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Am J Physiol Renal Physiol 278: F799-F808, 2000;
0363-6127/00 $5.00
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Chlorpropamide upregulates antidiuretic hormone receptors and unmasks constitutive receptor signaling

Jacques A. Durr1,2, Johannes Hensen3, Tobias Ehnis4
Mary S. Blankenship5
(With the Technical Assistance of C. Klein)

1 Division of Nephrology, Department of Veterans Affairs Medical Center, Bay Pines 33744; 2 Department of Medicine, University of South Florida College of Medicine, Tampa 33612; 3 Klinikum Hannover Nordstadt, Medizinische Klinik, Hannover 30167; 4 Department of Medicine IV, University of Erlangen-Nuernberg, Erlangen 8520, Germany; and 5 Medical Research and Development, Department of Veterans Affairs Medical Center, Bay Pines, Florida 33744


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

The mechanism by which chlorpropamide (CP) treatment promotes antidiuresis is unknown. CP competitively inhibited antidiuretic hormone (ADH) binding and adenylyl cyclase (AC) stimulation (inhibition constants Ki and K'i of 2.8 mM and 250 µM, respectively) in the LLC-PK1 cell line. CP (333 µM) increased the apparent Ka of ADH for AC activation (0.31 vs. 0.08 nM) without affecting a maximal response, suggesting competitive antagonism. Because CP lowers "basal" AC activity and the AC activation-ADH receptor occupancy relationship (A-O plots), it is an ADH inverse agonist. Twenty-four-hour CP exposure (100 µM) upregulated the ADH receptors without affecting affinity. This lowered Ka and increased basal AC activity and maximal response (1.86 vs. 1.35 and 14.9 vs. 10.6 fmol cAMP · min-1 · 103 cells-1, n = 6, P < 0.05). NaCl, which potentiates ADH stimulation, also increased basal AC activity. This, together with the CP-ADH inverse agonism and increased basal AC activity at higher receptor density, unmasks constitutive receptor signaling. The CP-ADH inverse agonism explains receptor upregulation and predicts the need for residual ADH with functional isoreceptors for CP-mediated antidiuresis. This could be why CP ameliorates partial central diabetes insipidus but not nephrogenic diabetes insipidus.

vasopressin; antidiuretic hormone; chlorpropamide; adenylate cyclase; receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

THE MECHANISM BY WHICH CHLORPROPAMIDE (CP) potentiates antidiuretic hormone (ADH) and ameliorates ADH-deficient diabetes insipidus (DI) has remained a mystery since its serendipitous discovery more than 30 years ago (1). Each element of the ADH-signaling cascade is indispensable for the antidiuretic effect of CP. First, CP is not an ADH ersatz, because some residual ADH is required (6, 34, 45, 48, 49, 56, 76). Second, CP also requires intact ADH receptors because it has no effect in X-linked nephrogenic DI (1), ADH-resistant polyuric states that result from mutations in the renal ADH isoreceptor gene (7, 44). Third, because exogenous cAMP fails to potentiate CP (34, 45), its site of action must be located before cAMP. Finally, because CP treatment potentiates the AC response to submaximal doses of ADH, but has no effect on postreceptor stimuli (52-54), CP may act via the ADH receptor itself.

Surprisingly, however, in vitro, CP behaves as a specific ADH antagonist because it inhibits adenylyl cyclase (AC) only when stimulated by ADH (3, 43, 54) but not by postreceptor stimuli (54). Consistent with an antagonist-induced receptor upregulation is our finding, utilizing a high-specific-activity ADH ligand (23, 24), that rats treated with CP had upregulated renal V2 ADH isoreceptors (32). Because these rats could not maximally dilute their urine even after a sustained 8-h water load, thus presumably after complete ADH suppression (32), we turned to the LLC-PK1 cell line. Indeed, the confounding effect of residual ADH that could escape sensitive radioimmunoassay detection is avoided in this model. This model also allows correlation of AC activity with ADH receptor occupancy. The present in vitro study reproduced our in vivo results that CP promotes ADH receptor upregulation (32). It also shows that CP meets the criteria for an inverse ADH agonist and suggests constitutive signaling activity of the renal V2 ADH receptors. ADH-independent constitutive receptor signaling activity was consistent with a higher basal AC activity associated with V2 receptor upregulation. The ADH-independent signaling activity of the ADH receptor explains why receptor upregulation impairs maximum diluting ability in the water-loaded rat (32). The hypothesis of hormone-independent signaling activity of the ADH receptor may also explain several other hitherto unresolved apparent paradoxes of the physiology of antidiuresis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Unless stated otherwise, chemicals were from Merck, Sigma Chemical, and Seromed. Iodinated p(OH)-phenyl-propionyl succinimidyl ester was from Amersham. CP (Sigma Chemical) was dissolved directly in the respective buffers. The CP-supplemented culture media and the sterile control media were filtered and handled similarly.

Cell cultures. LLC-PK1 cells (ATCC CRL 1392; 1.5 × 105 cells/ml) were seeded in 75-cm2 tissue culture flasks (Falcon) and grown at 37°C in a humidified CO2 incubator (95% air-5% CO2) in Ham's F-12 medium supplemented with 6% inactivated fetal bovine serum, 2 mmol/l L-glutamine, 100 UI/ml penicillin, and 100 µg/ml streptomycin. The culture medium was replaced every 48 h.

