Effects of in Vivo Treatment with Vasopressin and Analogues on Renal Adenylate Cyclase Responsiveness to Vasopressin Stimulation in Vitro* RABARY M. RAJERISON, DANIEL BUTLEN, AND SERGE JARD Laboratoire de Physiologie Cellulaire, College de France 11, Place Marcelin Berthelot, 75231Paris Cedex 05, France ate cyclase activation by vasopressin in vitro. Sodium chloride loading or chronic administration of DDAVP induced a 30% increase in enzyme responsiveness to vasopressin. The effect of the DDAVP injected was only apparent when the animals were killed for membrane preparation at least 40 h after the final injection. Administration of pharmacological doses of vasopressin or analogues induced rapid and reversible reduction in adenylate cyclase responsiveness to vasopressin stimulation in vitro. An almost complete loss in enzyme response was obtained within 1 h but was completely reversed 24 h after treatment. The modifications observed were specific for vasopressin-sensitive activity (stimulation of the enzyme by sodium fluoride or 5'-guanylyl-imidodiphosphate was maintained). When comparing the relative abilities of neurohypophysial peptides to induce a decrease in enzyme responsiveness to vasopressin, the following order of potency was observed: AVP > LVP > OT. The long acting vasopressin analogues, DC6-DAVP and DDAVP, were found to be more potent than AVP. (Endocrinology 101: 1, 1977)

ABSTRACT. Dose-dependent activation by vasopressin of renal adenylate cyclase was measured on medullo-papillary fractions obtained from rats which have an altered rate of endogeneous antidiuretic hormone (ADH) secretion. Rats were of the Brattleboro strain with hereditary hypothalamic diabetes insipidus and Wistar rats submitted to several treatments: water diuresis induced either by repeated water gastric loading of 7% of BW, or by drinking 5% glucose solution for two weeks; sodium chloride loading induced by giving 1.5% NaCl solution as a water source. Finally acute or chronic rises in blood antidiuretic activity up to pharmacological levels were obtained by im or iv administration of arginine-vasopressin (AVP) or other antidiuretic peptides, i.e., lysine-vasopressin (LVP), oxytoxin (OT) and the long acting vasopressin analogues, [l-deamino-8-D-arginine] vasopressin (DDAVP) and [6,l-/3-deamino-cystathionine-8D-arginine]vasopressin (DC6-DAVP). Reduction of ADH secretion in Brattleboro rats and in rats displaying induced water diuresis was accompanied by a 30% reduction of maximal adenyl-

R

(2) and rat pineal gland (3)), showed parallel

ECENT findings indicated that, for

several hormone target cells, the con- modifications of the number of receptors centration of specific receptors can be modi- and magnitude of maximal adenylate cyclase fied by manipulating the concentrations of activation. Dousa et al. (4) reported that in rats with homologous hormone to which these cells are exposed (for review, see Lesniak and diabetes insipidus of hypothalamic origin, Roth (1)). Depending on the system con- sensitivity to vasopressin of the medullosidered, an increase in the hormonal con- papillary adenylate cyclase was reduced, centration resulted either in a decrease compared to that obtained from normal rats (negative feedback regulation) or in an in- of the same hooded strain. Furthermore, crease (positive feedback regulation) of the chronic administration of pitressin to rats number of homologous hormonal receptors. with diabetes insipidus was found to restore Studies on receptors functionally coupled to normal sensitivity of renal adenylate cyclase membrane adenylate cyclase (j3 adrenergic to vasopressin stimulation in vitro (4). Thes. e receptors from the amphibian erythrocytes observations suggest that the responsiveness of renal adenylate cyclase to stimulation by vasopressin might also be regulated Received May 26, 1976. by the level of circulating vasopressin. * Supported by the Centre National de la Recherche In the present study, we investigated the Scientifique (R.C.P. 220 and LRA 219) and by grant effects of acute and chronic modifications of no. 73 31207 from the Delegation Generale a la blood antidiuretic activity on rat kidney Recherche Scientifique et Technique. 1

