 211: 255, 1966. Atherton, J. C, R. Green, and S. Thomas, Influence of lysine-vasopressin dosage on the time course of changes in renal tissue and urinary composition in the conscious rat, J Physiol (Lond) 213: 291, 1971. Morel, F., Action of neurohypophyseal hormones on the active transport of sodium, In de Graeff, J., and B. Leijnse (eds.), Water and Electrolyte Metabolism, vol. 2, Elsevier Publishing Co., Amsterdam, 1964, p. 91. Levitin, H., A. Goodman, G. Pigeon, and F. H. Epstein, Composition of the renal medulla during water diuresis, J Clin Invest 41: 1145, 1962. Perlmutt, J. H., Influence of hydration on renal function and medullary sodium during vasopressin infusion, Am J Physiol 202: 1098, 1962. Ruiz-Guinazii, A., E. E. Arrizurieta, and L. Yelinek, Electrolyte, water and urea content in dog kidneys in different states of diuresis, Am J Physiol 206: 725, 1964. Gardner, K. D., and J. M. Vierling, Solids, water and solutes in papillary region of the rat kidney, Am J Physiol 217: 58, 1969. Jaenike, J. R., Acute effects of the administration of vasopressin during water diuresis in the dog, J Clin Invest 42: 161,1963. Valtin, H., Sequestration of urea and non urea solutes in renal tissues of rats with hereditary hypothalamic diabetes insipidus: effect of vasopressin and dehydration on the countercurrent mechanism, J Clin Invest 45: 337, 1966. Schnermann, J., H. Valtin, K. Thurau, W. Nagel, M. Horster, H. Fischbach, M. Wahl, and G. Liebau, Micropuncture studies of the influence of antidiuretic hormone on tubular fluid reabsorption in rats with hereditary hypothalamic diabetes insipidus, Pfluegers Arch 306: 103, 1969. Antoniou, L. D., T. J. Burke, R. R. Robinson, and R. J. Clapp, Vasopressin related alterations of sodium reabsorption in the loop of Henle, Kidney Int 3: 6, 1973. Burg, M., J. Grantham, M. Abramow, and J. Orloff, Preparation and study of fragments of single rabbit nephrons, Am J Physiol 210: 1293, 1966. Imai, M., Effect of bumetanide and furosemide on the thick ascending limb of Henle's loop of rabbits and rats perfused in vitro, Eur J Pharmacol 41: 409, 1977.
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Vol. 102, No. 4 Printed in U.S.A.
Vasopressin-Dependent Adenylate Cyclase Activities in the Rat Kidney Medulla: Evidence for Two Separate Sites of Action* MARTINE IMBERT-TEBOUL,t DANIELLE CHABARDfiS, MADELEINE MONTfiGUT, ANDRE CLIQUE, AND FRANCOIS MOREL Laboratoire de Physiologie Cellulaire, College de France, 75231 Paris, France ABSTRACT. This study demonstrates 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. (Endocrinology 102: 1254, 1978)
F
ROM studies performed on kidney homogenates and membrane fractions it is now clearly established that vasopressin greatly enhances cAMP production in renal medulla, whereas it produces only a small effect in the cortex which does respond to parathyroid hormone (1, 2). On the basis of physiological data available in 1968, Chase and Aurbach considered (1) that the "localization of parathyroid hormone-sensitive adenyl cyclase to renal cortex and vasopressinsensitive adenyl cyclase to renal medulla is consonant with reports that cellular transfer of phosphate (3) and calcium (4) occurs primarily in the proximal portions of the nephron where parathyroid hormone seems to act, and that sodium transport and water permeability are induced by vasopressin primarily in the collecting tubule (5)." It is, thus, now generally admitted that vasopressin-sensitive adenylate cyclase units present in membrane prepara-
tions from kidney medulla correspond to those located along the collecting tubule. Recent work from our laboratory led us, however, to reconsider this interpretation. By using a microtechnique to measure adenylate cyclase (AC) activities on rabbit single tubules (6), we could demonstrate the presence of vasopressin receptors not only in the collecting tubule, but also in the ascending limb of the loop of Henle (7, 8). Such an observation was of some interest, from a biochemical point of view, because it suggested a possible heterogeneity for particulate membrane preparations of rat kidney medulla with regard to vasopressin-sensitive adenylate cyclase. From a physiological point of view, it provided the first evidence for a direct effect of vasopressin on the loop of Henle. The purpose of this paper was to check these results in the rat, a species currently used both in biochemical and physiological studies, to establish whether or not they could Received June 27, 1977. * This work was supported by a grant from the Centre be considered as a general feature and, if conNational de la Recherche Scientifique (LRA 219) and a firmed, to discuss their possible physiological grant from the Institut National pour la Sante et la significance in the light of in vivo data availRecherche Medicale (ATP 44 76 76). able in the rat but not in the rabbit. f To whom requests for reprints should be addressed. 1254 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:59 For personal use only. No other uses without permission. . All rights reserved.
