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Home , Nephron, Vasopressin

Kidney International, Vol. 31(1987), pp. 512—520 MOLECULAR MECHANISM OF ACTION OF

Receptors to and other in the mammalian

Flt&Ncols MOREL, MARTINE IMBERT—TEBOUL, and DANIELLE CHABARDES

Laboratoire de Physiologie cellulaire, College de France, 11, Pce. Marcelin Berthelot, 75231 Paris Cedex 05, France

In the course of the last decade, the systematic analysis ofhal cell types of the such as those in the thin ascending successive sites along the nephron where various hormones,limb (TAL), the medullary (MAL) and cortical (CAL) portions including vasopressin, may regulate cell function by stimulatingof the thick ascending limb, and the early distal convoluted the intracellular production of cAMP revealed an unexpectedtubule (eDCT). As long as these responsive segments corre- panorama, and thereby raised a series of new questions. Thisspond to physiological target sites for AVP, the biological contribution will discuss some of the observations we maderesponse induced by the hormone is expected to be different in when investigating this general field, those which, in our minds,each segment, since the response depends on the transport were the most directly related to some aspect of the mechanismproperties specific for the cell type itself, as pointed out before of concentration and dilution by the kidney. We focus[3, 4]. In order to assess the physiological relevance of the mainly on problems raised by the sites and conditions in whichresults given in Table 1, dose—response curves were established vasopressin was observed to induce in vitro effects in differentin several responsive segments of the rat kidney, that is, TAL species. Consequently, we have attempted neither to cover the[5], MAL [3], CAL (Fig. 1), CCT (Fig. 1) and MCT [3]. Half field of hormone receptors in the kidney, nor to quote exten-maximal responses corresponded to AVP concentrations gen- sively the literature. We are fully aware that, among theerally ranging between 5x10'° and 5 xl09 M, under the questions raised in this paper, many remain yet unanswered,conditions given in Table 1. When such curves were established whereas others may appear to be unwarranted in the future. for MAL and MCT segments isolated from the rat kidney, it was noted that the sensitivity to vasopressin was always greater Sites of ADH action along the rat nephron (KA three- to fivefold lower) in the collecting than in the thick ascending limb [3]. Similar observations were also made The development, in 1975, of a micromethod designed toin MAL and MCT samples from rabbit [6], hamster (unpub- measure the hormone—dependent adenylate cyclase activity inlished observation) and jerboa [7]. single and well localized pieces of tubule [1, 2] made it possible Figure 1 gives a dose—response curve to AVP established in to investigate the sites along the nephron where vasopressinsamples of cortical collecting and cortical thick ascend- exerts physiological effects via the intracellular production ofing limbs isolated from the same rat kidney. Adenylate cyclase cAMP as second messenger. activity was measured in both segments in the presence or Table 1 indicates the responses obtained with 10—6 M arginineabsence of l0 M exogenous guanosine triphosphate (GTP), vasopressin in the successive portions of the rat nephron. Thebecause GTP is known to increase cyclase responsiveness to hormone produced no stimulation of adenylate cyclase in thehormones by acting on the coupling mechanism [8]. The pres- early nephron segments, namely the proximal convoluted tu-ence of GTP clearly enhanced the magnitude of the maximal bule (PCT), the proximal straight tubule (PR) and the thinresponses to AVP in CAL, but much less so in CCT samples. In descending limb of the 1oop of Henle (TDL). By contrast, AVPaddition, GTP shifted the dose—response curves of both seg- stimulated the enzyme activity 10 to 40-fold compared to basalments towards low AVP concentrations, indicating an in- values in all nephron portions located beyond the hairpin turn ofcreased sensitivity to the hormone (apparent KA =2x 10—11 the . vs. 7 X 10_li M (CCT), and lObo vs. 3 x b_b M (CAL) with This approach, therefore, revealed that antidiuretic hormoneand without GTP added, respectively). These values also indi- might exert effects via V2 receptors (that is, antidiuretic ascate that CCT is three— to fivefold more sensitive to AVP than opposed to V1 or vasopressor receptors) coupled to cAMPis CAL, in the presence or absence of GTP, in agreement with generation not only in the cells of cortical (CCT) and medullarythe results obtained in MCT and MAL without GTP addition[3]. (MCT) collecting tubules, as expected, but also in other epithe-Furthermore, the responsiveness of CCT to AVP in this cell broken microassay of adenylate cyclase activity was compara- ble, in the presence of GTP, to that obtained by measuring the Received for publication July 9, 1986 endogenous production of cAMP in surviving collecting tubules © 1987 by the International Society of [9]: the 10_li M AVP, concentration, that is, a concentration

512 Renal receptors to AVP and other hormones 513

CCT CAL Fig.1. Sensitivity to vasopressin (A VP) of adenylate cyclase (AC) present in the cortical thick E500 500 ascending limb (CAL) and in the cortical collecting tubule (CCT) of the rat kidney. Each point corresponds to the mean value of 4 to 5 replicate samples. Symbols are: (0) values measured in the absence of added guanosine triphosphate (GTP); (•)valuesmeasured in the presence of l0 M GTP in the incubation solution. The AVP concentration corresponding to the 0)250 E half-maximal AC response, as estimated from the 0 curves, was about 4 to 5 fold higher in CAL than in a- CCT in the absence (CAL, 3x l0° M; CCT, 0.7x 10_b 4 at)or in the presence of added GTP (CAL, 10- 10 0 CCT, 0.2x 100at). Notethat GTP addition increased the sensitivity to AVP in both nephron portions but tt more so in CAL (Imbert—Teboul and Siaume—Perez, AVP,LogM unpublished data).