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

RAJERISON, BUTLEN AND JARD

adenylate cyclase. Vasopressin-dependent enzyme activity was measured on membranes obtained from rats submitted to treatments known to alter the endogenous ADH secretion rate. The effects of acute and chronic administration of AVP or analogues were also explored. It is shown that varying blood antidiuretic activity from very low to high levels resulted in a biphasic modification of maximal adenylate cyclase responsiveness to vasopressin. Materials and Methods Experimental procedure The experiments were performed on male rats (180-250 g) of the following types: Wistar rats from the Janvier Breeding Center (France), rats of the Brattleboro strain with hereditary hypothalamic diabetes insipidus (DI/DI) (5), normal rats from the same Long-Evans hooded strain (+/+), and heterozygous rats (+/DI). These latter rats were kindly donated by Dr. Mohring (University of Heidelberg). Mean ± SE urine outputs were 35.2 ± 5.0, 2.5 ± 0.3 and 3.3 ± 0.6 ml/100 g BW/day for DI/DI, +/+ and +/DI, respectively; the corresponding values for urine osmolality were: 110 ± 16, 2210 ± 90 and 2240 ± 40 mosmol/kg (6 determinations). All animals were given normal laboratory diet and free access to water, except when otherwise indicated. The rate of ADH secretion by the neurohypophysis was modified by the following treatments: 1) experimental water diuresis was induced either by gastric water load (7% of BW) every 6 h for 2 days, or by supplying 5% glucose solution for drinking at least 2 weeks (6). By the latter treatment, water intake was increased 4.8-fold in experimental rats, compared to the controls, and urine osmolality was reduced from 1550 ± 100 to 340 ± 40 mosmol/kg (33 determinations); 2) blood hypertonicity was induced by giving 1.5% NaCl as the drinking solution for the same period (7). Plasma osmolality in treated animals was 311 ± 3 as compared to 299 ± 2 mosmol/kg in control animals (the difference between the two groups was highly significant: t = 3.333, P < 0.01, 9 determinations). Acute modifications of blood antidiuretic activity were obtained by administering one of the

Endo * 1977 Vol 101 . No 1

following drugs: arginine-vasopressin (AVP), lysine-vasopressin (LVP), oxytocin (OT) or the long acting ADH analogues [l-deamino-8-Darginine]vasopressin (DDAVP) (8) and [6, 1-/3deamino-cystathionine-8-D-arginine] vasopressin (DC6-DAVP) (9). The peptides were administered either by im injection into conscious rats or by iv infusion (33.3 /tl/min for 1 h) into pentobarbital anesthetized rats (5 mg/100 g BW). The animals were killed for preparation of kidney membranes either at different intervals after the im injection (5 min to 16 h), or 1 h after the beginning of the iv infusion. Chronic modifications of blood antidiuretic activity were induced by a total of 18 im injections of the long acting DDAVP, administered twice daily. Animals were killed 24 or 40 h after the final injection. Experimental details are given in the legends to figures and table. Membrane fractions from the medullopapillary and cortical regions of the kidney were prepared as previously described (10,11). Adenylate cyclase activity of membrane fractions was measured by conversion of [a-32P]ATP into labeled cAMP. The incubation medium (60 fi\ final volume) contained Tris-HCl, 100 mM, pH 7.4; MgCl2, 0.75 mM; EDTA-Tris, 0.25 mM, pH 7.4; ATP, 0.25 mM; [a-32P]ATP, 0.65 /xCi; cAMP, 1 mM; creatine phosphate, 20 mM and creatine kinase, 100 fig. Activity was measured either under basal conditions or in the presence of the following stimulating agents: NaF, 10 mM; GPP(NH)P 10 fiM; LVP (1 nM to 10 pM) or PTH (60 nM to 20 /JLM). The reaction was started by the addition of enzyme (40 to 70 fig protein). It was allowed to proceed for 10 min at 37 C and stopped by addition of sodium dodecyl sulfate (2% final concentration). [32P]cAMP was separated from the mixture either by the method of Ramachandran and Lee (12) or that of Birnbaumer and Yang (13). Similar results were obtained by the two methods. Proteins were determined by method of Lowry et al. (14) using bovine serum albumin as a standard. When comparing basal or stimulated adenylate cyclase activities of membranes obtained from different animals submitted to the same treatment, it was observed (10) that fluctuations among individual determinations were minimal when the membranes were prepared in parallel during the course of the same experiment. Larger fluctuations were observed when comparing activi-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

VASOPRESSIN-SENSITIVE ADENYLATE CYCLASE ties determined on homologous membrane preparations obtained during the course of different independent experiments. Therefore, in order to minimize fluctuations each of the experiments to be described below included the experimental groups and the appropriate control one. Differences among paired data were tested using Student's t test (see legend to Fig. 1). Adenylate cyclase activities were usually expressed as per cent of control values.

100,-

Materials . [8-lysine]Vasopressin (LVP) (vasopressin chlorhydrate) and oxytocin (OT) (oxytocin chlorhydrate) were kindly donated by Dr. Boissonnas (Sandoz). [8-arginine]Vasopressin (AVP) was isolated from bovine neurophysin complex, as described by Prusik et al. (15), to whom we are grateful for supplying this compound. [1-deamino-8-D-arginine]Vasopressin (DDAVP) was synthetized according to the procedure of Zaoral et al. (8) and was the generous gift of Dr. Zaoral. [6, l-/3-deamino cystathionine-8-D-arginine]Vasopressin (DC6-DAVP) was prepared and kindly supplied by Dr. Jost (9). Other chemicals were obtained from the following sources: synthetic 1-34 sequence of bovine parathyroid hormone (1200 IU/mg) from Beckmann; Tris, EDTA and cAMP from Sigma; ATP (disodium salt) from B. L. Biochemical Inc.; creatine kinase, phosphocreatine (disodium salt) and 5'-guanylyl-imidodiphosphate (Gpp (NH)p) from Boehringer; neutral aluminum oxide (activity grade I, batch 183) from M. Woelm (Eschwege); Dowex AG 50 W-X8 from Bio Rad Laboratories; sodium dodecyl sulfate from Serlabo; bovine serum albumin, fraction V, from Pentex; [3H]cAMP (ammonium salt), 21 Ci/mmol from the Commissariat a l'Energie Atomique (Saclay, France); [a-32P]ATP (5-20 Ci/mmol) from New England Nuclear. Results