LLC-PK1 cells reach ~40% confluence after 2 days, 70% after 3 days, and 95% after 4-5 days, with dome formations by days 6-8. Exposure of matched subcultures to 100 µM CP for 24 h did not affect cell viability (trypan blue), growth pattern, or membrane protein content of confluent monolayers (0.32 ± 0.02 vs. 0.33 ± 0.02 mg/ml). Inhibition of cell growth required chronic exposure to CP concentrations that were 20-50 times higher (2-5 mM). Therefore, chronic CP exposure consisted of supplementing culture media of matched subcultures with 100 µM CP for 24 h. Cells were harvested in 50 mM Tris · HCl (pH 7.4), with 0.1% Na2-EDTA and 150 mM NaCl. Plasma membranes were obtained by suspending the cells in hypotonic 5 mM Tris · HCl buffer (3 mM MgCl2 and 1 mM Na2-EDTA, pH 7.4) and homogenizing them in a Porter-Elvehjam homogenizer. After centrifugation in a high-speed microcentrifuge, the pellet was resuspended once more in hypotonic buffer to yield the final pellet. Protein content was determined with the Bio-Rad protein kit by using IgG as the standard.

Radioligands. Conjugation labeling of ADH is described elsewhere (23, 24, 32). Owing to the large difference in HPLC retention times between unconjugated and Nepsilon -conjugated alpha -amino-protected and deprotected lysine vasopressin (LVP) derivatives, the specific activity of the tracer matches that of the carrier-free 125I label used in the preparation (23-25). In the presence of bacitracin, ligand binding to LLC-PK1 cell membranes was rapid, saturable, and reversible (24, 25).

Binding. For the "cold saturation" (55) studies, cells or membranes were incubated with a fixed amount of ADH tracer (~60,000 counts · min-1 · tube-1) and increasing concentrations of ADH, ranging from 0 to 5 × 10-8 M. Nonspecific binding was assessed in the presence of excess ADH (>= 10-6 M). The binding buffer consisted of 100 mM Tris · HCl, 5 mM MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.8. Each tube (150 µl) contained 50 µl LLC-PK1 membranes in binding buffer without BSA and bacitracin, 50 µl ADH (LVP) standard, and 50 µl radioligand, both in complete binding buffer.

The incubation proceeded for 90 min at room temperature and was stopped by the addition of 1 ml ice-cold binding buffer and centrifugation for 10 min at 4°C. This was repeated once after the supernatant was discarded. The bound radioactivity in each pellet was counted for 10 min. The presence of IBMX, at the concentration used in the AC assay (see below), had no effect on specific binding, measured after 90-min incubation.

AC. cAMP production rate (fmol cAMP · min-1 · cell or mg protein-1), as a function of ADH dose, was assessed under similar conditions as binding. Thus to 50 µl of cells suspended in binding buffer without BSA and bacitracin but containing 1.5 mM IBMX were added 50 µl binding buffer and 50 µl ADH standards in binding buffer. The reaction proceeded for 2 h at room temperature and was terminated by the addition of 280 µl iced ethanol, followed by centrifugation at 4,100 g at 4°C for 15 min. The pellet was extracted once more with 65% iced ethanol. Pooled supernatants were evaporated at 60°C under a gentle stream of air. The residue was dissolved in 0.05 M sodium acetate with 0.1% azide, pH 5.8, and cAMP was measured in appropriate dilutions in a standard cAMP RIA (Amersham) by using the same buffer.

Analysis. ADH binding and cAMP production rates were analyzed with the computer programs LIGAND (55) and ALLFIT (17). The ALLFIT program statistically tests whether two or more sigmoidal dose-response curves share common parameters (16, 17). Other software used were Excel (Microsoft), Framework (AshtonTate), Scientist (Micromath), and Sigma Plot (Jandel). The Mann-Whitney U-test and the Wilcoxon matched pairs test were used where appropriate. Computer modeling of ADH-responsive AC activity, based on the extended ternary complex model of hormone signaling, was performed by using the program ALLFIT. This analysis and the derivation of the A-O plots are provided in the APPENDIX.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

CP as an ADH antagonist. The present study confirms our earlier finding (24, 25) that the binding of our Nepsilon (lys)-conjugated ADH ligands to LLC-PK1 cell membranes is specific and saturable (Fig. 1). Here we show that CP displaces the ADH ligand from LLC-PK1 cell membranes in a competitive manner (Fig. 2) with an IC50 of 2.5 ± 0.5 mM. Analysis with LIGAND revealed an inhibition constant (Ki) of 2.8 ± 0.5 mM (n = 6). In addition to displacing ADH from its receptor, CP also competitively inhibited ADH-stimulated AC activity (Fig. 3). CP inhibited ADH-stimulated AC, with an inhibition constant K'i of 250 ± 6 µM (n = 6). The CP concentration of 333 µM was used because it is in the vicinity of the K'i for inhibition and within the therapeutic range (30). CP did not affect the maximal AC response [maximal velocity (Vmax)] to ADH but merely increased the ED50 for stimulation from 0.08 ± 0.02 to 0.31 ± 0.10 nM ADH (n = 6, P < 0.05) (Fig. 3). Analysis with the ALLFIT program (16) confirmed that the ADH-cAMP dose-response curves with and without CP (Fig. 3) shared the same Vmax (parameter d; see legend of Fig. 3) but rejected the additional hypothesis that baseline activity (parameter a) and the ED50 (parameter c) were the same in the presence and absence of CP. The competitive inhibition of ADH-stimulated AC by CP (i.e., unchanged Vmax but increased ED50) was further suggested by the Lineweaver-Burk plots (Fig. 3, inset).