Table 1. Effect of vasopressin on adenylate cyclase activity (AC) in maximal. In other words, GTP addition may either leave isolated segments of the rat nephron unchanged or increase the Vmax of cyclase activation by hor- cAMP fmol/mm/30 mm mones, depending on whether the maximal adenylate cyclase response obtained is limited by the full activation of the enzyme Segment B pa AVP N itself or by the full occupancy of the receptors. PCT 7.6 1.5 NS 9.6 1.3 4 PR 6.8 1.3 NS 8.6 1.3 3 Multi-hormonal control of adenylate cyclase TDL 19.9 5.3 NS 18.8 4.4 3 Before discussing the respective part played in the concen- TAL 5.1 1.4 <0.001 93.0 8.2 6 MAL 20.5 1.7 <0.001 844.2 50.3 19 trating mechanism by the various nephron segments responsive CAL 16.9 2.8 <0.001 244.6 29.5 5 to vasopressin, it is necessary to mention that other hormones eDCT 33.6 3.6 <0.001 228.1 14.0 7 were also observed to stimulate adenylate cyclase in some of CCT 33.6 3.6 <0.001 715.4 55.1 14 these segments. This situation is particularly striking for the MCT 35.4 6.6 <0.001 899.1 87.2 12 medullary and cortical portions of rat thick ascending limbs. Values are means SEM calculated from several experiments. Thus, the enzyme present in CAL responded to PTH [10—12], Abbreviations are: B, basal AC activity measured in the absence ofglucagon [10, 11, 13], [10—12], and, to a lesser extent, hormone; AVP, corresponding values measured in the presence of 10-6 M vasopressin; a Statistically different from control value. to /3 adrenergic [14] and vasopressin (Table 1). The cyclase present in MAL was highly responsive to [10, 11, 13] and vasopressin (Table 1) and, to a lesser extent, to calcitonin [10-42]. Since these nephron portions are relatively homogenous at the cell level, it was of importance to establish falling in the physiological range, induced a statistically signif-whether the receptors specific for these different hormones and icant stimulation of adenylate cyclase in both assays. for vasopressin are present in the membranes of the same cells The different sensitivity of collecting tubules and thick as-(in which case they should induce the same biological responses cending limbs to vasopressin does not imply the presence ofas vasopressin does), or are segregated in different cells (in two kinds of with a different affinity for the hormone inwhich case the response might be different). One way to answer the two segments. It might result from a more efficient couplingthis question was to investigate whether the maximal cyclase reaction between occupied receptors and adenylate cyclaseactivation elicited in these two nephron portions by each units in CCT and MCT cells than in MAL and CAL cells. If, forhormone separately is additive when the hormones are tested in example, the number of V2 receptors to AVP per cyclasecombination. Glucagon, calcitonin and PTH maximal effects moiety were greater in CCT than in CAL cell membranes, awere observed to be non-additive in CAL [5, 10, 11], indicating given hormone concentration, although resulting in a similarthat these three hormones stimulate a common pool of adenyl- fractional occupancy of the receptors in both structures, wouldate cyclase in the structure (Fig. 2). Full additivity was ob- induce a higher fractional cyclase activation in CCT where thetained, as expected, when low hormonal concentrations were number of receptors is assumed to be proportionally greater. used [11]. In these experiments, however, additivity of effects Such a situation might also explain why the Vmax of thecould not be tested for vasopressin in the presence of these cyclase response to AVP was increased by GTP in CAL but nothormones, because vasopressin alone induces only a limited in CCI (Fig. 1): in the absence of GTP addition, full receptormaximal cyclase activation in CAL without GTP addition [12]. occupancy (due to high AVP concentrations) may have resultedWhen tested in MAL samples, on the other hand, the effects of in maximal cyclase activation in CCT, but only in partialglucagon and vasopressin were only poorly additive, whereas cyclase activation in CAL. By increasing coupling efficiency,those of calcitonin and vasopressin were nearly fully additive, GTP addition could have increased further the enzyme re-due to the low maximal response induced by calcitonin in this sponse in CAL, but not in CCT in which it was alreadysegment [11]. 514 Morel et a!