Figure 1 shows the results of the experiments using rats with hereditary hypothalamic diabetes insipidus of the Brattleboro strain, normal rats from the same LongEvans hooded strain and heterozygous rats. The apparent K values for medullo-papillary adenylate cyclase activation by LVP, i.e.,

-10

-9

-8

Vasopressin

-7

-6

-5

(log M)

FIG. 1. Dose-dependent activation by LVP of medullopapillary adenylate cyclases from Brattelboro (DI/DI), normal Long Evans (+/+) and heterozygous rats (+/DI). The results of 8 experiments are summarized. For each set, one Brattleboro and one heterozygous rat were compared to one normal Long Evans rat. All activities (mean ± SE) were expressed as a percentage of the maximal vasopressin-stimulated activity measured on the corresponding normal Long Evans preparation. The latter was 600 ± 88 pmol cAMP formed/10 min/mg protein (n = 8). Basal adenylate cyclase activities were 72 ± 10, 76 ± 10, 90 ± 15, and NaF-stimulated activities were 565 ± 125, 586 ± 121 and 607 ± 120 pmol cAMP formed/10 min/mg protein (n = 8) in Brattleboro, heterozygous and Long-Evans rats, respectively. The differences among the three groups were tested as follows: all individual values (increase in cAMP production due to LVP: pmol/10 min/mg protein) obtained with +/+ animals were plotted against the corresponding paired values from DI/DI or +/DI rats obtained during the course of the same experiment. The calculated linear regression lines were: DI/DI vs. +/+, y = 0.69 x -8.93 (r = 0.95, n = 79); +/DI vs. +/+, y = 0.78 x +5.81 (r = 0.98, n = 79). The slopes of these regression lines were different from 1 (P < 0.01). Similar treatment of NaF-stimulated activities did not show significant differences among groups.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

RAJERISON, BUTLEN AND JARD the hormonal concentrations leading to half maximal activation, were similar in all three groups (about 30 nM) as were the basal and fluoride-stimulated enzyme activities (see legend to Fig. 1). T h e magnitude of the response to maximal hormonal concentrations was reduced by 30% in DI/DI animals compared to normal rats. T h e doseresponse curve determined for + / D I rats

UO.-

120

5? 80 •>

"40 "8

f 20 9

-8 Vasopressin

-7

-6

dog M)

-5

FIG. 2. Effects of endogeneous ADH level modifications on medullo-papillary adenylate cyclase activity. Adenylyl cyclase activity in the presence of increasing amounts of LVP was measured on medullopapillary membrane preparations from: waterloaded rats ( • , n = 3); rats drinking 5% glucose for 2 weeks (O, n = 6); rats drinking 1.5% NaCl for 2 weeks (A, n = 6) and normal rats drinking water ad libitum used as control animals (O, n = 5). Values are expressed in % of control enzyme activity measured in the presence of 10 /tiM LVP. This activity was 524 ± 40 pmol cAMP/10 min/mg protein (mean ± SE, n = 5). For the same groups of animals, the corresponding NaF-stimulated activities expressed in pmol cAMP/10 min/mg protein were 703 ± 97 (water loaded rats), 686 ± 60 (rats drinking 5% glucose), 668 ± 19 (rats

Endo 1977 Vol 101 No 1

was between curves for DI/DI and +/+ animals. Similarly, a 20 to 25% decrease in the maximal response to LVP was observed when chronic water diuresis was induced in Wistar rats (Fig. 2). The effect of drinking 5% glucose solution was progressive with time; it was apparent after one week and reached its maximum after two weeks treatment. Conversely, in animals given 1.5% NaCl as water source for two weeks, the response to maximal concentration of LVP increased by 25% (Fig. 2). Under all experimental conditions, basal, fluoride-stimulated enzyme activities and apparent K for LVP were unchanged (see legend to Fig. 2). In vivo treatment of rats with the long acting ADH analogue DC6-DAVP (1 nmol/ 100 g BW administered by im injection) was followed by rapid changes in the properties of the medullo-papillary adenylate cyclase studied in vitro (Fig. 3). A sharp decrease in the activity stimulated by 5 /JLM LVP was observed during the 30 min that followed DC6-DAVP treatment. After l h this activity was identical to that measured without hormone. The loss in LVP responsiveness was slowly reversible: 16 h after injection, the maximal hormonal response was 80% of that measured in control animals. Adenylate cyclase activity measured without added LVP was increased by DC6-DAVP treatment and lowered to control levels within 4 h.