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  		Fig. 1.   Antidiuretic hormone (ADH) saturation binding studies in membranes from control and chlorpropamide (CP)-treated cells. ADH receptor saturation studies were performed in membranes from matched pairs of control (open circle ) and 24-h CP-exposed () LLC-PK1 subcultures. Scatchard plots (inset) suggested a single class of binding sites that increased with 24-h exposure to 100 µM CP. Moreover, CP treatment did not alter affinity for ADH (identical slopes). This was confirmed by the program LIGAND, which, in this example, revealed a higher ADH receptor density [Bmax; 909 vs. 803 fmol/mg protein, or 140,432 vs. 124,048 copies of receptors per cell, respectively, in control and CP-exposed cells, but identical affinities (Kd), i.e., 1.2 vs. 1.3 nM, respectively]. B/F, ratio of bound to free ADH.



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  		Fig. 2.   ADH ligand displacement study with CP. CP competitively inhibited binding of ADH ligand to LLC-PK1 cell membranes. Binding was expressed in %binding in the absence of CP (% B0). Ki for inhibition of ADH binding, calculated with LIGAND, was 2.8 ± 0.5 mM (n = 6). LVP, lysine vasopressin.



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  		Fig. 3.   Acute effects of CP on ADH-stimulated cAMP production rate in LLC-PK1 cells. CP (333 µM, open circle ) displaced the normal () ADH-AC dose-response curve to the right without affecting maximum response (competitive inhibition). Indeed, CP increased ED50 from c1 = 0.075 ± 0.020 nM to c2 = 0.255 ± 0.059 nM but did not affect maximum response of adenylyl cyclase (AC) to ADH [d1 = d2 = maximum velocity (Vmax) = 6,906.96 ± 240.07 fmol cAMP · min-1 · mg protein-1]. CP also reduced basal AC activity (a2 = 716.98 ± 243.43 vs. a1 = 1,293.85 ± 284.91 fmol cAMP · min-1 · mg protein-1). Conventional Lineweaver-Burk plots (inset), corrected for basal activity, also suggest competitive inhibition (72) of ADH-stimulated AC activity by CP (unchanged y- intercept, i.e., 1/Vmax, but a different x-intercept, i.e., -1/Ka). Results obtained with ALLFIT; see APPENDIX.

CP as an ADH "inverse agonist." Because in addition to displacing ADH (Fig. 2) and inhibiting ADH-stimulated AC activity (Fig. 3), CP also inhibited ADH-independent basal AC activity (i.e., lowered parameter a in ALLFIT; see APPENDIX), it also behaved as an ADH inverse agonist rather than a pure, neutral, competitive antagonist. Moreover, LLC-PK1 cells exposed to 100 µM CP for 24 h revealed a 15.35 ± 3.29% higher ADH receptor density (Bmax = 1,040 ± 82 vs. 899 ± 58 fmol/mg, n = 14, P < 0.01) (Figs. 1 and 4) but the same ADH affinity (Kd = 1.14 ± 0.096 vs. 1.14 ± 0.113 nM, n = 14, not significant, LIGAND), consistent with an antagonist-mediated receptor upregulation. This receptor upregulation not only potentiated the subsequent maximal AC response to ADH in harvested cells (Vmax 14.9 ± 0.8 vs. 10.6 ± 1.0 fmol cAMP · min-1 · 103 cells-1, n = 6, P < 0.05) but increased the basal AC activity (1.86 ± 0.17 vs. 1.35 ± 0.18 fmol · min-1 · 103 cells-1, n = 6, P < 0.05) as well. The search with ALLFIT (17) of the parameters that were shared between the dose-response curves of control and CP-pretreated cells revealed that baseline activity (parameter a), Vmax (d), and the ED50 (c) were significantly different (16) (Fig. 5). Moreover, a nonessential mode of activation (72) of ADH-sensitive AC (i.e., up-arrow Vmax, down-arrow Ka), by 24-h exposure to 100 µM CP, was also suggested by the double reciprocal plots, corrected for basal activity (Fig. 5, inset). The normalized (i.e., in %maximum) fractional AC activation-fractional receptor occupation (A-O) plots constructed with the present results (APPENDIX) are depicted in Fig. 6.

As the A-O plots for both controls (Figs. 3 and 5) were identical, one single plot representing their arithmetical mean was constructed (open circle , middle curve). The top curve () depicts the A-O plot obtained for cells chronically exposed to CP (Fig. 5), and the bottom curve () represents the A-O plot in the presence of 333 µM CP (Fig. 3). The A-O plot pattern of the cells exposed for 24 h to CP suggests enhanced receptor-to-AC stoichiometry or increased receptor density, as predicted by the "random hit matrix model" proposed for ADH receptor-AC coupling (4), or an enhanced agonist "efficacy" (14). Conversely, the A-O plot pattern obtained in the presence of CP is characteristic of decreased coupling between receptor and cyclase units (4), or lower intrinsic "efficacy" (14). These results are expected if CP both behaves as an inverse agonist when added acutely to the medium and upregulates the ADH receptors when cells are chronically exposed to it (see the APPENDIX). Although CP-pretreated cells displayed a significant higher ADH-stimulated AC activity, the acute addition of a suprapharmacological dose of CP (1 mM) inhibited the ADH-stimulated AC activity by the same percentage in both the control and CP-treated cells. Thus the residual activity remaining after the acute dose of 1 mM CP was 44.6 ± 2.6 and 43.1 ± 3.4% (n = 3, not significant) of their respective controls. This finding is consistent with the notion that the increased ADH-sensitive AC seen in the CP-treated cells is mediated by the ADH receptor itself, rather than being due to a nonspecific effect of CP at a different step along the AC-signaling cascade. In other words, even at 24 h after chronic administration, CP still behaves as a specific ADH antagonist.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