CAL examples of such experiments for CAL and MAL respectively * (unpublished data from Imbert—Teboul and Siaume—Perez). The —— followingpoints are clearly demonstrated by these experi- 0 + ments: 1) when the maximal response of adenylate cyclase to a E given hormone was measured with or without GTP addition in 1000- 0I- (0 homologous tubular samples isolated from the same rat kidney, + the response was increased to a large extent for the poorly 0I- active hormones, that is, vasopressin and calcitonin in CAL and + calcitonin in MAL. The response was only slightly increased or 0 6) 500 - > not affected for the most active hormones (glucagon and PTH in E 4 0 CAL, glucagon and vasopressin in MAL). 2) In the presence of 0 added GTP, all hormones, except AVP in CAL, tended to 42 produce maximal cyclase stimulation of a similar magnitude C) (about 1100 to 1300 fmoles/mm/30 mm in CAL, and 600 to 800 fmoles/mm/30 mm in MAL in these experiments), an observa- 0 L1IB SCT GLU PTH IAVP tion suggesting that the Vmax of the cyclase was the limiting factor of the response under such conditions. 3) When various Fig. 2. Absence of additive effects of vasopressin, parathyroid hor- mone, glucagon and calcitonin on AC activity in cortical thick ascend- hormones were tested in combination, full additivity of effects ing limbs (CAL) of the rat. Results of an experiment in which ACwas not observed with AVP and calcitonin in MAL; moreover, activity contained in CAL samples from the same rat kidney wasthe response of the CAL cyclase to PTH + glucagon + measured either in the absence (open bars) or the presence of iO- M calcitonin was not further increased in the presence of AVP. addedGTP (dashed bars) under basal conditions (B) and in the presence of one or several hormones as indicated. Abbreviations are: SCT,These results, therefore, confirm that these different hormones salmon calcitonin, 100 ng. ml; Glu, porcine glucagon, 106 M;PTH, stimulate a common pool of adenylate cyclase in MAL and bovine (synthetic 1-34 fragment), 10 U ml1;CAL, respectively; consequently, they should elicit the same AVP, arginine vasopressin, 10-6 M.Eachbar corresponds to the mean biological effects in these structures [11]. value of 4 to 5 replicate samples. Symbols are: NS not statistically When the hormone—induced increase in endogenous cAMP different, and (*) p <0.05,when compared to the value measured in the absence of GTP addition. (Imbert—Teboul and Siaume—Perez, unpub- was measured in vitro by radioimmunoassay [9] in living single lished data). MAL tubules isolated from the rat outer medulla, it was also observed that the effects of 10-6 M AVP (114.7 9.4 fmol• mm' 4 min1 (N =8))and of 10 ng .ml'salmon MAL calcitonin (82.8 8.4 fmol .mm'4 min' (N =7))were 1000 - non-additive (116 7.1, N 7) when the two hormones were NS tested in combination (Chabardès and Montégut, unpublished data). NS -j 0 + Physiological effects of vasopressin in its responsive nephron 0 0 > segments 500 4 a ///. The results given in Table 1 indicate that the thin ascending E /, 0 /; limb (TAL), the thick ascending limb or diluting segment (MAL 0 + CAL), the (DCT) and the collecting 2 4C) tubule (CCT + MCT) represent the target segments for vaso- pressin in the rat nephron. Measurement of adenylate cyclase 0- activity is obviously not an appropriate method in order to B SCT AVP GLU study the biological action induced by vasopressin in these Fig.3. AbsenceJI of additive effects of vasopressin, calcitonin andsegments since, as mentioned before, the nature of the biolog- glucagon on AC activity in medullary thick ascending limbs (MAL) ofical actions results from cAMP-dependent phosphorylation the rat. Results of an experiment in which AC activity contained in reactions which are specific for each type of responsive cell. MAL samples from the same rat kidney was measured either in the presence (open bars) or the absence (dashed bars) of l0 Madded Other approaches are required, therefore, in order to investi- GTP. Hormone abbreviations and concentrations as indicated in the gate this problem. legend to Fig. 2 (Imbert—Teboul and Siaume—Perez, unpublished data). Thin ascending limb Therefore, additional experiments including the addition of There are no data available in the literature as regards an GTP to the incubation solution were performed in order toaction induced by vasopressin in microperfused thin ascending further establish: a) if GTP could increase the maximal re-limbs. It was only noted that the hormone did not increase the sponses of the cyclase to those hormones which induced only apermeability of this segment to [15]. A possible effect of low response without GTP addition (namely AVP in CAL andAVP on the permeability to and/or to should be calcitonin in MAL); and b) if all the hormonal effects becomeanalyzed in microperfused TAL, since the permeability of the non-additive under such conditions. Figures 2 and 3 givethin ascending segment to these solutes is considered to play a Renal receptors to AVP and other hormones 515

TAL Thick ascending limb Antidiuretic hormone is known to produce two effects in 150 skin and : it increases the osmotic permeability to water and it stimulates active sodium transport. In the kidney, the hormone also stimulates both water and sodium [19]. In the mammalian kidney, the dilut- E ing segment (thick ascending limb) and the segment where ADH 100 controls water reabsorption (collecting tubule) are clearly sep- arated from each other. In view of these anatomical differences,