drinking 1.5% NaCl) and 727 ± 57 (control rats). The correlation test for paired data for LVP-stimulated activities (see legend to Fig. 1), gave the following results: water loaded vs. control, y = 0.81x - 2.42 (r = 0.99, n = 21); 5% glucose vs. control, y = 0.65x - 0.44 (r = 0.94, n = 42); 1.5% NaCl vs. control, y = 1.21x + 9.85 (r = 0.94, n = 42). The slopes of these regression lines were different from 1 (P < 0.01). Similar treatment of NaF-stimulated activities did not show significant differences among groups. In a parallel experiment, one rat from each group received an im injection of DC6-DAVP (1 nmol/lOOg BW) l h before membrane preparation. Compared to the normal non-treated animal, maximal enzyme activations by LVP were 3 (water load), 9 (5% glucose), 13 (1.5% NaCl) and 6% (normal) of the paired control value.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

VASOPRESSIN-SENSITIVE ADENYLATE CYCLASE Vasopressin added No Vasopressin added FIG. 3. Time-course of DC6DAVP-induced decrease in adenylate cyclase responsiveS100 ness to vasopressin. At the indicated time after im injection of 0.2 ml 0.9% NaCl solution only (controls), or 0.9% saline solution containing DC6-DAVP (1 nmol/100 g BW), rats were killed and membranes prepared for 50 50 adenylate cyclase assay. Enzyme activity was measured under basal conditions (left panel) or in the presence of 5 /i.M LVP (right panel). All values are expressed as a per cent of the vasopressin-stimulated activity obtained from control animals. 6 — L ^ Tim* 4 (hours) 6 Tim« (hours) The latter value was 814 ± 64 pmol cAMP/10 min/mg protein (mean ± SE, n = 18). Mean ± SE for basal adenylate cyclase activity of control preparations is indicated by the two parallel dotted lines. The graphs include individual values obtained during a single experiment (•, A) and means ± SE of a series of 9 different experiments. (^), J ) .

I I

Similar results were obtained when rats were injected im with pharmacological amounts of LVP (10 nmol/100 g BW) (Fig. 4). Maximal desensitization of adenylate cyclase was obtained 30 min after treatment and was more rapidly reversible than that obtained in DC6-DAVP treated animals. Desensitization of adenylate cyclase was also observed in anesthetized animals when vasopressin or analogues were administered by iv infusion (Fig. 5). The observed order

of potency for AVP, LVP and OT in inducing desensitization is that found when measuring either the antidiuretic activity of these peptides in vivo (16) or when measuring their ability to induce adenylate cyclase activation in vitro (10,11). It should, however, be noted that the amount of peptide needed to obtain desensitization of adenylate cyclase (an amount equivalent to 90 mlU antidiuretic activity is considerably larger than the amount eliciting a doseVasopressin added

No Vasopressin added

FiG. 4. Time-course of LVPinduced decrease in adenylate cyclase responsiveness to vasopressin. Results are from 6 experiments similar to those described in Fig. 3. Each experiment included 1 control animal im injected with vehicle only and 2 animals injected im with 10 nmol/100 g BW LVP in 0.2 ml 0.9% NaCl. Mean ± SE for 5 fiM LVP-stimulated activity in control animals was 594 ± 61 pmol cAMP/10 min/mg protein.

^100-

50

SI 50

Control

L Time

6 (hours)

Time

U

6 (hours)

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

RAJERISON, BUTLEN AND JARD

Endo • 1977 Vol 101 • No 1

FIG. 5. Dose-dependency for adenylate cyclase desensitiza100 tion by neurohypophysial peptides and analogues. Increasing amounts of AVP (A, A), LVP (•, O), OT (T, V), DC6-DAVP (•, O) or DDAVP (•, • ) were administered in 0.9% NaCl solution. The peptides were given either as 0.2 ml im injections (left 50 panel) or 2 ml iv infusions: 33.3 pil/min for 60 min (right panel). In the former case, animals were killed either 30 min (AVP, LVP, OT) or 60 min (DC6-DAVP) after injection. In the latter case, animals were killed at the end of the 60 min iv infusion period. These 30 min and 60 min 0 0.1 10.0 100.0 1.0 0.01 0.1 1.0 10.0 treatment periods were those for Peptide administered (nmol / TOOg body weight) which maximal loss in enzyme responsiveness was observed (see Fig. 3: DC6-DAVP and Fig. 4: LVP). In both cases, control rats were treated with saline solution only. Open symbols: basal adenylate cyclase activities; closed symbols: activities measured in the presence of 5 fiM LVP. Means ± SE (n = 4) for 5 /xM LVP-stimulated activities in control animals receiving vehicle only were: 579 ± 25 (im injection) and 449 ± 59 pmol cAMP/10 min/mg protein (iv infusion).