The mechanism by which CP potentiates ADH-mediated antidiuresis (1, 6, 7, 34, 44, 45, 48, 49, 52-54, 56, 76) has eluded any explanation for the past 30 years. Our findings that CP competitively inhibits ADH binding and AC stimulation in LLC-PK1 cells (Figs. 2 and 3) were surprising, although the paradoxical in vitro inhibition by CP of ADH binding (57) and AC stimulation (3, 43, 54) were previously reported in rat renal membranes. Becasuse CP has no intrinsic ADH-like activity (1, 6, 34, 45, 48, 49, 52, 53, 56, 76) and inhibits ADH- but not fluoride-stimulated AC (54), it behaves as a specific competitive ADH antagonist. And as the in vivo (6, 45, 48, 49, 52-54, 56, 76) and in vitro (Figs. 1-6) (52-54, 57) CP concentrations were identical (30), the seemingly irreconcilable ADH potentiation in vivo but ADH antagonism in vitro cannot merely be explained by a dose effect. Any inherent differences between the in vivo and in vitro models can be excluded because the paradox of ADH potentiation (6, 48, 49, 52-54, 76) and antagonism (3, 43, 54, 57) is now documented within the same model (Figs. 1-6). Thus although Lineweaver-Burk plots suggest competitive inhibition of ADH-sensitive AC (unchanged Vmax, higher apparent Ka) when CP is added acutely to the assay, nonessential activation (72) of ADH-sensitive AC (up-arrow Vmax, down-arrow Ka), is seen when CP is added to the culture medium for 24 h before cell harvesting (Figs. 3 and 5, insets). The same nonessential mode of activation of ADH-sensitive AC was observed in renal membranes of CP-treated rats (52, 53), a model found by us to be also associated with ADH receptor upregulation (32).

ADH receptor upregulation in rats (32) and LLC-PK1 cells (Figs. 1 and 4) alone, however, cannot explain, at least within the frame of the classical G protein-coupled receptor-signaling theory, the enhanced basal AC activity in both models (Fig. 5) (52, 53) and the inability of CP-treated rats to dilute their urine after a sustained oral water load (6 × 10 ml/8 h), the latter of which was designed to suppress ADH completely (32). An inability to fully dilute the urine after a sustained water load was also observed in CP-treated normal volunteers, and even in patients with ADH-deficient DI (28, 48, 60). Moreover, sensitive radioimmunoassays for ADH and neurophysine also suggest that the impaired free water clearances after CP treatment in rats and humans are ADH independent (60). Finally, the fact that impaired maximal free water clearance is also observed in CP-treated DI rats (39) is compelling evidence that it is ADH independent. Thus, although CP-mediated antidiuresis requires residual ADH (6, 34, 45, 48, 49, 56), the impaired diluting ability after CP treatment (32, 39, 48, 60) is ADH independent.

This puzzling phenomenon is reminiscent of the antidiuretic state that arises during chronic infusions of peptidic ADH antagonist in DI rats (37, 46, 74). Indeed, ADH receptor upregulation also occurs in this model (10) where the antidiuresis, by definition, is ADH independent, because it appears in rats unable to produce ADH (DI rat). That it is also independent of other putative endogenous ADH agonists such as oxytocin is further suggested by the fact that this antidiuresis arises during infusion of saturating doses of "neutral" ADH antagonists (i.e., in a situation where there are no free ADH receptors available). In fact, this antidiuresis correlated with the relative potency of the antagonists used; therefore, intrinsic partial agonism is also unlikely. Moreover, their half-life is several orders of magnitude shorter (<20 min) than the protracted (days) antidiuresis (10) that persisted after their infusion was discontinued. That this paradoxical antidiuresis could be due to an acquired intrinsic ADH agonist activity of the ADH antagonist is also unlikely, because these antagonists have no demonstrable agonistic activity when tested acutely in the same rats (74). Such confounding questions could be avoided with the present in vitro model, where the same constellation of increased basal AC activity and ADH receptor upregulation was elicited by CP (Figs. 1 and 5), which also behaved as a competitive antagonist, for both ADH binding (Fig. 2) and AC stimulation (Fig. 3). ADH receptor upregulation (Fig. 4) (10, 32) is the common finding in the above states and is characterized by increased basal ADH-like signaling activity, i.e., either an increased basal AC activity (Fig. 5) and/or an impaired free water clearance (10, 32, 52, 53) during ADH suppression. Thus constitutive, hormone-independent receptor-signaling activity (14, 18) has to be considered for the antidiuretic V2 ADH isoreceptor.


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  		Fig. 4.   Effect of CP on ADH receptor density in 14 LLC-PK1 cell subcultures. ADH receptor density was assessed in preparations from 14 matched pairs of LLC-PK1 subcultures exposed (+CP; hatched bars) or not exposed (-CP; open bars) to 100 µM CP for 24 h before harvesting. Individual saturation studies (Fig. 1) revealed that membranes from CP-exposed subcultures had a higher ADH receptor density (1,040 ± 82 vs. 899 ± 58 fmol/mg protein, n = 14, P < 0.01) but the same ADH affinity (1.14 ± 0.096 vs. 1.14 ± 0.113 nM, n = 14, not significant).



