Receptors to vasopressin and other hormones in the mammalian kidney

Kidney International, Vol. 31(1987), pp. 512—520

MOLECULAR MECHANISM OF ACTION OF ANTIDIURETIC HORMONE

Receptors to vasopressin and other hormones in the mammalian kidney 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 of hal cell types of the nephron 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 stimulating of the thick ascending limb, and the early distal convoluted the intracellular production of cAMP revealed an unexpected tubule (eDCT). As long as these responsive segments correpanorama, and thereby raised a series of new questions. This spond to physiological target sites for AVP, the biological contribution will discuss some of the observations we made response 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 mechanism properties specific for the cell type itself, as pointed out before of urine 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 which results given in Table 1, dose—response curves were established vasopressin was observed to induce in vitro effects in different in 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 the erally ranging between 5x10'° and 5 xl09 M, under the questions raised in this paper, many remain yet unanswered, whereas others may appear to be unwarranted in the future. Sites of ADH action along the rat nephron

The development, in 1975, of a micromethod designed to measure the hormone—dependent adenylate cyclase activity in single and well localized pieces of tubule [1, 2] made it possible

to investigate the sites along the nephron where vasopressin exerts physiological effects via the intracellular production of cAMP as second messenger. Table 1 indicates the responses obtained with 10—6 M arginine

vasopressin in the successive portions of the rat nephron. The hormone produced no stimulation of adenylate cyclase in the early nephron segments, namely the proximal convoluted tu-

conditions given in Table 1. When such curves were established

for MAL and MCT segments isolated from the rat kidney, it was noted that the sensitivity to vasopressin was always greater (KA three- to fivefold lower) in the collecting tubule than in the thick ascending limb [3]. Similar observations were also made

in MAL and MCT samples from rabbit [6], hamster (unpublished observation) and jerboa [7]. Figure 1 gives a dose—response curve to AVP established in samples of cortical collecting tubules and cortical thick ascending limbs isolated from the same rat kidney. Adenylate cyclase activity was measured in both segments in the presence or absence of l0 M exogenous guanosine triphosphate (GTP), because GTP is known to increase cyclase responsiveness to hormones by acting on the coupling mechanism [8]. The presence of GTP clearly enhanced the magnitude of the maximal

bule (PCT), the proximal straight tubule (PR) and the thin responses to AVP in CAL, but much less so in CCT samples. In descending limb of the 1oop of Henle (TDL). By contrast, AVP stimulated the enzyme activity 10 to 40-fold compared to basal values in all nephron portions located beyond the hairpin turn of the loop of Henle. This approach, therefore, revealed that antidiuretic hormone

addition, GTP shifted the dose—response curves of both seg-

ments towards low AVP concentrations, indicating an in-

creased sensitivity to the hormone (apparent KA = 2 x 10—11 vs. 7 X 10_li M (CCT), and lObo vs. 3 x b_b M (CAL) with and without GTP added, respectively). These values also indimight exert effects via V2 receptors (that is, antidiuretic as cate that CCT is three— to fivefold more sensitive to AVP than opposed to V1 or vasopressor receptors) coupled to cAMP is CAL, in the presence or absence of GTP, in agreement with generation not only in the cells of cortical (CCT) and medullary the 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 comparable, 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 [9]: the 10_li M AVP, concentration, that is, a concentration © 1987 by the International Society of Nephrology 512

Renal receptors to AVP and other hormones

E 500

0)

Fig. 1. Sensitivity to arginine vasopressin (A VP) of

CAL

CCT

adenylate cyclase (AC) present in the cortical thick 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); (•) values measured in the presence of l0 M GTP in the incubation solution. The AVP concentration corresponding to the half-maximal AC response, as estimated from the curves, was about 4 to 5 fold higher in CAL than in CCT in the absence (CAL, 3x l0° M; CCT, 0.7x 10_b at) or in the presence of added GTP (CAL, 10- 10 CCT, 0.2x 100 at). Note that GTP addition increased the sensitivity to AVP in both nephron portions but

500

250

E

0

a-

40 t t

more so in CAL (Imbert—Teboul and Siaume—Perez, unpublished data).

