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
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ABSTRACT |
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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 · min1 · 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 N-conjugated
-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 · min1 · 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.
AC.
cAMP production rate (fmol
cAMP · min1 · 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.
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RESULTS |
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CP as an ADH antagonist.
The present study confirms our earlier finding (24, 25) that the
binding of our N(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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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 · min1 · 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.,
Vmax,
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.
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DISCUSSION |
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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 (Vmax,
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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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
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
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 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
R*) (67, 68), or models that overexpress the wild-type receptor
(R
R*) in vitro (2,
12, 75) and in transgenic mice (9), i.e., models that stochastically increase the active receptor conformation
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 (baseline; i.e., parameter
a) and displace the curve to the right
(
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 (
baseline,
Vmax) and shifts the curve to the left
(
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
R
R*. A pure neutral
competitive antagonist should not affect the A-O plot.
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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 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
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 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
R*). It is now well accepted that receptor transitions (R
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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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
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.
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APPENDIX |
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The Extended Model of G Protein-Coupled Receptor Signaling
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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
R* is merely replaced with the equivalent equilibrium IR
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
(R
R*) or induced by external,
nonhormonal factors such as NaCl (R
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
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
![]() |
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
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|
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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