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  		Fig. 5.   Chronic effects of CP on ADH-stimulated cAMP production rate in LLC-PK1 cells. Exposure to 100 µM CP for 24 h potentiated subsequent AC response to ADH. In this study, ED50 of ADH for AC activation was c1 = 0.112 ± 0.025 nM in control cells () compared with c2 = 0.053 ± 0.009 nM for cells preexposed to CP (open circle ). Maximal stimulation of AC (i.e., parameter d = Vmax) was significantly higher in 24-h CP exposed cells than in control (d2 = 8,414.77 ± 219.80 vs. d1 = 5,748.22 ± 238.67 fmol cAMP · min-1 · mg protein-1, respectively). Conventional double reciprocal plots (1/cAMP vs. 1/LVP, inset), corrected for basal activity, suggest nonessential activation (72) of ADH-sensitive AC by CP.

This constellation induced by CP and ADH antagonists in vivo (10, 32) and CP in vitro (Figs. 2, 3, 5) may be explained by the extended G protein-coupled receptor-signaling hypothesis (14, 42). This model challenges the traditional view that inactive receptors R are converted, on hormone (H) binding, into active HR* complexes (H +R right-left-harpoons  HR*) (18). Rather, agonists merely stabilize the active state R* of receptors that spontaneously isomerize into their inactive (silent) R and active (signaling) R* conformations (R left-right-harpoons  R*), as they display higher affinity for R* than R (APPENDIX).

Pure neutral competitive antagonists, on the other hand, have no R/R* preferences and hence do not affect the equilibrium R left-right-harpoons  R*, i.e., basal receptor-signaling activity; they only compete with agonist for binding. Finally, those antagonists that display a higher affinity for R, and thus stabilize the inactive receptor conformation, are termed inverse agonists (71). Inverse agonists inhibit "basal" signaling activity because they decrease the number of receptors that spontaneously reside in the productive conformation R*. However, as the inactive state R predominates in the absence of agonists, hormone-independent signaling activity (due to R*) is difficult to detect. Initially, computer simulations were used to predict the behavior of such models (14, 41).

Eventually, receptor mutations that spontaneously favor the active transition state (R right-left-harpoons  up-arrow R*) (67, 68), or models that overexpress the wild-type receptor up-arrow (R left-right-harpoons  R*) in vitro (2, 12, 75) and in transgenic mice (9), i.e., models that stochastically increase the active receptor conformation up-arrow R*, were found to consistently display increased basal, i.e., hormone-independent receptor-signaling, activity (41, 50). Although the significance of tonic receptor-signaling activity in normal physiology is still debated (8, 50, 71), it is increasingly recognized in vitro in cell cultures (40), where inverse agonists have become invaluable probes for unmasking this phenomenon (13).

Several independent lines of evidence suggest constitutive signaling activity of the renal ADH isoreceptor. First, the association of receptor upregulation with ADH-independent, ADH-like effects in vivo (10, 32) and in vitro (52) (Fig. 5) point to some constitutive signaling activity of unliganded ADH receptors. Tonic ADH receptor-signaling activity is further suggested by the unique inhibitor properties of CP. Indeed, the inhibition of ADH binding and activation of AC in LLC-PK1 cells by CP is of a competitive nature because CP increases the apparent Kd and Ka (Figs. 2 and 3) but has no effects on Bmax and Vmax and is restricted to the effect of ADH. Indeed, CP inhibits AC only when stimulated by ADH (3, 43, 54) (Fig. 3) but not by postreceptor stimuli such as fluoride, GTP, or 5'-guanylyl imidodiphosphate (43, 52-54). Because the competitive ADH inhibitor CP also inhibits basal, i.e., ADH-independent AC, activity in rat renal membranes (3, 43) and LLC-PK1 cells (Fig. 3) and, similarly, increases urine flow rate or free water clearance in the absence of endogenous ADH in dogs (76) and rats (52), it acts as an ADH inverse agonist. These findings imply that the ADH receptor displays constitutive signaling activity in vivo as well as in vitro. Moreover, the computer simulations of agonist dose-response curves in the allosteric receptor model (14, 41) predict that inverse agonists lower the initial plateau (down-arrow baseline; i.e., parameter a) and displace the curve to the right (up-arrow ED50; i.e., parameter c) but do not affect the upper plateau (Vmax; parameter d) and that receptor upregulation elevates both the lower and the upper plateaus (up-arrow baseline, up-arrow Vmax) and shifts the curve to the left (down-arrow ED50) (41). This is exactly the pattern seen in the present study (Figs. 3 and 5) and confirmed by analysis with both LIGAND and ALLFIT. Finally, the fractional AC activation-receptor occupation plots (Fig. 6) further suggest ADH inverse agonism of CP because for the same AC activation, more bound ADH is required with CP, consistent with an effect on the allosteric equilibrium up-arrow R left-right-harpoons  down-arrow R*. A pure neutral competitive antagonist should not affect the A-O plot.


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  		Fig. 6.   Fractional (%Vmax vs. %Bmax) AC activation-ADH receptor occupation (A-O) plots. Middle curve, open circle : normal A-O relationship in LLC-PK1 cells as derived from 2 control experiments ( in Figs. 3 and 5) (4) (see APPENDIX); , A-O plot pattern in presence of 333 µM CP (Fig. 3); , A-O plot pattern for cells grown in 100 µM CP (Fig. 5), a treatment that results in receptor upregulation (Fig. 4). Line of identity (dashed line; fractional A = fractional O) represents 1/1 A-O coupling pattern. For points above this line, receptor "reserve" is invoked.