dependent antidiuretic effect in waterloaded rats (20-40 fxlU) (16). DC6-DAVP and DDAVP, which exhibit very high antidiuretic activity in vivo (8,9,17), were more potent than AVP in inducing desensitization of adenylate cyclase. Desensitization resulted from a decrease in the maximal response of adenylate cyclase to vasopressin whereas the apparent K value for the hormone was unchanged (Fig. 6 and Fig. 8, left panel). As indicated by Table 1, injection of DC6-DAVP (1 nmol/100 g BW) reduced enzyme responsiveness to vasopressin by 90%, whereas responsiveness to NaF was reduced by 31%. The responsiveness of cortical adenylate cyclase to bPTH 1-34 was not impaired by DC6-DAVP treatment (Fig. 7). Complete desensitization can be induced both in water-loaded animals and in those drinking 5% glucose solution or 1.5% NaCl solution (see legend to Fig. 2). The medullo-papillary enzyme from control and/or desensitized animals was also stimulated by Gpp(NH)p (Table 1). The nucleotide effect was greater in desensitized preparations than in control ones (Fig. 8). It can be noted that the sum of the activities

measured in the presence of maximal amounts of LVP on the one hand, and of Gpp(NH)p on the other, was almost constant.1 The loss in enzyme responsiveness to vasopressin was not due to the presence of a soluble inhibitor in the membrane fractions used for adenylate cyclase assay in vitro. Thus, when membrane preparations from desensitized and control animals were mixed, the response to vasopressin was that expected from their relative specific activities (Fig. 9). 1 Such reciprocal modifications of sensitivity to vasopressin and Gpp(NH)p were not observed in anesthetized and control animals. The magnitude of the nucleotide effect was similar in desensitized and control animals and approached the magnitude of the effect measured in desensitized, non-anesthetized animals. Preliminary experiments indicated that pentobarbital, when added in vitro to membrane preparations, enhanced the magnitude of adenylate cyclase activation by Gpp(NH)p. The pentobarbital effect was obtained in a concentration range (10 fiM100 fx,M) which is low enough to make it conceivable that circulating pentobarbital in anesthetized animals is responsible for complete unmasking of the nucleotide-sensitive activity, irrespective of the degree of desensitization of vasopressin-sensitive activity.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

VASOPRESSIN-SENSITIVE ADENYLATE CYCLASE The long-term effects of exogeneous antidiuretic peptide administration were tested by measuring the stimulation by LVP of medullo-papillary enzyme obtained from animals killed 24 or 40 h after the last of a total of 18 im injections of DDAVP (0.01 or 1 nmol/100 g BW) administered twice daily. As indicated by Fig. 10, chronic treatment with 1 nmol/100 g BW DDAVP enhanced by about 30% the maximal response to LVP of membrane adenylate cyclase obtained from animals killed 40 h after the last peptide injection. When tested 24 h after the final injection, the maximal enzyme response was found to be close to the control level. The difference between the two experimental groups might be accounted for by the incomplete reversal after 24 h of the desensitization induced by the final injection. It was observed that repeated injections of an antidiuretic peptide did not prevent transient desensitization of medullopapillary adenylate cyclase. Maximal activation by LVP of membranes obtained from animals given 1 nmol/100 g BW DC6-DAVP twice daily for three days and killed 90 min after the final injections was similar to that TABLE 1. Characteristics of medullo-papillary adenylate cyclases from control and DC6-DAVP desensitized rats

Adenylate cyclase activity (pmol cAMP formed/10 min/ mg protein) Control Basal activity 80 ± 6 Increase in activity due to: 361 ± 28 + 5 fiM LVP 392 ± 63 + 10 mM NaF + 10 fiM Gpp(NH)p 344 ± 25

Desensitized (9)

112 ± 11 (11)*

(9) 34 ± 7 (9) 271 ± 37 (11) (6) 533 ± 27 (8)*

Membranes were prepared from the medullo-papillary region of the kidneys from control rats (im injection of 0.2 ml 0.9% NaCl solution) and desensitized rats (im injection of DC6-DAVP, 1 nmol/100 g BW). The animals were killed 1 h after treatment. Adenylate cyclase activities were measured under the experimental conditions indicated. Values are means ± SE of basal adenylate cyclase activities and of increases in cAMP production due to the indicated amount of stimulating agents. The number of determinations are in brackets. * Statistically different from control value, P < 0.02.

-9

-8 -7 -6 Vasopressin (logM)

-5

FIG. 6. Dose-dependent activation by LVP of adenylate cyclases from normal and partially desensitized rats. Dose-dependent activation by LVP was determined on medullo-papillary adenylate cyclases from pentobarbital-anesthetized rats, infused for 1 h either with 2 ml 0.9% NaCl solution (•) or 2 ml saline containing DDAVP (100 pmol/lOOg BW) (O). All values are expressed as % of maximal activity measured on control preparations. Data on the graph are means ± SE of 5 experiments. The arrows indicate apparent K values for vasopressin in normal (continuous line, K = 36 ± 8 nM) and desensitized rats (dashed line, K = 29 ± 6 nM), respectively. These values are not statistically different. Maximal enzyme activity measured in the presence of 5 fiM LVP in control animals was 563 ± 90 pmol cAMP/10 min/mg protein (mean ± SE, n = 5). 10 mM NaF-stimulated activities (pmol cAMP/10 min/mg protein) were 1150 ± 325 and 850 ± 230 for control and desensitized rats, respectively. Corresponding values measured in the presence of 10 /iM Gpp(NH)p were 1090 ± 210 and 1090 ± 230. None of these paired values are .statistically different when tested with the method mentioned in the legend to Fig. 1.