0) it was suggested years ago that ADH might stimulate both salt E and water reabsorption in the kidney of , each effect 0 being segregated in a different nephron portion, that is, the 50 4 collecting tubule for the effect on water and the thick ascending limb for the effect on active sodium transport [20]. The obser- vation that vasopressin actually stimulated adenylate cyclase in both of these nephron portions provided some support to this hypothesis [3]. Finally, direct evidence that ADH may stimu- 0 ______lateactive salt transport by the diluting segment in mammals —7 —6 was obtained when Hall and Varney showed, in microperfused, AVP concentration medullary thick ascending limbs isolated from mouse kidneys, Fig. 4. Sensitivity to vasopressin of AC activity contained in thinthat vasopressin increases chloride transport without altering ascending limbs (TAL) isolated from the rat inner medulla. In thisthe permeability to water [211. These aspects will not be experiment, adenylate cyclase activity was measured in the absence discussed here since they are covered in detail in another (basal, B) and in the presence of increasing vasopressin (AVP) concen- chapter in this special issue (de Rouffignac et a!, this issue). Let trations on single pieces of TAL microdissected from the same rat kidney. Each point corresponds to the mean value of 4 to 5 replicate us simply mention two expectations of physiological interest, samples. A statistically significant activation of AC was already ob- which result from the above described properties of the tained with 10-10 M AVP. The half maximal response corresponded to hormone—dependent adenylate cyclase in MAL and CAL. 6. l0 M AVP in this experiment, assuming that l0 M AVP elicitedFirst, the ADH effect on ion transport by the diluting segment the maximal response (redrawn from [5]). should require (to occur in vivo) higher hormonal concentra- tions in plasma than the antidiuretic effect in collecting tubules, since adenylate cyclase is less sensitive to vasopressin major role in the concentrating mechanism along the innerin the former than in the latter segment. Second, various kidney medulla [16]. Let us point out that AVP (10-6 M)hormones, such as PTH, glucagon and calcitonin, should pro- increased adenylate cyclase activity more than 15-fold in ratduce, in the rat thick ascending limb, the same effects on ion TAL, whereas it was completely devoid of action in descendingtransport as vasopressin does [22, 23], since the maximal limbs (TDL) from the same animals (Table 1). As shown instimulations of adenylate cyclase induced by these different Figure 4, the sensitivity of the cyclase to AVP was similar in thehormones in MAL and CAL respectively were not additive in rat TAL (KA =6x l0 M, Fig. 4) to that measured in MALvitro. The cogency of these arguments is considered in the (average value: 6 x io, N =6[3]). The thin ascending limb incontribution by de Rouffignac et al. the rabbit was also responsive to AVP [17]; the average values SEM (fmol .mm'.30min') were respectively: controls 9.3 Distal convoluted tubule l.9(N =9)versus AVP (10—6 M), 82.6 5.8 (N =12)in TAL, = Distal convoluted tubules exhibit a great axial heterogeneity and, control, 9.52.3(N 7)versus AVP (10—6 M), 12.0 1.8 in cell type composition in the rat. Moreover, they are difficult (N =8)in TDL. Adenylate cyclase activity was measured in theto isolate over their full length in this species, even when presence of AVP in three adjacent samples (TDL, TAL andmicrodissected from collagenase treated kidneys. Finally, MAL) microdissected from the same rabbit long loop with themarked species differences were noted in adenylate cyclase following results (fmol .mm.30mm .'): TDL,2.9; TAL,responsiveness to vasopressin along the successive distal con- 113; MAL, 142 [17]. Such an observation suggests that thevolutions between the mouse [24], rabbit [4, 24, 25], rat [4, 25] change in response to the hormone takes place close to theand human kidneys [26]. For these reasons, it is premature to hairpin turn of the loop. Thus, the transition between thespeculate about the possible action(s) of vasopressin along this descending and ascending thin portions of long loops, which issegment from the data obtained by measuring cyclase activity. present at (or near) their turning point, concerns not only ultrastructural organization and permeability properties, but Collecting tubule responsiveness to hormones as well, a situation that Homer The antidiuretic action of vasopressin along collecting tu- Smith claimed to dislike [18]. bules results from adenylate cyclase activation via V2 receptors In our opinion, these different results suggest that vasopres-is so well established that it does not require additional com- sin exerts in thin ascending limbs, via V2 receptors and cAMP,ment. The modulation of this action by various regulators, on effects of physiological relevance which require further inves-the other hand, is discussed later on in this contribution. Let us tigation. mention here that the adenylate cyclase activity contained in rat 516 Morelet a! cortical collecting tubules (CCT) is responsive not only to 1000 vasopressin, but also to salmon calcitonin [11] and, to a lesser extent, to glucagon [13]. The maximal effects of AVP and C calcitonin given in combination were observed to be partly 0E additive or non-additive, depending on the experiment [11]. E When GTP was added to the incubation solution, the Vmax E 500 0 response induced by calcitonin was clearly enhanced, so that E the absence of additivity of effects of the two hormones was0 clearly confirmed under such conditions (Imbert—Teboul et al, 0 unpublished data). Such an observation suggests that AVP and -1 calcitonin stimulate the same pool of adenylate cyclase in the LLL rat CCT. Therefore, both hormones should induce the same Rat Mouse Jerboa Rabbit Man biological response in this segment, namely an increase in II. Fig. 5. Responsiveness to vasopressin of adenylate cyclase (AC) pres- osmotic permeability to water. Note that calcitonin did notent in medullary thick ascending limbs from different mammalian stimulate the enzyme contained in medullary collecting tubulesspecies. AC responses to 1O M vasopressin are expressed here as (MCT) [4, 25]. According to these data, injection of salmoncAMP formed (stimulated—basal AC activity); dashed bars are re- calcitonin to rats undergoing water or to diabetessponses measured in medullary thick ascending limbs (MAL); open bars are responses measured in adjacent medullary collecting tubules insipidus (DI) Brattleboro rats should increase urine osmotic(MCT). Note that the AC response of MCT to vasopressin was of pressure up to isoosmolality (but not to hyperosmolality as similar magnitude in all species studied, except the jerboa. By contrast, vasopressin does). This aspect is considered in another contri-the responses measured in MAL varied over a wide range, depending bution (de Rouffignac et al, this issue). on the species studied [3, 6, 7, 24, 26] and unpublished results by Le Bouffant and Morel for the golden hamster).