ADH TARGET SITES IN RAT KIDNEY MEDULLA
Materials and Methods Methods Experiments were performed on 19 adult male Wistar rats, 244 ± 40 g (SD), starved 14 h before the experiments but with free access to water. Animals received an iv injection of heparin (Liquemine, Roche, 50 IU) before being bled under pentobarbital anesthesia (Nembutal, 5 mg/100 g). The blood contained in the left kidney was flushed out by in situ perfusion of 5 ml cold "maceration" solution (vide infra) via the abdominal aorta (rate, 0.5 ml/min). The renal vein was then clamped and 5 additional ml were perfused at a higher rate (1.5 ml/min), to swell the kidney. The kidney was then quickly removed, decapsulated, and sliced along the corticomedullary axis. Pieces of tissue were incubated 30-40 min at 35 C in aerated "maceration" solution. They were then washed out in ice-cold "microdissection" solution and transferred into Petri dishes for microdissection. About 100-250 anatomically localized pieces of tubules were isolated in each experiment. Each tubular sample was photographed in order to measure its length and stored for 24 h at 0 C in microdissection medium before enzymatic assay. Such a procedure was adopted after verification that AC responses to vasopressin (10~6 M) achieved in the collecting tubule and the thick ascending limb were not significantly different (P > 0.5 in both cases, according to Student's t test) when measured on the same day as microdissection or after 24-h storage of the samples at 0 C, with the other experimental conditions remaining the same. AC activity contained in a single piece of tubule (0.5-1.0 mm in length) was measured by using a method derived from that described in (9) and (10). Pretreatment of the tubules and conditions of the assay were the same as those described previously (6, 11). Briefly, all samples were submitted to an osmotic shock and to a freezing step in order to ensure permeability of the tubular cells to nucleotides. Incubation was performed at 30 C in a final volume of 2.5 fd in the presence of [a-32P]ATP, unlabeled ATP (0.25-0.30 HIM), CAMP (1 HIM), Mg ++ (4 DIM), and an ATP-regenerating system. The reaction was stopped after 30 min. cAMP was separated from other 32P-labeled compounds by double filtration on Dowex and aluminium oxide columns, according to Salomon et al. (12). AC activities were expressed as femtomoles (10~15 mol) of cAMP formed per 30-min incubation period/mm tubular length. We previously determined in the rabbit that, under the conditions used,
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the amount of cAMP formed was proportional to the incubation time (up to 60 min) and to the length of tubule contained in the samples (6). Composition of the media The "microdissection" solution was a modified Hanks' medium as described previously (6). The "maceration" solution was of the same composition as the microdissection solution except that its calcium concentration was 2 mM and it contained bovine serum albumin (BSA) at a final concentration of 0.1% (wt/vol), collagenase at 0.07-0.1% (wt/vol) according to the activity of the batch, and hyaluronidase at 0.1% (wt/vol). Compounds Collagenase from Clostridium histolyticum was purchased from Worthington Biochemical Corporation; hyaluronidase (bovine testes), B grade, was from Calbiochem. Pure arginine-vasopressin (AVP) (13) and [ 1,6 - a - deaminocystathionine ] oxytocin ([deamino-6-carba]oxytocin) were kindly donated by Dr. T. Barth. Pure synthetic vasotocin was kindly donated by Dr. P. Cohen. Other compounds were the same as those specified by Bockaert et al. (10). Radiochemicals [a-32P]ATP (5-20 Ci/mmol) was purchased from New England Nuclear; [3H]cAMP (ammonium salt, 21 Ci/mmol) was obtained from the Commissariat a l'Energie Atomique (Saclay, France).