measured in rats given a single peptide injection (10 and 6% of that measured in control animals injected with saline, respectively). Chronic administration of smaller amounts of DDAVP (0.01 nmol/100 g BW)

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

Endo • 1977 Vol 101 • No 1

RAJERISON, BUTLEN AND JARD CORTEX

MEDULLA

NaF 6pp(NH)p

600

Control

Trtattd

825

519

372

621

Control NaF

c

800

Gpp(NH)p

Trtattd

2S9

228

216

169

0)

^ a.500

600

01

c £ o

c 400 E 5 300

ot 400
600

6

o

(L

400

Desensitiztd

T .a

I 200 o Q.

0L -9

-8 -7 -6 Vasopressin dogM)

-5

FIG. 9. Effect of LVP on mixed membrane preparations from control and desensitized animals. Dosedependent enzyme activation was determined on each of the two preparations indicated on the graph as control and desensitized and on a 1/1 mixture of these two preparations. 109, 88 and 98.5 /xg protein were present for control, desensitized and mixed preparations, respectively. Assuming that the presence in the incubation medium of one membrane preparation does not interfere with the activity of the other, the activity of the mixed preparation was calculated from the specific activities of each of the two preparations measured independently. The calculated curve (dotted line) was found to be very close to the experimental curve obtained for the mixed preparation. The experiment was repeated twice and gave similar results.

-9

-8 -7 Vasopressin (tog M)

-6

FIG. 10. Effect of chronic treatment with a longacting AVP analogue. Four groups of rats were compared. Animals from the first group were given twice daily injections of 0.01 nmol/100 g BW DDAVP for 9 days. They were killed for membrane preparation 24 h after the final injection (O). Animals from the second and third groups were given a similar treatment except for the amount of DDAVP administered (1 nmol/100 g BW). They were killed 40 h (second group, • ) and 24 h (third group, A) after the last injection. Animals from the fourth group were controls receiving saline injections (O). The dose-dependent adenylate cyclase activation by LVP was determined on each membrane preparation. Values on the graph are means ± SE of 4 independent determinations. They are expressed in per cent of those obtained in the presence of 5 ju.M LVP in control animals (468 ± 9 pmol cAMP/10 min/mg protein; n = 4). The correlation test for paired data (see legend to Fig. 1), gave the following results: 0.01 nmol DDAVP 24 h vs. control, y = 1.41x - 42.0 (r = 0.99, n = 36); 1 nmol DDAVP 40 h vs. control, y = 1.23x + 53.4 (r = 0.99, n = 36); 1 nmol DDAVP 24 h vs. control, y = 1.08x - 45.4 (r = 0.99, n = 36). The slopes of the two first regression lines are different from unity (P < 0.01) whereas that of the third is not.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

10

RAJERISON, BUTLEN AND JARD

cose solution for drinking for two weeks led to similar modifications in the adenylate cyclase response to vasopressin. Conversely, drinking 1.5% NaCl increased adenylate cyclase responsiveness to vasopressin. It seems unlikely that those changes are due to reduction in aldosterone secretion associated with NaCl loading. Actually, previous studies (10) in the rat indicated that aldosterone was able to increase slightly the renal adenylate cyclase responsiveness to vasopressin stimulation. The effects of NaCl treatment are most probably related to an enhancement of vasopressin secretion due to the observed blood hypertonicity (see Materials and Methods). The observation that repeated injections of a long acting AVP analogue (DDAVP) increased enzyme responsiveness to vasopressin in normal rats supports such an interpretation. Similar effects following repeated pitressin injections in Brattleboro rats were recently reported by Dousa et al. (4). The above results are compatible with the hypothesis that, within the range of physiological modifications, the blood ADH level might exert a positive feedback regulation on renal adenylate cyclase responsiveness to vasopressin stimulation. On the other hand an increase in the circulating ADH level to concentrations far higher than the physiological values induced a rapid reduction in adenylate cyclase responsiveness to vasopressin whatever the physiological state of the animal. This loss in sensitivity was rapidly reversible as the injected hormone was cleared from the body fluids. The desensitization phenomenon can hardly be accounted for by non-specific alterations in the catalytic activity of adenylate cyclase. Thus, the desensitized enzyme remained responsive to stimulation by NaF or Gpp(NH)p. The reciprocal variations observed in sensitivities to nucleotide and hormone suggest that the interrelation between these two agents in the enzyme activation process is not impaired (18). In addition, cortical sensitivity to PTH was unchanged by in vivo ADH administration.