Species differences in adenylate—cyclase responsiveness to vasopressin differences exist as regards tubular outer diameters and there- The distribution of the hormone—dependent adenylate cy-fore protein content per mm of tubule, the absolute response of clase activity exhibits marked species differences in certainMCT to AVP was almost identical in five among the six species nephron portions [25]. We focus here on the main segmentsstudied, ranging between 700 (in rabbit) and 1000 fmol mm' involved in the mechanism of urine concentration and dilution,30 min (in human). The only exception was the desert rodent, namely the diluting (MAL and CAL) and collecting segmentsJerboa (Jaculus orientalis), which exhibited a relatively poor (CCT and MCT). response in MCT (300 fmol .mm30 min'). On the other As regards glucagon (porcine hormone), adenylate cyclase ishand, the MAL response to 10-6 M AVP varied greatly among nearly unresponsive to this hormone in the rabbit, whereas thespecies: in the hamster and the rat, the response was very high enzyme is highly responsive to glucagon in MAL from the rat,and comparable in absolute value to that induced in MCT (800 hamster and jerboa for example. Salmon calcitonin stimulatesto 1000 fmol mm1 .30min1). In the other species, the MAL adenylate cyclase activity to a greater extent in CAL than MALresponse decreased in the following order (fmol .mm30 in the rat [11, 12], but to a greater extent in MAL than in CALmin'): mouse (about 300), jerboa (about 200), rabbit (about in the rabbit [27]. The hormone is equally active in MAL and100), and human (no response). Note that the lack of AVP effect CAL of the human nephron [26]. In the mouse, salmonin the human MAL is probably not a technical artifact due to calcitonin is inactive in MAL and only poorly active in CALkidney storage, since, under the same experimental conditions: [281. PTFI, finally, stimulates the enzyme in CAL but not ina) the MAL cyclase remained quite responsive to PTH and MAL for all species studied (rat, rabbit, mouse, hamster),calcitonin; and b) the adjacent collecting tubules (MCT) were as except for man where it stimulates equally these two nephronsensitive to vasopressin as MCT samples isolated from freshly portions [25, 26]. In accordance with this difference in respon-prepared rat kidneys for example. Finally, Ruggles et al [32] siveness between MAL and CAL enzyme, PTH stimulatesconfirmed recently the absence of AVP effect on adenylate (and magnesium) reabsorption in microperfused rabbitcyclase activity in MAL samples microdissected from freshly CAL [29], but not in MAL [30]. On the other hand, calcitoninexcised human kidney . increases calcium reabsorption in microperfused rabbit MAL Examination of Figure 5 suggests the existence of a correla- [31]. However, the physiological meaning of these speciestion between MAL responsiveness to vasopressin and the differences in hormone responsiveness of the cyclase betweenconcentrating ability of the kidney. Thus, hamsters and rats are CAL and MAL is unknown. It is also not established in allknown to concentrate urine up to 3500 mOsm per liter or more species whether the different hormone receptors in these seg-during , whereas rabbits (about 2000 mOsm per ments are coupled to a common pool of enzyme as they are inliter), and, especially, humans (about 1300 mOsm per liter) have the rat. a relatively poor concentrating capacity, even during water Species differences were also noted regarding the responsive-deprivation. By increasing active salt reabsorption across the ness of MAL adenylate cyclase to vasopressin. Figure 5 illus-water—impermeable, epithelial cell layer of thick ascending trates the increase in adenylate cyclase per mm of tubulelimbs, vasopressin might contribute to the accumulation of induced by 106 MAVPin medullary thick ascending limbssolutes in the medullary interstitium and thereby might allow (MAL) and outer medullary collecting tubules (MCT) isolatedvery high medullary gradients of osmotic pressure to be created from the kidneys of six different species. Although large specieswhen required. Such a role of vasopressin in the concentrating Renal receptors to AVP and other hormones 517 mechanism was suggested for the rat kidney by Thomas and A Brattleboro DI rat C Jerboa associates [33, 34] several years ago. If this view were correct, the jerboa—which is a highly—concentrating true desert rodent DI tDI DI tDI species—would constitute a remarkable exception, because the 100 (0 adenylate cyclase response to AVP was rather low in this 0 species, both in MAL and MCT samples (Fig. 5). Whether this 0 0 poor responsiveness may represent some "down—regulation" U ti1 due to high AVP concentrations in is discussed in 0 0 the next section. 