Results The presence of target sites of vasopressin was investigated in the medullary portion of thick ascending limbs and collecting tubules. Under the experimental conditions used, tubular segments located in the outer medulla could be easily identified and microdissected. Thick ascending limbs were characterized by their small diameter (about 22 /mi, as determined from photographs), their sharp outlines, and their junctions with thin ascending limbs at the transition between outer and inner medulla. Collecting tubules were characterized by their larger diameter (about 37 jtim), their occasional branchings, and their transparent aspect.
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IMBERT-TEBOUL ET AL.
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TABLE 1. Effect of AVP on AC activities in its medullary target sites in the rat nephron
Segment
cAMP formed (fmol/mm/30 min) mean ± SE Control
AVP (10- 6
M)
15.1 ± 1.8 465.3 ± 42.8 MAL MCT 680.6 ± 55.8 31.6 ± 4.4 AC activities (femtomole cAMP formed/30-min incubation time/mm tubular length) were measured in the absence (control) or presence of AVP in MAL and MCT. Mean values reported here were calculated from data obtained in 19 rats. In each rat, AC activities were calculated from three to five replicate samples.
MAL Basal
1) 2) 3)
AVP (10~6 M)
1) 421.8 2) 337.4 3) 353.6
14.9 11.7 9.6
MCT
2.8 (4) 3.0 (4) 2.5 (4)
37.1 36.0 30.7
96.5 (5) 49.2 (4) 104.7 (4)
745.7 834.3 756.3
-H -H -H
All data presented in this paper were obtained from AC activity measurements performed on MAL and MCT samples isolated from the same rats. Mean AC activities measured in the absence (control) and presence of AVP (10~6 M) are reported in Table 1. Results show that both segments contain an AVP-sensitive AC. The amount of cAMP formed under AVP stimulation (stimulated minus control) was lower in MAL than in MCT (P < 0.001). However, in relative terms, it corresponded to a greater AC stimulation (31 and 22 times in MAL and MCT, respectively). This observation was reproducible in spite of large variations observed from one experiment to another in basal activities (MAL, 6-27 fmol/mm/30 min; MCT, 12-40), as well as in maximal activation by AVP (see the range of absolute values in Table 3). Such variations, which were also observed with kidney homogenates (2,14), are unlikely to result only from individual differences between animals. As shown in Table 2, when AC activities were measured in the same experiment in three different rats, the range of variation for basal and AVP-stimulated values was much more limited for both MAL and MCT.
AC activity (fmol/mm/30 min) ± SD (n)
Condition
-H -H -H
Evidence for an AC sensitive to vasopressin in the thick ascending limb
TABLE 2. Variations observed from animal to animal in AC responses to AVP
•H -H -H
Samples of thick ascending limb (MAL) were isolated from the inner strip of outer medulla just after their junctions with thin ascending limbs. Medullary samples of collecting tubule (MCT) were isolated at the same level.
Endo • 1978 Vol 102 • No 4
3.1 (4) 9.6 (4) 8.7 (4)
± 220.3 (3) ± 167.7 (5) ± 125.2 (4)
The amount of cAMP formed in MAL and MCT was measured under basal and AVP-stimulated conditions in the same experiment in three different rats (numbered 1, 2, and 3). For all three rats, AC activities were measured under standard conditions.