AVP,LogM

Table 1. Effect of vasopressin on adenylate cyclase activity (AC) in isolated segments of the rat nephron cAMP fmol/mm/3 0 mm

Segment PCT PR TDL TAL MAL CAL eDCT CCT MCT

pa

B 7.6 6.8

1.5 1.3

19.9 5.1

5.3

20.5 16.9

33.6 33.6 35.4

1.4 1.7

2.8 3.6 3.6 6.6

NS NS NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

513

N

AVP 9.6 8.6

1.3 1.3

18.8

4.4 8.2

93.0 844.2 244.6 228.1

50.3

29.5 14.0

715.4

55.1

899.1

87.2

4 3 3 6 19 5 7 14 12

maximal. In other words, GTP addition may either leave unchanged or increase the Vmax of cyclase activation by hormones, depending on whether the maximal adenylate cyclase response obtained is limited by the full activation of the enzyme itself or by the full occupancy of the receptors. Multi-hormonal control of adenylate cyclase

Before discussing the respective part played in the concentrating mechanism by the various nephron segments responsive to vasopressin, it is necessary to mention that other hormones were also observed to stimulate adenylate cyclase in some of

these segments. This situation is particularly striking for the medullary and cortical portions of rat thick ascending limbs. Thus, the enzyme present in CAL responded to PTH [10—12],

Values are means SEM calculated from several experiments. Abbreviations are: B, basal AC activity measured in the absence of glucagon [10, 11, 13], calcitonin [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.

falling in the physiological range, induced a statistically significant stimulation of adenylate cyclase in both assays. The different sensitivity of collecting tubules and thick as-

to /3 adrenergic agonists [14] and vasopressin (Table 1). The cyclase present in MAL was highly responsive to glucagon [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 whether the receptors specific for these different hormones and for vasopressin are present in the membranes of the same cells (in which case they should induce the same biological responses

cending limbs to vasopressin does not imply the presence of as vasopressin does), or are segregated in different cells (in two kinds of receptor with a different affinity for the hormone in the two segments. It might result from a more efficient coupling

which case the response might be different). One way to answer

this question was to investigate whether the maximal cyclase reaction between occupied receptors and adenylate cyclase activation elicited in these two nephron portions by each units in CCT and MCT cells than in MAL and CAL cells. If, for hormone separately is additive when the hormones are tested in

example, the number of V2 receptors to AVP per cyclase combination. Glucagon, calcitonin and PTH maximal effects moiety were greater in CCT than in CAL cell membranes, a were observed to be non-additive in CAL [5, 10, 11], indicating given hormone concentration, although resulting in a similar fractional occupancy of the receptors in both structures, would induce a higher fractional cyclase activation in CCT where the number of receptors is assumed to be proportionally greater. Such a situation might also explain why the Vmax of the cyclase response to AVP was increased by GTP in CAL but not

that these three hormones stimulate a common pool of adenyl-

ate cyclase in the structure (Fig. 2). Full additivity was obtained, as expected, when low hormonal concentrations were used [11]. In these experiments, however, additivity of effects

could not be tested for vasopressin in the presence of these hormones, because vasopressin alone induces only a limited

in CCI (Fig. 1): in the absence of GTP addition, full receptor maximal cyclase activation in CAL without GTP addition [12]. occupancy (due to high AVP concentrations) may have resulted When tested in MAL samples, on the other hand, the effects of in maximal cyclase activation in CCT, but only in partial glucagon 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 already

segment [11].

514

Morel et a! CAL

examples of such experiments for CAL and MAL respectively

* — —

0

+

E 1000-

I0 (0

+

I0 +

6)

E

0 > 4

500 -

0

0 2

L1I

4 C)

0

I

(unpublished data from Imbert—Teboul and Siaume—Perez). The following points are clearly demonstrated by these experiments: 1) when the maximal response of adenylate cyclase to a given hormone was measured with or without GTP addition in homologous tubular samples isolated from the same rat kidney,

the response was increased to a large extent for the poorly active hormones, that is, vasopressin and calcitonin in CAL and calcitonin in MAL. The response was only slightly increased or not affected for the most active hormones (glucagon and PTH in CAL, glucagon and vasopressin in MAL). 2) In the presence of

added GTP, all hormones, except AVP in CAL, tended to produce maximal cyclase stimulation of a similar magnitude (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-

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 horhormones were tested in combination, full additivity of effects mone, glucagon and calcitonin on AC activity in cortical thick ascending limbs (CAL) of the rat. Results of an experiment in which AC was not observed with AVP and calcitonin in MAL; moreover, B