50- 0a) Does ADH level in blood plasma regulate adenylate cyclase responsiveness in the kidney? It has been established for many hormones that the number of 0- free receptors in target cell membranes, as well as the stimula- MCI MAL MCI MAL MCI MAL tion of adenylate cyclase activity, coupled to the occupancy of these receptors may be reduced when the tissue has been Fig.6. Relationship between in vivo ADH levels in blood plasma and in vitro AC responsiveness to A VP of medullary collecting tubules previously exposed, either in vivo or in vitro, to a high(MCT) and medullary thick ascending limbs (MAL). The AC responses concentration of the corresponding hormone before the mea-(average values) to 106 M AVP obtained in MCT and MAL samples surements are made. This phenomenon, called down—regulationfrom the kidneys of the treated animals are expressed as per cent of the or desensitization, results from an increased rate of receptorcorresponding responses measured in homologous samples from the internalization when the hormone concentration has beenkidney of control animals studied in the same experiments. A. DI, Brattleboro rats with ; tDI, DI rats microperfused for greatly enhanced, that is, when the fractional occupancy of6 to 8 weeks with physiological doses of dDAVP; normal Long Evans receptors is high. Rajerison, Butlen and Jard [35] showed in therats were used as controls in these experiments (data recalculated from rat that the vasopressin—sensitive adenylate cyclase activity in[38, 391. 13. Wistar rats were desensitized by a single injection of membrane fractions from the kidney medulla is greatly reducedpitressin (1 U) 20 mm before the experiment. Wistar rats injected with the solvant were used as controls in this type of experiment. From [361. after the injection or perfusion of pharmacological doses ofC. Two groups of5jerboas (Jaculus orientalis) received for 4 to 7 weeks AVP to the animals. The degree of desensitization obtained wasbefore the experiments either a dry diet (controls) or the same diet dose and time dependent. Experiments using microdissectedsupplemented with a large supply of water in the form of lettuce and tubules were performed under experimental conditions produc-carrots (experimental animals). Data recalculated from [7]. ing a moderate desensitization (injection of! unit pitressin i.m., 20 mm before kidney removal), in order to analyze whether the decrease in cyclase response concerns the two main targetdata indicate that the presence of a normal level of ADH in segments to AVP in the medulla, namely, the MAL and MCTblood plasma is required, not only to regulate the number of [36]. The results of such an experiment are shown on Figure 6B.MAL receptors to AVP, but to ensure the full morphological They indicate a similar 40 to 50 percent decrease in cyclaseand functional development of the structure as a whole, as response in both segments of the AVP-injected rat compared topreviously demonstrated by Kriz and Bankir [40], Trinh—Trang— the control animal. Tan et al [41], and Bouby et al [42]. These observations explain On the other hand, Rajerison and associates [35] observedwhy long—term treatment of Brattleboro DI rats with ADH is that the response of AC to AVP was also diminished in thenecessary to fully restore the concentrating ability [43]. They medullary membranes prepared from Brattleboro rats withdemonstrate, therefore, that AVP plays a major role in the diabetes insipidus (DI) when compared to those prepared frommechanism of urine concentration via an action exerted on the Long Evans control rats, in confirmation of the results ofmedullary thick ascending limbs. The molecular mechanism Dousa, Hui and Barnes [37]. When the cyclase response towhereby AVP produces a long—term effect on MAL develop- AVP was measured in microdissected MAL and MCT samplesment is not known. separately, however, a clear dissociation of effects was ob- We mentioned before that the responsiveness of adenylate served [38]: a 50 percent decrease in Vmax (without change incyclase was relatively low in both MCT and MAL from the KA) was obtained in MAL from DI rats compared to MAL fromjerboa. It has been reported that freshly captured jerboas, or controls, whereas the Vrnax of the MCT samples were similar injerboas fed a dry diet, have extremely high ADH concentrations DI and control rats (Fig. 6A). When DI Brattleboro ratsin their blood plasma (in the range of several hundred picograms received low doses of AVP from a minipump for six to eightper ml) [44], Experiments were performed using jerboas fed for weeks, the responsiveness of the MAL cyclase to AVP wasseveral weeks a water—deprived diet, or the same diet supple- restored to a large extent, whereas no effect was measured inmented with a large amount of water in the form of fresh MCT (Fig. 6A). This chronic treatment of DI Brattleboro ratslettuce, to establish if such high concentrations of ADH may with AVP was observed in MAL to increase not only the AChave physiologically "desensitized" the kidney target sites [7]. response to AVP, but also the AC response to glucagon (by 80%At the time of the experiment, the animals on a dry diet compared to the non-treated DI controls), the Na-K-ATPaseexcreted a highly hypertonic urine (3800 mOsm .l)and their activity (by nearly 60%) and, finally, the tubular volume itselfblood plasma contained an exceedingly high ADH concentra- (by about 65%) [39]. All these stimulatory effects were limitedtion (372 pg. ml' as an average). The animals fed the to the medullary portion (MAL) of thick ascending limbs. Thesewater—enriched diet were (average urine osmotic pres- 518 Morelet a! sure, 650 mOsm 1'), and the plasma ADH concentration (76 MCT ___AVP pg. ml'), although still very high, was decreased by fivefold. In membrane fractions prepared from the kidney medulla, the [3H]-LVP binding capacity and the adenylate cyclase response 50 to AVP were both increased in the hydrated jerboas compared to the water—deprived controls, an observation compatible with receptor desensitization in the animals fed the dry diet. When the responsiveness of adenylate cyclase to AVP was measured * in MCT and MAL samples separately, as shown in Figure 6C, Q no effect of the diet