The proportionality between cAMP production and the length of the segments used was analyzed in the experiment depicted by Fig. 1. MAL and MCT segments (from 0.3-3.5 mm/sample) were isolated from the same kidney and exposed to moderate concentrations of AVP (lO"10 and 10"9 M for MCT and MAL, respectively) in order to determine whether or not there was detectable hormone degradation during the time course of the 30-min incubation period. It is shown that the amount of cAMP formed increased proportionally to the length of the tubule in MAL and MCT, which suggests that under our experimental conditions none of these segments caused much degradation of the vasopressin (and ATP) present in the medium. Linearity also shows that it is valid to express AC activities/U length in the case of MAL and MCT samples. It was calculated from this experiment that 159 ± 34 and 205 ± 42 (SD) fmol cAMP were formed per 30-min incubation time, per millimeter tubular length in MCT (n = 22) and MAL (n = 24), respectively, SD values (±20% of the mean in both cases) give a rough estimate of the reproducibility of our micromethod for measuring AC activities. Such a value is in good agreement with that previously calculated from a large number of data (11). It should be noted that part of the scatter must be attributed here to functional heterogeneity and not to technical factors, because by using this technique, AC determinations are necessarily performed on separate tubular
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ADH TARGET SITES IN RAT KIDNEY MEDULLA
-800
MCT
MAL
-800
o
-400
0/ /
o
-400
•-' / O
2
3 0 Tubular length (mm)
1
segments originating from different nephrons and not on replicate samples from a homogeneous preparation.
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FIG. 1. AVP-stimulated cAMP production as a function of tubule length. The left-hand panel corresponds to MCT stimulated by AVP (1(T10 M); the right-hand panel corresponds to MAL stimulated by AVP (1(T9 M). The amount of cAMP formed/30-min incubation time was closely correlated to the length of the sample in both MAL (y = 198x + 14; n = 24; r = 0.95) and MCT (y = 186X - 54; n - 24; r = 0.93). AC activities were measured as described under Materials and Methods. Each point corresponds to a single AC activity measurement.
AC stimulation (per cent of the max.)
100-
Comparison of effects of AVP on the collecting tubule and the thick ascending limb Dose-response curves were performed between 10" n and 10~6 M AVP in six rats. One of these experiments is illustrated by Fig. 2. With respect to MCT, a significant (P < 0.001) 3-fold AC stimulation was observed in the presence of AVP at 10"n M, whereas maximal activation occurred at 10~8 M. Half-maximal stimulation corresponded to 2 X 10~10 M AVP. In MAL, AC activation was nearly maximal at 10"8 M, as in MCT, but the threshold AC response occurred only at 10"10 M (3-fold stimulation, P < 0.001) and the apparent Km was about 9 X 10"10 M, suggesting a lower sensitivity to AVP in MAL than in MCT. As shown in Table 3, similar results were obtained from the five other experiments. As for basal activities, large variations in maximal AC responses and in apparent Km were observed from experiment to experiment. It should be noted, however, that in all rats the apparent Km of AC activation by AVP was higher in MAL than in MCT. Fig. 3 compares the magnitude of responses induced by the same concentration of AVP in MAL and MCT samples isolated from the same rats, in nine different experiments. All individual experimental points fall above the line of identity, reflecting the fact that mean
-9 AVP
(log M)
-7
FIG. 2. AVP dose-response curves in MAL and MCT. MAL (O) and MCT (•) from the same rat were incubated as described under Materials and Methods, in the presence of increasing concentrations of AVP. Maximal AC activations achieved in each of these segments were taken as 100% activation (for absolute values see Table 3, exp 4). For each concentration tested, AC stimulations were plotted on the ordinate as a percentage of the maximal effect. Each point is the mean of three to five replicate AC activity measurements. Apparent Km values were 2 x 10"'° and 9 X 10"10 M for MCT and MAL, respectively.