Constitutive, ADH-independent ADH receptor-signaling activity may explain other paradoxes of antidiuresis. Thus the antidiuresis arising with receptor upregulation in the DI rats chronically infused with peptidic ADH antagonists was resistant, by definition, to ADH antagonists, because it occurred during their infusion (10, 37, 46, 74). Constitutive receptor-signaling activity is resistant, by definition, to neutral competitive antagonists (APPENDIX). If these antagonists were also inverse agonists, then receptor upregulation alone may not have led to the antidiuresis and, like for CP, residual ADH would have been required for the antidiuresis. The need for residual ADH release in the CP-mediated antidiuresis is well known but has hitherto never been explained (6, 48, 49, 52-54, 56, 76). Similarly, the impaired ability to maximally dilute the urine after water loading, hence ADH suppression, in CP-treated rats (32, 39, 60) and humans (48, 60), has not yet received an adequate explanation. The allosteric receptor model predicts that receptor upregulation alone (32) could explain the impaired diluting ability uncovered by ADH suppression and medullary washout (48, 32, 28, 60) of CP during water loading, if CP had weak ADH inverse agonist properties, or its corollary, if the ADH receptor displayed ADH-independent activity.

The set point of the allosteric receptor equilibrium (R left-right-harpoons  R*), crucial in hormone signaling, depends on the ionic strength (41, 42), and triggering of G protein-coupled receptor-signaling by salts (35) has recently been attributed to an allosteric receptor transition R right-harpoon-up  R* (14). This phenomenon is of particular relevance to the renal ADH isoreceptor, strategically located in the renal medulla, in an ionic milieu affected by the state of hydration and the antidiuresis itself.

Potentiation of the ADH-sensitive AC by NaCl occurs in the rat (15, 19, 27), rabbit (21, 22), pig (63-66, 73), and bovine (31, 58) kidneys, where NaCl not only enhances ADH-stimulated but also basal AC activity (15, 19, 21, 22, 27, 31, 58, 63-66, 73). Dose-response curves in LLC-PK1 membranes (Fig. 7) reveal an increase in parameters a and d (i.e., basal AC activity and Vmax) but not in c (EC50), consistent with an R right-harpoon-up  R* transition. At each dose of ADH the ratio of productive (R* + HR*) to total receptor Rt, (R* + HR*)/Rt, an index of activity, is increased in the presence of NaCl. NaCl promotes the high-affinity state of the ADH receptor in LLC-PK1 cells but has no effect on Rt (64, 65), a phenomenon coined "receptor transition" (64, 65). That this may be an allosteric effect is further suggested by the finding that mannitol inhibits the salt effect in LLC-PK1 cell membranes (73). Indeed, mannitol is known to act as a "compatible" solute that stabilizes the native conformation of proteins and prevents their salting out by chaotropic agents like NaCl. The "salting out" phenomenon is nothing more than an extreme allosteric transition. Furthermore, the rapid reversibility of the NaCl effect (65), together with the fact that NaCl increased basal and ADH-stimulated AC activities by 50 and 100%, respectively, but affected fluoride-stimulated activity by <10% (73), also points to an activating allosteric transition at a step preceding AC and G proteins, hence by exclusion, that takes place at the ADH receptor itself (R right-harpoon-up  R*). It is now well accepted that receptor transitions (R right-harpoon-up  R*) that favor the high-affinity receptor conformation R* not only potentiate the AC response to hormone but also increase the hormone-independent basal AC activity (41).


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  		Fig. 7.   Effect of NaCl on LVP-cAMP dose-response in intact LLC-PK1 monolayers. Culture medium (Ham's F-12) of confluent monolayers in 24-well plates was aspirated and replaced with 1 ml culture medium supplemented with 1 mM IBMX and 0.1 g% bacitracin, and plates were incubated at 37°C for 10 min. This medium was then aspirated and replaced with 0.5 ml of similar medium that contained serial dilution of LVP and was obtained by adding to 9 vol of preincubation medium 1 vol of either 150 () or 1,500 (open circle ) mM NaCl. After 15 min at 37°C, plates were placed on ice and 1 ml of iced ethanol-acetic acid (99:1) was added to each well. After gentle stirring, solution was aspirated and diluted in assay buffer. cAMP standards contained the same final ratio of ethanol and acetic acid. Protein content of each well was measured by the Lowry method directly on "ethanol-acetic acid-fixed" monolayer. cAMP production rate was expressed in fmol · µg-1 · min-1. Normal Ham's F-12 medium contains 130 mM NaCl. Basal activity (no LVP added) was 1.168 ± 0.109 and 2.427 ± 0.174 fmol · µg-1 · min-1 in control and NaCl-supplemented wells, respectively (P < 0.001).