SCT

GLU

PTH

AVP

activity contained in CAL samples from the same rat kidney was measured either in the absence (open bars) or the presence of iO- M

the response of the CAL cyclase to PTH + glucagon +

calcitonin was not further increased in the presence of AVP. 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 parathyroid hormone (synthetic 1-34 fragment), 10 U ml1; CAL, respectively; consequently, they should elicit the same AVP, arginine vasopressin, 10-6 M. Each bar 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 MAL tubules isolated from the rat outer medulla, it was also lished data). added GTP (dashed bars) under basal conditions (B) and in the presence

observed that the effects of 10-6 M AVP (114.7

fmol• mm' 4 min1 (N = NS

0 500

a

E

0

0 2

4

JI

9.4

and of 10 ng . ml' salmon

8.4 fmol . mm' 4 min' (N = 7)) were calcitonin (82.8 non-additive (116 7.1, N 7) when the two hormones were tested in combination (Chabardès and Montégut, unpublished data).

MAL

1000 -

NS

8))

-j 0 + 0 > 4

/. // /,/;

Physiological effects of vasopressin in its responsive nephron segments

The results given in Table 1 indicate that the thin ascending limb (TAL), the thick ascending limb or diluting segment (MAL + CAL), the distal convoluted tubule (DCT) and the collecting

tubule (CCT + MCT) represent the target segments for vasopressin in the rat nephron. Measurement of adenylate cyclase 0activity is obviously not an appropriate method in order to B AVP GLU SCT study the biological action induced by vasopressin in these segments since, as mentioned before, the nature of the biolog3. Absence of additive effects of vasopressin, calcitonin and Fig. glucagon on AC activity in medullary thick ascending limbs (MAL) of ical 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 M added Other approaches are required, therefore, in order to investiGTP. Hormone abbreviations and concentrations as indicated in the gate this problem. C)

legend to Fig. 2 (Imbert—Teboul and Siaume—Perez, unpublished data).

Thin ascending limb

There are no data available in the literature as regards an Therefore, additional experiments including the addition of GTP to the incubation solution were performed in order to action 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 a permeability of this segment to water [15]. A possible effect of low response without GTP addition (namely AVP in CAL and AVP on the permeability to sodium and/or to urea should be calcitonin in MAL); and b) if all the hormonal effects become analyzed in microperfused TAL, since the permeability of the non-additive under such conditions. Figures 2 and 3 give thin ascending segment to these solutes is considered to play a

515

Renal receptors to AVP and other hormones

Thick ascending limb

TAL 150

Antidiuretic hormone is known to produce two effects in amphibian skin and urinary bladder: it increases the osmotic

100

permeability to water and it stimulates active sodium transport. In the frog kidney, the hormone also stimulates both water and sodium reabsorption [19]. In the mammalian kidney, the diluting segment (thick ascending limb) and the segment where ADH controls water reabsorption (collecting tubule) are clearly separated from each other. In view of these anatomical differences, it was suggested years ago that ADH might stimulate both salt and water reabsorption in the kidney of mammals, each effect

E

0)

E 0

4

being segregated in a different nephron portion, that is, the 50

collecting tubule for the effect on water and the thick ascending limb for the effect on active sodium transport [20]. The observation that vasopressin actually stimulated adenylate cyclase in

0

___________ —7

—6

AVP concentration

Fig. 4. Sensitivity to vasopressin of AC activity contained in thin ascending limbs (TAL) isolated from the rat inner medulla. In this experiment, adenylate cyclase activity was measured in the absence (basal, B) and in the presence of increasing vasopressin (AVP) concen-

trations on single pieces of TAL microdissected from the same rat kidney. Each point corresponds to the mean value of 4 to 5 replicate samples. A statistically significant activation of AC was already obtained with 10-10 M AVP. The half maximal response corresponded to

6. l0 M AVP in this experiment, assuming that l0 M AVP elicited the maximal response (redrawn from [5]).

both of these nephron portions provided some support to this hypothesis [3]. Finally, direct evidence that ADH may stimulate active salt transport by the diluting segment in mammals was obtained when Hall and Varney showed, in microperfused, medullary thick ascending limbs isolated from mouse kidneys, that vasopressin increases chloride transport without altering

the permeability to water [211. These aspects will not be discussed here since they are covered in detail in another chapter in this special issue (de Rouffignac et a!, this issue). Let us simply mention two expectations of physiological interest,

which result from the above described properties of the hormone—dependent adenylate cyclase in MAL and CAL. First, the ADH effect on ion transport by the diluting segment should require (to occur in vivo) higher hormonal concentrations in blood plasma than the antidiuretic effect in collecting tubules, since adenylate cyclase is less sensitive to vasopressin