AC responses achieved in MCT between 10"H and 10~7 M AVP (see inset) were significantly higher than those in MAL (for the level of significance, see legend to Fig. 3). The effect elicited by two structural ana-
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TABLE 3. Km of AC activation by AVP in MAL
and
MCT Exp no. Exp 1 MAL MCT Exp 2 MAL MCT Exp 3 MAL MCT
Maximal effect (fmol/mm/30 min)
Apparent Km (M)
143.3 161.4
3.0 x 10"9 1.0 x 10~9
249.6 365.9
1.7 x 10"8 2.8 x 10~9
462.9 577.1
9.0 X 10"9 1.4 X 10~9
323.8 602.9
9.3 X 10"10 1.8 X 10"10
406.9 651.5
1.0 X 10"9 2.0 X 10"10
842.6 1043.1
3.0 x 10"9 1.0 X 10"9
Exp 4
MAL
MCT Exp 5 MAL MCT Exp 6 MAL
MCT
Endo • 1978 Vol 102 • No 4
IMBERT-TEBOUL ET AL.
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along the collecting tubule and the thick ascending limb. Discussion This study demonstrates the presence of an AVP-sensitive AC in the thick ascending limb in addition to the collecting tubule of the rat kidney medulla. Its sensitivity to low AVP concentrations (10~10 M), as well as the magnitude of its maximal activation by the hormone (465 fmol cAMP formed/mm/30 min, i.e., a 31-fold stimulation factor), suggest that the thick ascending limb is actually a target structure involved in the physiological action of vasopressin in the kidney. Such a conclusion is in agreement with our previous study in the rabbit (7). It should be noted, however, that AVP responses
Paired AVP dose-response curves were determined for MAL and MCT. AVP concentrations between 10"" and 10~6 M were used. The magnitude of the maximal effect was expressed in femtomoles of cAMP formed/30-min incubation time/mm tubular length, in response to AVP (stimulated minus control). Apparent Km values, determined from the curves, correspond to the AVP concentrations inducing the half-maximal effect.
logues of AVP on AC contained in the thick ascending limb and the collecting tubule was also investigated and compared to that induced by AVP in the same experiments. [Deamino-6-carba]oxytocin and vasotocin ([Arg8]oxytocin) were chosen because of their low antidiuretic activity and their high natriuretic properties (15, 16, 17). [Deamino-6carba]oxytocin, which had a lower affinity than lysine-vasopressin for pig kidney plasma membranes (18), was tested between 10~8 and 10~5 M, whereas vasotocin was tested between 10~10 and 10"5 M. In MAL and MCT, AVP and its analogs were found to induce comparable AC stimulations when the vasotocin concentration used was about 10-fold that of AVP and the [deamino-6-carba]oxytocin concentration was 2000-fold that of AVP. When plotted as in Fig. 3, where the hormone concentrations used are not taken into account, the results show that the experimental points corresponding to these two analogs fall within the range of AVP data. On the basis of these experiments, there is, thus, no evidence for a different stereospecificity of receptors located
2 5 5 0 7 5 AC Stimulation in MAL (per cent of the max I
100
FIG. 3. Comparison of effects of AVP and analogues on MAL and MCT. Paired measurements of AC activities were performed, on MAL and MCT using AVP (•, from 10"u-10~6 M, nine rats), vasotocin (D, from 10"10-10"5 M, one rat) and [deamino-6-carba]oxytocin (O, from 10"8-10~5 M, one rat). In both segments, AC activations by AVP (stimulated minus control) were expressed as a percentage of the corresponding maximal response; AC activations by vasotocin and [deamino-6-carba]oxytocin were also expressed as a percentage of the maximal effect elicited by AVP in the same experiment. Each point corresponds to three to five replicate AC activity measurements in each segment. The line represents the line of identity (slope = 1). The inset gives the mean value (± SE) calculated from all rats at each AVP concentration tested (10~12-10~6 M); mean AC activations (% max) achieved in MCT were significantly higher than in MAL in the presence of AVP 10"11 M (P < 0.05, n - 6), 10"10 M (P < 0.01, n = 9), 10~9 M (P < 0.025, n = 9), 10"8 M (P < 0.025, n = 7), and 10"7 M (P < 0.025, n = 8).