A salt-induced allosteric transition of the ADH receptor in favor of ADH-independent signaling activity may explain why ADH-deficient rats concentrate their urine during dehydration (26), a state that elevates the ionic strength of the renal medulla. A receptor transition in the opposite direction (R ↼ R*) has been postulated to account for the initial ADH resistance of water-replete DI rats (64). The effect of NaCl on the ADH-AC dose-response curve in LLC-PK1 cells supports this hypothesis (Fig. 7). Our present knowledge of the physiology of antidiuresis suggests that the renal ADH receptor may have evolved into an exquisitely salt-sensitive allosteric transducer. This could compensate for the lack of redundancy in the antidiuretic mechanism (5), which rests primarily on one single hormone and one single ADH-regulated water channel (36), compared with the diversity of the renal antinatriuretic mechanisms (20). Clearly, although outside the scope of the present report on CP, similar studies based on the allosteric receptor isomerization model will be required to further assess the effects of NaCl on ADH signaling (Fig. 7). Of relevance to both CP (Figs. 1-6) and NaCl (Fig. 7) is the recent report of ADH-independent constitutive signaling activity and ADH inverse agonism of the antagonist SR-121 463A in the D136A mutated human V2 ADH receptor (51). Relevance to CP resides in the fact that its chemical structure, with a benzenesulfunamide residue at one end, linked, via the aminocarbonyl bridge, to a hydrophobic moiety at the other, closely resembles the structure of this nonpeptidic ADH inverse agonist. Reminiscent of the salt effect in ADH signaling (Fig. 7) is the general rule that mutations that confer constitutive activity (R right-left-harpoons  up-arrow R*) destabilize receptors, hence rendering them susceptible to denaturation (29, 51). Substitution of the aspartic acid in the conserved DRH/Y sequence, like in this case (51), consistently activates receptors (69, 70). This anionic residue has the potential of forming "constraining salt brides" that stabilize the inactive conformation R of receptors (61, 69, 70). Moreover, aspartic acid residues are known to play a key role in the allosteric modulation of receptor activity by Na+ (38, 59, 62). That the salt effect (Fig. 7) occurs specifically at the level of the V2 receptor within the V2 receptor-G protein-adenylyl cyclase signaling cascade in LLC-PK1 is further suggested by the fact that although NaCl markedly magnifies (Fig. 7, and Ref. 73) cAMP stimulation by ADH, it has very little (<10%) effect on postreceptor stimulation of AC by NaF (73). Moreover, although in rat renal papillary collecting tubule cells NaCl markedly potentiates the cAMP response to ADH, it has no effect on cAMP stimulation by forskolin or prostaglandin E2 (47).

In summary, this study provides evidence that CP is a weak inverse ADH agonist for the V2 ADH renal receptor. This explains why CP upregulates ADH receptors in vivo (32) and in vitro (this study) and why CP treatment ameliorates water handling in partial central DI but has no effect in patients with nephrogenic DI due to mutated ADH receptors incapable of constitutive signaling activity (1). Because the corollary of inverse ADH agonism is constitutive ADH receptor activity, the inability of rats and humans to maximally dilute their urine after CP treatment (32, 39, 48, 60), and presumably upregulated ADH receptors (32), suggests the presence of ADH-independent, but ADH receptor-dependent, signaling in normal renal water handling.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

The Extended Model of G Protein-Coupled Receptor Signaling

  

<AR><R><C>R</C></R><R><C>+</C></R><R><C>H</C></R><R><C>↓↑</C></R><R><C>HR</C></R></AR> <AR><R><C>⇋</C></R><R><C> </C></R><R><C> </C></R><R><C> </C></R><R><C>⇌</C></R></AR> <AR><R><C>R*</C></R><R><C>+</C></R><R><C>H</C></R><R><C>⇓↑</C></R><R><C>HR*</C></R></AR>
In this model (2, 42) the receptor spontaneously assumes an allosteric equilibrium (transitions) between a silent R and an active R* conformation. The signal [i.e., adenylyl cyclase (AC) activity] is a function of the ratio (R* + HR*)/Rt, where R* and HR* are the active conformations of the free and hormone-bound receptor that interact productively with G proteins, and where total receptor Rt is given by the mass conservation Rt = R + R* + HR + HR*. G protein interactions are not depicted. The agonist has a higher affinity for the active conformation R* and hence will stabilize it. In the absence of hormone H, the silent conformation R predominates, and thus basal signaling activity is minimal.

Any nonhormonal perturbation that increases R* will enhance basal and ADH-responsive AC activity. Because neutral antagonists (I) bind equally well to the R and R* conformations, they do not affect the allosteric equilibrium or the total amount of active species of the receptor in the absence of H. At the extreme, in the presence of an excess of neutral antagonist I, the allosteric receptor equilibrium R right-left-harpoons  R* is merely replaced with the equivalent equilibrium IR right-left-harpoons  IR*, and because IR* is active, the basal signaling activity is not affected. Thus neutral antagonists neither inhibit basal signaling activity nor prevent the increase in signaling activity seen with receptor upregulation up-arrow (R right-left-harpoons  R*) or induced by external, nonhormonal factors such as NaCl (R right-left-harpoons  up-arrow R*). However, as they compete with H for binding to R and R*, they inhibit the stimulation mediated by H. On the other hand, antagonists that have a higher affinity for R than for R*, i.e., inverse agonists, decrease basal signaling activity in the absence of agonists. Computer simulations of this model (14, 41) predict that receptor upregulation should be associated with 1) an enhanced maximal agonist response (Vmax) to agonist, 2) an increased basal signaling activity, and 3) a lower ED50 for agonist stimulation. Moreover, computer simulations based on this model also predict that inverse agonists should 1) decrease basal activity, 2) increase the ED50 for agonist, and 3) leave unaltered the maximal response to agonists. This was the pattern observed for the ADH-AC signaling system in LLC-PK1 cells and for the antagonistic characteristics of CP. As inverse agonists stabilize preferentially the inactive receptor conformation R, i.e., perturbate the allosteric equilibrium R left-right-harpoons  R* in favor of R, they may also affect the trajectory of the fractional activation-occupation (A-O) plots. This was observed for CP, and may in part account for the finding that its Ki for inhibition of ADH binding was higher than its K'i for inhibition of ADH-stimulated AC. Clearly, studies will be required to elucidate the molecular mechanism.