major role in the concentrating mechanism along the inner in 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 proincreased adenylate cyclase activity more than 15-fold in rat duce, in the rat thick ascending limb, the same effects on ion TAL, whereas it was completely devoid of action in descending transport as vasopressin does [22, 23], since the maximal limbs (TDL) from the same animals (Table 1). As shown in stimulations of adenylate cyclase induced by these different Figure 4, the sensitivity of the cyclase to AVP was similar in the hormones in MAL and CAL respectively were not additive in rat TAL (KA = 6 x l0 M, Fig. 4) to that measured in MAL vitro. The cogency of these arguments is considered in the (average value: 6 x io, N = 6 [3]). The thin ascending limb in contribution by de Rouffignac et al. the rabbit was also responsive to AVP [17]; the average values SEM (fmol . mm' . 30 min') were respectively: controls 9.3 l.9(N = 9) versus AVP (10—6 M), 82.6 5.8 (N = 12) in TAL, and, control, 9.5 2.3 (N = 7) versus AVP (10—6 M), 12.0 1.8 (N = 8) in TDL. Adenylate cyclase activity was measured in the presence of AVP in three adjacent samples (TDL, TAL and MAL) microdissected from the same rabbit long loop with the following results (fmol . mm . 30 mm . '): TDL, 2.9; TAL,

Distal convoluted tubule Distal convoluted tubules exhibit a great axial heterogeneity in cell type composition in the rat. Moreover, they are difficult

to isolate over their full length in this species, even when microdissected from collagenase treated kidneys. Finally, marked species differences were noted in adenylate cyclase

responsiveness to vasopressin along the successive distal con113; MAL, 142 [17]. Such an observation suggests that the volutions between the mouse [24], rabbit [4, 24, 25], rat [4, 25] change in response to the hormone takes place close to the and human kidneys [26]. For these reasons, it is premature to hairpin turn of the loop. Thus, the transition between the speculate about the possible action(s) of vasopressin along this descending and ascending thin portions of long loops, which is segment from the data obtained by measuring cyclase activity.

present at (or near) their turning point, concerns not only ultrastructural organization and permeability properties, but responsiveness to hormones as well, a situation that Homer Smith claimed to dislike [18]. In our opinion, these different results suggest that vasopressin exerts in thin ascending limbs, via V2 receptors and cAMP, effects of physiological relevance which require further investigation.

Collecting tubule

The antidiuretic action of vasopressin along collecting tubules results from adenylate cyclase activation via V2 receptors is so well established that it does not require additional comment. The modulation of this action by various regulators, on the other hand, is discussed later on in this contribution. Let us mention here that the adenylate cyclase activity contained in rat

516

Morel et 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 calcitonin given in combination were observed to be partly additive or non-additive, depending on the experiment [11]. When GTP was added to the incubation solution, the Vmax response induced by calcitonin was clearly enhanced, so that the absence of additivity of effects of the two hormones was clearly confirmed under such conditions (Imbert—Teboul et al, unpublished data). Such an observation suggests that AVP and calcitonin stimulate the same pool of adenylate cyclase in the

C

E

0 E E

0 E

0 0

-1

500

II.

LLL

rat CCT. Therefore, both hormones should induce the same Rat Mouse Jerboa Rabbit Man biological response in this segment, namely an increase in 5. Responsiveness to vasopressin of adenylate cyclase (AC) presosmotic permeability to water. Note that calcitonin did not Fig. ent in medullary thick ascending limbs from different mammalian stimulate the enzyme contained in medullary collecting tubules

(MCT) [4, 25]. According to these data, injection of salmon calcitonin to rats undergoing water diuresis or to diabetes insipidus (DI) Brattleboro rats should increase urine osmotic pressure up to isoosmolality (but not to hyperosmolality as vasopressin does). This aspect is considered in another contribution (de Rouffignac et al, this issue).

species. AC responses to 1O M vasopressin are expressed here as cAMP formed (stimulated—basal AC activity); dashed bars are responses measured in medullary thick ascending limbs (MAL); open bars are responses measured in adjacent medullary collecting tubules