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ADH TARGET SITES IN RAT KIDNEY MEDULLA achieved in the rat thick ascending limb are, both in absolute and relative values, higher and of a more constant magnitude than those { generaUy achieved in the rabbit (only 90 fmol cAMP formed/mm/30 min, which corres p o n d s to a mean 9-fold stimulation in the /presence of AVP 10~6 M, as described earlier Because the thick ascending limb and the collecting tubule have different diameters, comparison of AC activities/mm tubular length does not give accurate information on their respective ability to generate cAMP in response to vasopressin. From determinations of the outer diameters of these segments (MAL, 22 /xm; MCT, 37 /mi), it can be roughly calculated, however, that there is about 1.7 times more tubular surface/mm tubular length in the collecting tubule than in the thick ascending limb. As compared to the ratio for mean absolute AC responses/mm tubular length (MCT:MAL, 1.5), this value suggests that the amount of cAMP formed/U membrane surface must be rather comparable in the thick ascending limb and the collecting tubule. From our observations, there are about five thick ascending limbs/collecting tubule in the outer medulla of the rat; it can thus be calculated that thick ascending limbs would account for about 80% of the effect of vasopressin on a membrane preparation from outer medulla. This is certainly an overestimation in the case of a whole medulla homogenate, because thick limbs are absent from the inner medulla. Nevertheless, the bulk of plasma membranes present in a whole medulla particulate fraction is obviously provided by outer medulla, so that thick ascending limbs must, in any case, play a greater part than collecting tubules in the overall AC response of an homogenate to vasopressin. Such a conclusion is in agreement with the fact that the apparent Km of AC activation by AVP, measured on kidney medulla homogenates (from 2 X 10~9-2 X 10"8 M; (1, 2, 19-22) is very close to that measured on the thick ascending limb (from 10~9-2 X 10~8 M) and higher than that measured in the collecting tubule (2 X 10~10-3 X 10"9 M) in our experiments.
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The shift observed in MAL and MCT doseresponse curves (Fig. 2) is unlikely to be the result of a greater hormone degradation by MAL than by MCT samples. If such an inactivation process occurred, the data of Fig. 1 should indicate a drop in the response of the thick ascending limb to AVP with increasing amounts of tissue in the sample. On the contrary, cAMP production as a function of tubular length increased linearly in both segments, even in the presence of relatively low AVP concentrations in the medium. Neither can it be explained by the existence of molecular receptors of a different stereospecificity towards AVP along the thick ascending limb and the collecting tubule because, in our experiments, these two segments had apparently the same ability to discriminate between AVP and structural analogues such as vasotocin and [deamino-6-carba]oxytocin (Fig. 3). In addition, previous kinetic studies of [ 3 H]vasopressin binding to plasma membranes from kidney medulla did not provide any evidence for heterogeneity of the receptor population (22, 23). Consequently, a step beyond the hormone-receptor interaction must be involved to account for the difference in the shape of MAL and MCT dose-response curves; for example, the number of hormone-receptor sites per cyclase unit may differ in the two structures or a difference in coupling efficiency may exist in MCT and MAL. The data do not allow us to discriminate between these two hypotheses. At any rate, the difference observed in vitro for the threshold of AC activation suggests that the in vivo concentrations of circulating vasopressin necessary to induce the final biological response could be higher for the thick ascending limb than for the collecting tubule. If results obtained in the rabbit can be extrapolated to the rat, the fact that phosphodiesterase activity was reported to be higher in the thick ascending limb than in the collecting tubule (24) would tend to reinforce this conclusion. On the basis of physiological data, it is now well established that the thick ascending limb actively reabsorbs sodium chloride and has a low water permeability (25, 26); both of these properties account for the delivery of a hy-