could be observed in medullary collecting - tubules, whereas the MAL response was increased by 40% in + the animals adapted to the water—enriched diet (the average AVP SCT GLU ISO values with 10 M AVP were 22112 fmol. mm' .30min' 50 (N =35)vs. 15813 (N =33),P <0.001, in the MAL samples E from hydrated and dehydrated jerboas, respectively). This E diet—induced alteration in MAL responsiveness was AVP- specific, since the MAL response to glucagon (10-6 M), mea- * sured in the same animals, varied in opposite direction: it was greater in the dehydrated (365 52 fmol .mm'.30min 1) 0NE —± — + — CLO than in the hydrated jerboas (23122 fmol .mm'.30min'). A vasopressin—specific desensitization of the AVP receptorsFig.7. Effect of a2-adrenergic agonists on the hormone—dependent was also noted in the cell membranes from the jerboas fedproduction of cAMP by single pieces of medulla,y (MCT) or cortical the water—deprived diet [45]. Altogether, these data suggest thatcollecting tubule (CCT) microdissected from rat kidneys. Intracellular cAMP generated from endogenous ATP in the presence of IBMX was some down—regulation of the number of receptors to vasopres-measured by radioimmunoassay as described in [91. Each bar corre- sin occurs physiologically in the jerboa, in particular when thesponds to the mean valueSE of 6 to 8 replicate samples. Norepineph- water supply is restricted, because AVP concentration in bloodrifle (NE, io M) was used as a2 adrenergic (in the presence of plasma appears to be unusually high in this species. Note that10 M propranolol) in all conditions, except for testing the inhibitory effect of a2-agonists on the response induced by isoproterenol in CCT. the kidney retains a full capacity to concentrate urine underIn this case, propranolol was omitted and the specific a2 agonist, these conditions. The results of Figure 6C suggest that MALclonidine (Clo, l0 si) was used. Yohimbine (Yo, 10 M) was used as would be more sensitive than MCT to such a down—regulation.a2 blocker and Prazosin (PR, i0 M) as a1 blocker in the experiments Furthermore, down—regulation alone is not sufficient to account shown in the upper pannels. Arginine—vasopressin (AVP, 10b0 M), for another observation in this species [7]: in response to acutesalmon calcitonin (SCT, 100 ng .ml-'),porcine glucagon (GLU, 10 M) and isoproterenol (ISO, 10_6 M) were used as cyclase activators in overhydration by gastric gavage, some jerboas exhibited athese experiments. In the absence of added hormone, the basal produc- delayed water diuresis characterized by the of hypo-tion of cAMP was too low to be detected under the conditions of the osmotic urine (average osmotic pressure, 1576 mOsm- 1I,microassay used (data redrawn from [91). Note (upper pannels) that 6 animals). The average AVP concentration in the blood plasmanorepinephrine almost completely suppressed the MCT response to 8.4 vasopressin via a2-adrenergic receptors, When tested against the dif- sample collected immediately afterwards was 41.5 ferent hormones known to stimulate AC in CCT (bottom pannel), pg -mlin these six animals. The production of dilute urine bya2-adrenergic agonists inhibited specifically the response induced by the kidney in spite of the presence of such huge ADH concen-vasopressin. trations in blood plasma suggests that some additional factor blunted the action of the hormone on the permeability of collecting tubules to water. nism involving receptors of the a2 type and Ni coupling subunits. Figure 7 demonstrates that , through Regulation of adenylate cyclase responsiveness at the this mechanism inhibits almost completely the vasopressin— transduction level induced cAMP generation in collecting tubules isolated from Several factors modulate the biological response elicited bythe rat [9]. The experiments depicted in the upper part of Figure AVP in different systems. Some of them, like gluco- and7 show that the inhibition of AVP effects produced by norepi- mineralo-corticoids, for example, increase the hydro-osmoticnephrine was suppressed by blocking a2 receptors with effect of ADH [46, 471. Other agents, such as a2-adrenergicyohimbine, whereas blocking a1 receptors with prazosin had no agonists [481, [49], [50], exert aneffect. The inhibitory action of the alpha agonist, therefore, was inhibitory action. mediated through receptors of the a2 subtype. The experiment Among the mechanisms whereby regulatory agents mightdepicted in the bottom part of Figure 7 shows the effects modulate the cellular response to vasopressin are the following:obtained with a2 agonists on the increase in cAMP generation a) an adenylate—cyclase inhibition involving the so—called Niproduced in CCT by different hormones known to stimulate subunit of the GTP-dependent coupling protein complex; b) aadenylate cyclase in this nephron portion. The effect of AVP decrease in cAMP cell content resulting from phosphodies-was clearly inhibited, as in MCT samples, whereas those of terase activation; c) a modulation of the cell response due tocalcitonin, glucagon and isoproterenol were not altered under alterations in —free calcium concentration. the same conditions. This observation suggests that the cells are known to inhibit hormone—induced ade-responsive to vasopressin contain a2-adrenergic receptors, nylate cyclase activation in various cell systems via a mecha-whereas the cells responsive to the other three hormones are Renal receptors to AVP and other hormones 519 devoid of such receptors, as cortical collecting