(MCT). Note that the AC response of MCT to vasopressin was of similar magnitude in all species studied, except the jerboa. By contrast, the responses measured in MAL varied over a wide range, depending 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 thereThe 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 certain MCT to AVP was almost identical in five among the six species nephron portions [25]. We focus here on the main segments studied, 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 segments Jerboa (Jaculus orientalis), which exhibited a relatively poor response in MCT (300 fmol . mm 30 min'). On the other (CCT and MCT). As regards glucagon (porcine hormone), adenylate cyclase is hand, the MAL response to 10-6 M AVP varied greatly among nearly unresponsive to this hormone in the rabbit, whereas the species: 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 stimulates to 1000 fmol mm1 . 30 min1). In the other species, the MAL adenylate cyclase activity to a greater extent in CAL than MAL response decreased in the following order (fmol . mm 30 in the rat [11, 12], but to a greater extent in MAL than in CAL min'): mouse (about 300), jerboa (about 200), rabbit (about in the rabbit [27]. The hormone is equally active in MAL and 100), and human (no response). Note that the lack of AVP effect CAL of the human nephron [26]. In the mouse, salmon in the human MAL is probably not a technical artifact due to calcitonin is inactive in MAL and only poorly active in CAL kidney storage, since, under the same experimental conditions: [281. PTFI, finally, stimulates the enzyme in CAL but not in a) 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 nephron portions [25, 26]. In accordance with this difference in responsiveness between MAL and CAL enzyme, PTH stimulates calcium (and magnesium) reabsorption in microperfused rabbit CAL [29], but not in MAL [30]. On the other hand, calcitonin increases calcium reabsorption in microperfused rabbit MAL [31]. However, the physiological meaning of these species differences in hormone responsiveness of the cyclase between CAL and MAL is unknown. It is also not established in all species whether the different hormone receptors in these segments are coupled to a common pool of enzyme as they are in the rat. Species differences were also noted regarding the responsiveness of MAL adenylate cyclase to vasopressin. Figure 5 illus-

sensitive to vasopressin as MCT samples isolated from freshly prepared rat kidneys for example. Finally, Ruggles et al [32]

confirmed recently the absence of AVP effect on adenylate cyclase activity in MAL samples microdissected from freshly excised human kidney tissue. Examination of Figure 5 suggests the existence of a correlation between MAL responsiveness to vasopressin and the concentrating ability of the kidney. Thus, hamsters and rats are known to concentrate urine up to 3500 mOsm per liter or more during dehydration, whereas rabbits (about 2000 mOsm per liter), and, especially, humans (about 1300 mOsm per liter) have

a relatively poor concentrating capacity, even during water deprivation. By increasing active salt reabsorption across the water—impermeable, epithelial cell layer of thick ascending trates the increase in adenylate cyclase per mm of tubule limbs, vasopressin might contribute to the accumulation of induced by 106 M AVP in medullary thick ascending limbs solutes in the medullary interstitium and thereby might allow (MAL) and outer medullary collecting tubules (MCT) isolated very high medullary gradients of osmotic pressure to be created from the kidneys of six different species. Although large species when required. Such a role of vasopressin in the concentrating

517

Renal receptors to AVP and other hormones A Brattleboro DI rat

mechanism was suggested for the rat kidney by Thomas and associates [33, 34] several years ago. If this view were correct,

DI tDI

the jerboa—which is a highly—concentrating true desert rodent species—would constitute a remarkable exception, because the

adenylate cyclase response to AVP was rather low in this species, both in MAL and MCT samples (Fig. 5). Whether this poor responsiveness may represent some "down—regulation" due to high AVP concentrations in blood plasma is discussed in the next section.

(0

DI tDI

100

0 0 0 U 0 0

0a)

C Jerboa

ti1 50-

Does ADH level in blood plasma regulate adenylate cyclase responsiveness in the kidney?

It has been established for many hormones that the number of free receptors in target cell membranes, as well as the stimulation of adenylate cyclase activity, coupled to the occupancy of

0MCI

MAL

MCI MAL

MCI MAL

these receptors may be reduced when the tissue has been Fig. 6. Relationship between in vivo ADH levels in blood plasma and vitro AC responsiveness to A VP of medullary collecting tubules previously exposed, either in vivo or in vitro, to a high in (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—regulation

or desensitization, results from an increased rate of receptor internalization when the hormone concentration has been greatly enhanced, that is, when the fractional occupancy of

from the kidneys of the treated animals are expressed as per cent of the corresponding responses measured in homologous samples from the

kidney of control animals studied in the same experiments. A. DI, Brattleboro rats with diabetes insipidus; tDI, DI rats microperfused for 6 to 8 weeks with physiological doses of dDAVP; normal Long Evans rats were used as controls in these experiments (data recalculated from

receptors is high. Rajerison, Butlen and Jard [35] showed in the 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 reduced pitressin (1 U) 20 mm before the experiment. Wistar rats injected with solvant were used as controls in this type of experiment. From [361. after the injection or perfusion of pharmacological doses of the C. Two groups of5jerboas (Jaculus orientalis) received for 4 to 7 weeks