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poosmotic fluid to the distal tubule in the presence as well as absence of vasopressin (27). This renders it most unlikely that the final effect of vasopressin on this segment is the regulation of its permeability to water, as is the case in the collecting tubule (see, for example, 28). It has been reported by Atherton et al. (29) that the in vivo perfusion of low ADH concentrations in rats undergoing water diuresis induces an anti-diuretic effect without much change in the medullary content of sodium, whereas the perfusion of higher hormone concentrations permits the sequestration of sodium in the interstitium. To interpret these data, these authors proposed the existence of two types of vasopressin target sites in kidney medulla: 1) high affinity sites, located along the collecting tubule and involved in the control of water permeability; and 2) low affinity sites responsible for the accumulation of sodium ions in interstitial tissue, either by a decrease of the blood flow entering the medulla (vasa recta hypothesis) or, as previously suggested by Morel (30), by an increase of sodium chloride reabsorption in the thick ascending limb of the loop. The presence in the rat thick ascending limb of an AC with a lower sensitivity to vasopressin than in the adjacent collecting tubule fits perfectly with this latter hypothesis (without excluding, of course, a possible concomitant action of the hormone via vasa recta). It should be noted, however, that an increased interstitial accumulation of sodium chloride in response to vasopressin, although confirmed by some authors (31-34), has been negated by others (35, 36) and thus remains to be further substantiated. Such an indirect observation is, in any case, insufficient to allow a definite conclusion as to the nature of vasopressin action on the thick ascending limb. Micropuncture experiments were performed by two different groups in order to answer this question, but, unfortunately, the results obtained in the rat (37) and the dog (38) were at variance and did not lead to a clearcut response. Thus, it is clear that more direct techniques have to be used to establish the nature of vasopressin action on the thick ascending limb
Endo • 1978 Vol 102 • No 4
of Henle's loop. The technique for in vitro microperfusion of rabbit isolated tubules developed by Burg et al. (39) is, a priori, adequate although limited by the fact that, in the rabbit, responses to vasopressin in the thick ascending limb are generally low. In this respect, the recent report that the microperfusion method can also be applied to the rat (40) appears quite promising. References 1. Chase, L. R., and G. D. Aurbach, Renal adenyl cyclase: anatomically separate sites for parathyroid hormone and vasopressin, Science 159: 545, 1968. 2. Rajerison, R., J. Marchetti, C. Roy, J. Bockaert, and S. Jard, The vasopressin sensitive adenylate cyclase of the rat kidney; effect of adrenalectomy and cortico steroids on hormonal receptor-enzyme coupling, J Biol Chem 249: 6390, 1974. 3. Pitts, R. F., R. S. Gurd, R. H. Kessler, and K. Hierholzer, Localization of acidification of urine, potassium and ammonia secretion and phosphate reabsorption in the nephron of the dog, Am J Physiol 194: 125, 1958. 4. Duarte, C. G., and J. F. Watson, Calcium reabsorption in proximal tubule of the dog nephron, Am J Physiol 212: 1355, 1967. 5. Berliner, R. W., and C. M. Bennett, Concentration of urine in the mammalian kidney, Am J Med 42: 777, 1967.
6. Imbert, M., D. Chabardes, M. Montegut, A. Clique, and F. Morel, Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments, Pfluegers Arch 354: 213, 1975. 7. Imbert, M., D. Chabardes, M. Montegut, A. Clique, and F. Morel, Vasopressin-dependent adenylate cyclase in single segments of rabbit kidney tubule, Pfluegers Arch 357: 173, 1975. 8. Imbert, M., D. Chabardes, M. Montegut, A. Clique, and F. Morel, Presence d'une adenyl cyclase stimulee par la vasopressine dans la branche ascendante des anses des nephrons du rein de lapin, C R Acad Sci (Paris) 280: 2129,1975. 9. Krishna, G. B., B. Weiss, and B. B. Brodie, A simple sensitive method for the assay of adenyl cyclase, J Pharmacol Exp Ther 163: 379, 1968. 10. Bockaert, J., C. Roy, and S. Jard, Oxytocin-sensitive adenylate cyclase in frog bladder epithelial cells. Role of calcium, nucleotides and other factors in hormonal stimulation, J Biol Chem 247: 7073,1972. 11. Morel, F., D. Chabardes, and M. Imbert-Teboul, Methodology for enzymatic studies of isolated tubular segments: adenylate cyclase, In Martinez-Maldonado, M. (ed.), Methods in Pharmacology, vol 4B, Renal Pharmacology, Plenum Press, New York, chap. 7, in press. 12. Salomon, Y., C. Londos, and M. Rodbell, A highly
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ADH TARGET SITES IN RAT KIDNEY MEDULLA
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