Endo • 1977 Vol 101 • No 1

In some of the experiments described above, dose-dependency for adenylate cyclase activation by vasopressin was determined using preparations obtained from animals killed shortly after in vivo administration of hormone or analogues at high doses. It must be noted that the amount of free hormone which might remain within the final enzyme fraction is very likely negligible on account of the considerable dilution of the tissue homogenate (about 2 x 107 times) involved in the experimental procedure used for membrane preparation. However the presence of hormone-receptor complexes formed in vivo in the membrane preparation is not excluded. Previous studies indicated that lowering the temperature to 4 C (temperature used for membrane preparation) strongly slows down dissociation of the hormone-receptor complex (halflife of the hormone-receptor complex is 3-6 h at 4 C compared to 6 min at 37 C (19)). Furthermore, the duration of the final incubation at 37 C is not sufficient to allow complete dissociation of the hormonereceptor complexes formed in vivo. This can support the observations that in animals killed shortly after administration of ADH, adenylate cyclase activity measured in vitro in the absence of added LVP is higher than that measured on control preparations (see Figs. 3 and 4). The change as a function of time after DC6-DAVP or LVP im injection (Figs. 3 and 4) of adenylate cyclase activity measured without added vasopressin most probably reflects a combination of residual stimulation of the enzyme by injected peptide and gradual desensitization. Conversely, the duration of the final incubation in vitro is sufficient to ensure complete saturation of the free receptors when LVP is present in the incubation medium at high concentration, so that the magnitude of maximal adenylate cyclase activation can very likely be considered as a correct estimate of the actual capacity of the system to respond to hormonal stimulation. In most experiments (see Figs. 6 and 8), the residual enzyme

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

VASOPRESSIN-SENSITIVE ADENYLATE CYCLASE activation due to partial receptor occupancy at the beginning of adenylate cyclase assay was small as compared to maximal activation induced by LVP added in vitro and therefore did not markedly interfere with determination of the apparent K value of adenylate cyclase for LVP. In fact the difference in apparent K values determined in partially desensitized and control animals fell within the range of experimental errors (Fig. 6). It seems unlikely that the desensitization phenomenon is triggered by the magnitude of the final antidiuretic effect of the ADH or analogues administered. In all cases, the amounts of peptide needed to induce desensitization are far higher than those needed to induce a maximal antidiuretic effect (16). However, it is not impossible that desensitization is in fact triggered by relative receptor occupancy if the so-called reserve receptor phenomenon is operative, i.e., if a maximal biological response is achieved by occupancy of a small fraction of the total number of ADH receptors. Several arguments indicate that this might be the case: 1) the dissociation constant KD (1 nM) for in vitro binding of AVP to its medullo-papillary receptors (10,11) is far higher than the mean concentration of circulating ADH (in the range of 0.01 nM) (20,21) and 2) the existence of spare receptors for ADH in amphibian bladder epithelial cells is well documented (22,23). In fact, by use of AVP to induce total desensitization, it was shown that the ADH blood concentration2 at the time the animal was killed (50 nM) was comparable to that which might have been required to ensure complete saturation of membrane ADH receptors in vitro (10,11). Similarly the relative abilities of LVP and OT to induce desensitization are well correlated with 1) their relative affinities for the mem2

In one experiment, almost complete desensittzation was induced by infusing AVP (67 pmol/100 g BW/min) iv for 1 h. The level of circulating AVP at the time the animal was killed for membrane preparation was determined by radioimmunoassay (24). It was found to be 50 nM.

11

brane ADH receptor (10,11), and 2) the relative activities in stimulating adenylate cyclase in vitro (10,11). DC6-DAVP and DDAVP were shown to be less effective than natural vasopressin in activating rat kidney adenylate cyclase (25). It is suggested that the high antidiuretic potency of these peptides is related to their enhanced metabolic stability in vivo (8,9,17). Therefore, one can expect that a high degree of receptor occupancy by these analogues can be obtained in vivo by administration of relatively smaller amounts of peptide. This could account for the high potency of DC6DAVP and DDAVP in inducing desensitization. One or more of the four following mechanisms might be responsible for the reduction in maximal enzyme responsiveness to vasopressin in vitro which was observed in desensitized animals: 1) presence in the membrane preparation of agents inhibiting hormonal binding and/or enzyme response; 2) occupation of the hormonal receptor by a slowly reversible antagonist; 3) reduction in the number of hormonal receptors; 4) decrease in the efficiency of hormone receptor/enzyme coupling. The first supposition appears unlikely, since the experiment reported in Fig. 9 shows that mixing membrane preparations from normal and desensitized animals does not impair the response of normal preparations to vasopressin. It is not yet possible to discriminate among the other three possible mechanisms. However, by analogy with the results obtained for other systems, (cf., review by Lesniak and Roth (1)) and studies on catecholaminesensitive adenylate cyclases from amphibian erythrocytes (2) or rat pineal gland (3), it might well be that the observed reduction in the maximal response of kidney adenylate cyclase to vasopressin stimulation reflects a loss of active ADH receptors and that the magnitude of the maximal enzyme response may be considered as a relative estimate of the number of hormonal receptors. From the experiments described by Fig. 4 one can estimate the half-time for maximal