AVP to the animals. The degree of desensitization obtained was

dose and time dependent. Experiments using microdissected tubules were performed under experimental conditions producing 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 target segments to AVP in the medulla, namely, the MAL and MCT

before the experiments either a dry diet (controls) or the same diet supplemented with a large supply of water in the form of lettuce and carrots (experimental animals). Data recalculated from [7].

data indicate that the presence of a normal level of ADH in blood 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 cyclase and functional development of the structure as a whole, as response in both segments of the AVP-injected rat compared to previously 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] observed why long—term treatment of Brattleboro DI rats with ADH is that the response of AC to AVP was also diminished in the necessary to fully restore the concentrating ability [43]. They

medullary membranes prepared from Brattleboro rats with demonstrate, therefore, that AVP plays a major role in the

diabetes insipidus (DI) when compared to those prepared from mechanism of urine concentration via an action exerted on the Long Evans control rats, in confirmation of the results of medullary thick ascending limbs. The molecular mechanism Dousa, Hui and Barnes [37]. When the cyclase response to whereby AVP produces a long—term effect on MAL developAVP was measured in microdissected MAL and MCT samples ment is not known. separately, however, a clear dissociation of effects was obWe mentioned before that the responsiveness of adenylate served [38]: a 50 percent decrease in Vmax (without change in cyclase was relatively low in both MCT and MAL from the KA) was obtained in MAL from DI rats compared to MAL from jerboa. It has been reported that freshly captured jerboas, or controls, whereas the Vrnax of the MCT samples were similar in jerboas fed a dry diet, have extremely high ADH concentrations DI and control rats (Fig. 6A). When DI Brattleboro rats in their blood plasma (in the range of several hundred picograms received low doses of AVP from a minipump for six to eight per ml) [44], Experiments were performed using jerboas fed for weeks, the responsiveness of the MAL cyclase to AVP was several weeks a water—deprived diet, or the same diet supplerestored to a large extent, whereas no effect was measured in mented with a large amount of water in the form of fresh MCT (Fig. 6A). This chronic treatment of DI Brattleboro rats lettuce, to establish if such high concentrations of ADH may with AVP was observed in MAL to increase not only the AC have 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-ATPase excreted a highly hypertonic urine (3800 mOsm . and their activity (by nearly 60%) and, finally, the tubular volume itself blood plasma contained an exceedingly high ADH concentra(by about 65%) [39]. All these stimulatory effects were limited tion (372 pg. ml' as an average). The animals fed the to the medullary portion (MAL) of thick ascending limbs. These water—enriched diet were diuretic (average urine osmotic pres-

l)

Morel et a!

518

sure, 650 mOsm 1'), and the plasma ADH concentration (76 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 to AVP were both increased in the hydrated jerboas compared

___ AVP

MCT

50

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, 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 values with 10 M AVP were 221 12 fmol. mm' . 30 min' (N = 35) vs. 158 13 (N = 33), P <0.001, in the MAL samples

from hydrated and dehydrated jerboas, respectively). This diet—induced alteration in MAL responsiveness was AVPspecific, since the MAL response to glucagon (10-6 M), measured in the same animals, varied in opposite direction: it was greater in the dehydrated (365 52 fmol . mm' . 30 min 1) than in the hydrated jerboas (231 22 fmol . mm' . 30 min'). A vasopressin—specific desensitization of the AVP receptors was also noted in the liver cell membranes from the jerboas fed the water—deprived diet [45]. Altogether, these data suggest that some down—regulation of the number of receptors to vasopressin occurs physiologically in the jerboa, in particular when the water supply is restricted, because AVP concentration in blood plasma appears to be unusually high in this species. Note that the kidney retains a full capacity to concentrate urine under these conditions. The results of Figure 6C suggest that MAL would be more sensitive than MCT to such a down—regulation. Furthermore, down—regulation alone is not sufficient to account for another observation in this species [7]: in response to acute

* Q

AVP

SCT

GLU

+ ISO

50

E E

*

0NE —±

—+

—

CLO

Fig. 7. Effect of a2-adrenergic agonists on the hormone—dependent

production of cAMP by single pieces of medulla,y (MCT) or cortical collecting tubule (CCT) microdissected from rat kidneys. Intracellular cAMP generated from endogenous ATP in the presence of IBMX was measured by radioimmunoassay as described in [91. Each bar corresponds to the mean value SE of 6 to 8 replicate samples. Norepinephrifle (NE, io M) was used as a2 adrenergic agonist (in the presence of 10 M propranolol) in all conditions, except for testing the inhibitory effect of a2-agonists on the response induced by isoproterenol in CCT.