Computer Modeling of ADH-Dependent AC Activity

The program ALLFIT, which allows to test statistically the hypothesis that families of dose-response curves share common parameters (16, 17), was used to verify in the ADH-responsive AC system of LLC-PK1 cells, the predictions of the extended ternary complex model (2, 8, 9, 12-14, 40-42, 50, 67, 68, 71, 75) of hormone signaling.

ALLFIT fits the empirical four-parameter logistic equation to the data. The advantage of this method is that no assumptions are required concerning the mechanism(s) underlying the phenomenon under observation. Thus by testing whether dose-response curves share common parameters, ALLFIT allows extraction of objective information not obtainable by standard graphical assessment of dose-response curves, including the double reciprocal plots (Lineweaver-Burk plots) (72).

The conventional notation for the four-parameter logistic equation of a dose (X)- response (Y) relationship is
<IT>Y</IT> = <FR><NU><IT>a − d</IT></NU><DE>1 + <FENCE><FR><NU><IT>X</IT></NU><DE><IT>c</IT></DE></FR></FENCE><SUP><IT>b</IT></SUP></DE></FR> + <IT>d</IT>
where a is the lower plateau (X = 0) or basal activity, d is the upper plateau (X right-arrow infinity ) or maximal response (Vmax), c is the ID50, and b is the slope factor. In the last version of ALLFIT (16), the roles of a and d have been exchanged to ensure that the fraction is positive; hence b assumes a negative value (16).

A-O Plots

The dependency of AC activation (A = %Vmax) on receptor occupancy (O = %Bmax) has been extensively assessed for the renal ADH receptor (4, 31, 58). Computer modeling of this relationship has led to a random-hit matrix model of hormone signaling in which ADH receptors are assumed to interact with a set of vicinal AC units (4). Although the possibility of an ADH-independent, spontaneous ADH receptor-signaling activity has been considered initially, this then-novel concept was not pursued in this model (4). This idea, however, has recently been revived for G protein-coupled receptors (2, 8, 9, 12-14, 40-42, 50, 67, 68, 71, 75). To model the A-O relationship, A and O have to be recorded under similar experimental conditions (i.e., same buffer and at equilibrium) (4, 31, 58). Therefore, all the AC stimulation studies were performed in binding buffer under the same conditions as ADH binding, with the sole exception that 0.5 mM IBMX was present during AC stimulation. IBMX, however, had no affects on ligand binding.

Because the A-O plots for the renal ADH-signaling system, published by others, were constructed with the basal AC activity subtracted (4, 31, 58), for comparison we used the same representation (Fig. 8). Thus A, the fractional activation of AC, expressed as the %maximal activation (d = Vmax), corrected for basal activity a, was obtained by the equation A = 100(Y - a)/(d - a). By substituting Y with the four-parameter logistic equation above, we obtain, after simplification and rearrangement
A = 100 ⋅ <FR><NU><IT>X<SUP>b</SUP></IT></NU><DE><IT>c<SUP>b</SUP> + X<SUP>b</SUP></IT></DE></FR>
Similarly, a normalized expression for receptor occupancy (O = %Bmax) was derived from the saturation-binding equation for a ligand X in the presence of an inhibitor I (23), by writing
O = 100 ⋅ <FR><NU><IT>X</IT></NU><DE><IT>K<SUB>d</SUB></IT> ⋅ <FENCE>1 + <FR><NU><IT>I</IT></NU><DE><IT>K</IT><SUB>i</SUB></DE></FR></FENCE> + <IT>X</IT></DE></FR>
The parameters c, b, Kd, and Ki were determined with the programs LIGAND and ALLFIT. Note that although the independent variable X, in the four-parameter logistic equation, represents the total ADH concentration ([ADH]t) dose used to stimulate AC, in the normalized binding equation it represents free ADH concentration ([ADH]f). Thus the equation relating A to O [i.e., A = f(O); A-O plots] may not be derived by simple elimination of X between the two equations. However, because the amount of receptor used in the AC stimulation studies was small, and given the concentrations of ADH used and the Kd of 1.14 nM, the approximation [ADH]f ~ [ADH]t was allowed. Indeed, as calculated from the binding isotherm, the ratio [ADH]f/[ADH]t ranged from 0.9790 to 0.9997 (i.e., ~1.0); thus [ADH]t ~ [ADH]f. When the ratio of receptor to hormone during stimulation is such that only a small fraction of total hormone is bound, a valid expression of A as a function of O, I, and the parameters b, c, Kd, and Ki can be derived by eliminating X between the above two equations. As can be seen, the equation obtained this way produced curves that matched the experimental data points very closely (Fig. 6).


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  		Fig. 8.  


    ACKNOWLEDGEMENTS

This work was supported in part by a Deutsche Forschungsgemeinschaft Grant (He 1472/3-1) and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, VA Merit Review Grant.


    FOOTNOTES

The technical help of C. Klein is greatly acknowledged.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. A. Durr, Mail Code (111), Medical Service, Div. of Nephrology, Bay Pines VA Medical Center, PO Box 5005, Bay Pines FL 33744.

Received 11 August 1999; accepted in final form 30 November 1999.


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DISCUSSION
APPENDIX
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Am J Physiol Renal Fluid Electrolyte Physiol 278(5):F799-F808



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