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

RAJERISON, BUTLEN AND JARD

12

desensitization induced by vasopressin to be 20 min and the half-time for recovery to be 3 h. Such time constants make it unlikely that the desensitization and/or recovery might reflect changes in the population of vasopressin-responsive kidney cells. On the other hand, one cannot exclude that such a mechanism might be involved in the changes of enzyme responsiveness resulting from manipulation of the circulating endogenous hormone secretion rate. It must be noted, however, that a decrease in enzyme response to vasopressin can be demonstrated after 48 h water diuresis induced by repeated gastric water loads (Fig. 2). Taking into account the very high hormonal concentrations needed to induce a reduction in adenylate cyclase response, it is clear that the observed desensitization phenomenon is of little or no physiological or physiopathological significance, except perhaps for the syndrome of inappropriate ADH secretion (26) and when large amounts of vasopressin are administered. Finally, it is concluded that the vasopressin-sensitive adenylate cyclase from the rat kidney is under the influence of both apparent positive and negative feedback controls. The positive feedback regulation is operating in the range of physiological blood ADH levels, whereas the apparent negative feedback regulation can only be demonstrated when increasing blood ADH levels to highly pharmacological levels. Acknowledgments The authors are indebted to Dr. J. Marchetti for radioimmunoassay of blood arginine-vasopressin and to Miss M. Dreyfus for her help in the preparation of the manuscript. References 1. Lesniak, M. A., and J. Roth, J Biol Chem 251: 3720, 1976. 2. Mukherjee, C , M. G. Caron, and R. J. Lefkowitz, Endocrinology 99: 347, 1976.

Endo • 1977 Vol 101 • No 1

3. Kebabian, J. W., M. Zatz, J. A. Romero, and J. Axelrod, Proc Natl Acad Sci USA 72: 3735, 1975. 4. Dousa, T. P., Y. F. S. Hui, and L. D. Barnes, Endocrinology 97: 802, 1975. 5. Valtin, H., and H. A. Schroeder, Am J Physiol 206: 425, 1964. 6. Andriole, V. T., and F. H. Epstein,/ Clin Invest 44: 73, 1965. 7. Jones, C. W., and B. T. Pickering, J Physiol (Lond) 203: 449, 1969. 8. Zaoral, M., J. Kolc, and F. Sorm, Coll Czech Chem Commun 32: 1250, 1967. 9. Jost, K., Z. Prochazka, J. H. Cort, T. Barth, J. Skopkova, Z. Prusfk, and F. Sorm, Coll Czech Chem Commun 39: 2835, 1974. 10. Rajerison, R. M., J. Marchetti, C. Roy, J. Bockaert, and S. Jard,/ Biol Chem 249: 6390, 1974. 11. Rajerison, R. M., D. Butlen, and S. Jard, Mol Cell Endocrinol 4: 271, 1976. 12. Ramachandran, J., and V. Lee, Biochem Biophys Res Commun 41: 358, 1970. 13. Birnbaumer, L., and P. Ch. YangJ Biol Chem 249: 7848, 1974. 14. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. RandallJ Biol Chem 193: 265, 1951. 15. Prusfk, Z., E. Sedlahova, and T. Barth, Hoppe Seylers Z Physiol Chem 353: 1837, 1972. 16. Sturmer, E., In Berde, B. (ed.), Handbiich der Experimentellen Pharmakologie, vol. XXIII, Springer Verlag, Berlin, 1968, p. 130. 17. Vavra, I., A. Machova, and I. Krejci, J Pharmacol Exp Ther 188: 241, 1974. 18. Rodbell, M., M. C. Lin, Y. Salomon, C. Londos, J. P. Hardwood, B. R. Martin, M. Rendell, and M. Berman, In Drummond, G. I., P. Greengard, and G. A. Robison (eds.), Advances in Cyclic Nucleotide Research, vol. 5, Raven Press, New York, 1975, p. 3. 19. Roy, C., R. M. Rajerison, J. Bockaert, and S. Jard, / Biol Chem 250: 7885, 1975. 20. Little, J. B., and E. P. Radford,/ Appl Physiol 19: 179, 1964. 21. Tata, P. S., and O. H. Gauer, Pfliigers Arch 290: 279, 1966. 22. Eggena, P., I. L. Schwartz, and R. Walter,/ Gen Physiol 56: 250, 1970. 23. Roy, C., J. Bockaert, R. M. Rajerison, and S. Jard, FEBS Lett 30: 329, 1973. 24. Marchetti, J., Experientia 29: 351, 1973. 25. Barth, T., R. M. Rajerison, C. Roy, and S. Jard, Mol Cell Endocrinol 2: 81, 1975. 26. Bartter, F. C., In Berson, S. A., and R. S. Yalow (eds.), Methods in Investigative and Diagnostic Endocrinology, vol. 2A, North-Holland, Amsterdam, 1973, p. 672.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 22:14 For personal use only. No other uses without permission. . All rights reserved.

Effects of in vivo treatment with vasopressin and analogues on renal adenylate cyclase responsiveness to vasopressin stimulation in vitro.

Effects of in Vivo Treatment with Vasopressin and Analogues on Renal Adenylate Cyclase Responsiveness to Vasopressin Stimulation in Vitro* RABARY M. R...
1MB Sizes 0 Downloads 0 Views