In this case, propranolol was omitted and the specific a2 agonist, clonidine (Clo, l0 si) was used. Yohimbine (Yo, 10 M) was used as a2 blocker and Prazosin (PR, i0 M) as a1 blocker in the experiments shown in the upper pannels. Arginine—vasopressin (AVP, 10b0 M), salmon 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 a these experiments. In the absence of added hormone, the basal produc-

delayed water diuresis characterized by the excretion of hypoosmotic urine (average osmotic pressure, 157 6 mOsm- 1I, 6 animals). The average AVP concentration in the blood plasma 8.4 sample collected immediately afterwards was 41.5 in these six animals. The production of dilute urine by pg the kidney in spite of the presence of such huge ADH concentrations in blood plasma suggests that some additional factor

ml

tion of cAMP was too low to be detected under the conditions of the microassay used (data redrawn from [91). Note (upper pannels) that norepinephrine almost completely suppressed the MCT response to vasopressin via a2-adrenergic receptors, When tested against the dif-

ferent hormones known to stimulate AC in CCT (bottom pannel), a2-adrenergic agonists inhibited specifically the response induced by vasopressin.

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 norepinephrine, through this mechanism inhibits almost completely the vasopressin— induced cAMP generation in collecting tubules isolated from Several factors modulate the biological response elicited by the rat [9]. The experiments depicted in the upper part of Figure AVP in different systems. Some of them, like gluco- and 7 show that the inhibition of AVP effects produced by norepimineralo-corticoids, for example, increase the hydro-osmotic nephrine was suppressed by blocking a2 receptors with effect of ADH [46, 471. Other agents, such as a2-adrenergic yohimbine, whereas blocking a1 receptors with prazosin had no agonists [481, prostaglandins [49], bradykinin [50], exert an effect. The inhibitory action of the alpha agonist, therefore, was mediated through receptors of the a2 subtype. The experiment inhibitory action. Among the mechanisms whereby regulatory agents might depicted 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 Ni produced in CCT by different hormones known to stimulate subunit of the GTP-dependent coupling protein complex; b) a adenylate 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 to calcitonin, glucagon and isoproterenol were not altered under the same conditions. This observation suggests that the cells alterations in cytosol—free calcium concentration. Catecholamines 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 Regulation of adenylate cyclase responsiveness at the transduction level

Renal receptors to AVP and other hormones

devoid of such receptors, as cortical collecting tubules are known to consist of more than one type of cell. In the case of calcitonin, however, this explanation cannot easily be reconciled with the absence of full additivity of AVP and SCT effects

in this nephron portion [11]. If the receptors specific for AVP and for calcitonin are coupled to a common pool of cyclase moities, that is, are present in the membranes of the same cells, then the adrenergic a2 receptors also contained in these membranes would inhibit selectively the adenylate cyclase activa-

tion mediated by vasopressin. Additional experiments are needed to solve this particular problem and, more generally, to establish if inhibition of vasopressin effects by a2 agonists is present: a) in collecting tubules in species other than the rat and b) in vasopressin—responsive nephron segments other than collecting tubules (as regarding rat MAL, a2 stimulation pro-

duced no inhibition of the response to AVP, according to Umemura et a! [51]).

Prostaglandins, mainly PGE2, have also been observed to blunt vasopressin effects by inhibiting cAMP production in kidney tubules, in particular rat papillary collecting tubules [52] or thick ascending limbs from mouse [53] and rat [54]. In conclusion, it appears clearly established that various

519

5. MOREL F, CI-IABARDES D, IMBERT—TEBOUL M: Distribution of

adenylate cyclase activity in the nephron, in Current Topics in Membranes and Transport, (vol 13), edited by BRONNER F, KLEINZELLER A. New York, Academic, 1980, pp. 415—426 6. IMBERT M, CHABARDES D, MONTGUT M, CLIQUE A, MOREL F:

Vasopressin—dependent adenylate cyclase in single segments of rabbit kidney tubule. Pflugers Arch 357:173—186, 1975