tubules are 5. MOREL F, CI-IABARDES D, IMBERT—TEBOUL M: Distribution of known to consist of more than one type of cell. In the case of adenylate cyclase activity in the nephron, in Current Topics in Membranes and Transport, (vol 13), edited by BRONNER F, calcitonin, however, this explanation cannot easily be recon- KLEINZELLER A. New York, Academic, 1980, pp. 415—426 ciled with the absence of full additivity of AVP and SCT effects 6. IMBERT M, CHABARDES D, MONTGUT M, CLIQUE A, MOREL F: in this nephron portion [11]. If the receptors specific for AVP Vasopressin—dependent adenylate cyclase in single segments of and for calcitonin are coupled to a common pool of cyclase rabbit kidney tubule. Pflugers Arch 357:173—186, 1975 moities, that is, are present in the membranes of the same cells, 7. BADDOURI K, BUTLEN D, IMBERT—TEBOUL M, LE BOUFFANT F, MARCHETTI J, CHABARDES D, MOREL F: Plasma antidiuretic hor- then the adrenergic a2 receptors also contained in these mem- mone levels and kidney responsiveness to vasopressin in the branes would inhibit selectively the adenylate cyclase activa- Jerboa, Jaculus orientalis. Gen Comp Endocrinol 54:203—215, 1984 tion mediated by vasopressin. Additional experiments are 8. COOPER DMF: Bimodal regulation of adenylate cyclase. FEBS Lett needed to solve this particular problem and, more generally, to 138:157—163, 1982 9. CHABARDES D, MONTEGUT M, IMBERT—TEBOUL M, MOREL F: establish if inhibition of vasopressin effects by a2 agonists is Inhibition of az-adrenergic agonists on AVP-induced cAMP accu- present: a) in collecting tubules in species other than the rat and mulation in isolated collecting tubule of the rat kidney. Mol Cell b) in vasopressin—responsive nephron segments other than Endocrinol 37:263—275, 1984 collecting tubules (as regarding rat MAL, a2 stimulation pro- 10. TORIKAI S. WANG MS, KLEIN KL, KUROKAwA K: Adenylate duced no inhibition of the response to AVP, according to cyclase and cell cyclic AMP of rat cortical thick ascending limb of Henle. Kidney mt 20:649—654, 1981 Umemura et a! [51]). 11. MOREL F, CHABARDES D, IMBERT—TEBOUL M, LE BOUFFANT F, Prostaglandins, mainly PGE2, have also been observed to HUS—CITHAREL A, MONTEGUT M: Multiple hormonal control of blunt vasopressin effects by inhibiting cAMP production in adenylate cyclase in distal segments of the rat kidney. Kidney mt 21 kidney tubules, in particular rat papillary collecting tubules [52] (suppi l1):S-55—S-62, 1982 orthick ascending limbs from mouse [53]and 12. IMBERT—TEBOUL M, CHABARDE5 D, CLIQUE A, MONTEGUT M, rat [54]. MOREL F: Ontogenesis of hormone-dependent adenylate cyclase in Inconclusion, it appears clearly established that various isolated rat nephron segments. Am J Physiol 247:F316—F325, 1984 physiological agents and hormones may alter the response 13. BAILLY C, IMBERT—TEBOUL M, CHABARDE5 D, HUS—CITHAREL A, elicited by vasopressin in defined systems and under specific MONTEGUT M, CLIQUE A, MOREL F: The distal nephron of rat conditions. To what extent these results apply to other vaso- kidney: A target site for glucagon. Proc Nat! Acad Sci USA 77:3422—3424, 1980 pressin-sensitive systems or to other species, and may be 14. MOREL F, CHABARDE5 D, IMBERT—TEBOUL M: Effect of catechol- extrapolated to the conditions prevailing in the kidney in vivo is amines on adenylate cyclase activity along the rat nephron. (ab- far from established. As a consequence, the exact role played stract) Upsala J Med Sd (suppl) 26:53, 1979 by adrenergic agonists and prostaglandins in the overall func-15. IMAI M, KOKKO JP: Sodium choride, urea and water transport in the thin ascending limb of Henle. Generation of osmotic gradients tion of urine concentration and dilution by the kidney is difficult by passive diffusion of solutes. J Clin Invest 53:393—402, 1974 to evaluate. 16. KOKKO JP, RECTOR FC: Countercurrent multiplication system On the other hand, the mechanisms through which other without in inner medulla. Kidney mt 2:214—223, hormones, like , glucocorticoids, and hor- 1972 mones, exert their well established role on the urinary concen- 17. IMBERT M, CHABARDE5 D, MONTEGUT, CLIQUE A, MOREL F: Presence d'une adenyl—cyclase stimulée par Ia vasopressine dans Ia trating mechanism are still poorly understood at the cellular branche ascendante des anses des néphrons du rein de Lapin. C R level, even though some aspects have been recently clarified, Acad Sci (Paris) 280:2129—2132, 1975 for example the exact nephron portions in which these hor- 18. SMITH HW: The fate of sodium and water in the renal tubules. Proc mones regulate Na-K-ATPase activity. NYAcad Med 35:293—316, 1959 19. JARD 5, MOREL F: Actions of and some of its analogues on salt and water excretion by the frog. Am J Physiol 204:222—226, Acknowledgments 1963 This work was supported by a grant of CNRS to LA 219. We are 20. MOREL F: Action of neurohypophyseal hormones on the active greatly indebted to S. Siaume—Perez for her technical assistance. transport of sodium, in Water and Metabolism II, edited by DE GRAEFF J, LEINSE B. Amsterdam, Elsevier Publishing Co. 1964, pp. 91—104. Reprint requests to F. Morel, M.D., Laboratoire de Physiologie 21. HALL DA, VARNEY D: Effect of vasopressin on electrical potential cellulaire, College de France, ii, Pce. Marcelin Berthelot, 75231 Paris difference and chloride transport in mouse medullary thick ascend- Cedex 05, France. ing limb of Henle's loop. J Clin Invest 66:792—802, 1979 22. 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