7. BADDOURI K, BUTLEN D, IMBERT—TEBOUL M, LE BOUFFANT F, MARCHETTI J, CHABARDES D, MOREL F: Plasma antidiuretic hor-

mone levels and kidney responsiveness to vasopressin in the Jerboa, Jaculus orientalis. Gen Comp Endocrinol 54:203—215, 1984 8. COOPER DMF: Bimodal regulation of adenylate cyclase. FEBS Lett 138:157—163, 1982 9. CHABARDES D, MONTEGUT M, IMBERT—TEBOUL M, MOREL F:

Inhibition of az-adrenergic agonists on AVP-induced cAMP accumulation in isolated collecting tubule of the rat kidney. Mol Cell Endocrinol 37:263—275, 1984

10. TORIKAI S. WANG MS, KLEIN KL, KUROKAwA K: Adenylate cyclase and cell cyclic AMP of rat cortical thick ascending limb of Henle. Kidney mt 20:649—654, 1981 11. MOREL F, CHABARDES D, IMBERT—TEBOUL M, LE BOUFFANT F,

HUS—CITHAREL A, MONTEGUT M: Multiple hormonal control of adenylate cyclase in distal segments of the rat kidney. Kidney mt 21 (suppi l1):S-55—S-62, 1982 12. IMBERT—TEBOUL M, CHABARDE5 D, CLIQUE A, MONTEGUT M,

MOREL F: Ontogenesis of hormone-dependent adenylate cyclase in isolated rat nephron segments. Am J Physiol 247:F316—F325, 1984

physiological agents and hormones may alter the response elicited by vasopressin in defined systems and under specific conditions. To what extent these results apply to other vasopressin-sensitive systems or to other species, and may be

13. BAILLY C, IMBERT—TEBOUL M, CHABARDE5 D, HUS—CITHAREL A,

extrapolated to the conditions prevailing in the kidney in vivo is

amines on adenylate cyclase activity along the rat nephron. (ab-

MONTEGUT M, CLIQUE A, MOREL F: The distal nephron of rat

kidney: A target site for glucagon. Proc Nat! Acad Sci USA 77:3422—3424, 1980 14. MOREL F, CHABARDE5 D, IMBERT—TEBOUL M: Effect of catechol-

stract) Upsala J Med Sd (suppl) 26:53, 1979 far from established. As a consequence, the exact role played by adrenergic agonists and prostaglandins in the overall func- 15. IMAI M, KOKKO JP: Sodium choride, urea and water transport in

tion of urine concentration and dilution by the kidney is difficult to evaluate.

On the other hand, the mechanisms through which other hormones, like aldosterone, glucocorticoids, and thyroid hormones, exert their well established role on the urinary concentrating mechanism are still poorly understood at the cellular level, even though some aspects have been recently clarified, for example the exact nephron portions in which these hormones regulate Na-K-ATPase activity. Acknowledgments This work was supported by a grant of CNRS to LA 219. We are greatly indebted to S. Siaume—Perez for her technical assistance.

Reprint requests to F. Morel, M.D., Laboratoire de Physiologie

the thin ascending limb of Henle. Generation of osmotic gradients by passive diffusion of solutes. J Clin Invest 53:393—402, 1974 16. KOKKO JP, RECTOR FC: Countercurrent multiplication system without active transport in inner medulla. Kidney mt 2:214—223, 1972

17. IMBERT M, CHABARDE5 D, MONTEGUT, CLIQUE A, MOREL F: Presence d'une adenyl—cyclase stimulée par Ia vasopressine dans Ia

branche ascendante des anses des néphrons du rein de Lapin. C R Acad Sci (Paris) 280:2129—2132, 1975 18. SMITH HW: The fate of sodium and water in the renal tubules. Proc NYAcad Med 35:293—316, 1959 19. JARD 5, MOREL F: Actions of vasotocin and some of its analogues on salt and water excretion by the frog. Am J Physiol 204:222—226, 1963

20. MOREL F: Action of neurohypophyseal hormones on the active transport of sodium, in Water and Electrolyte Metabolism II, edited by DE GRAEFF J, LEINSE B. Amsterdam, Elsevier Publishing Co. 1964, pp. 91—104.

cellulaire, College de France, ii, Pce. Marcelin Berthelot, 75231 Paris Cedex 05, France.

21. HALL DA, VARNEY D: Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle's loop. J Clin Invest 66:792—802, 1979

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