A molecular model of agonist and nonpeptide antagonist binding to the human V(1) vascular vasopressin receptor

The affinity of the nonpeptide antagonist OPC-21268 is greater for the rat V(1) arginine vasopressin (AVP) receptor (V(1)R) than for the human V(1)R. Site-specific mutagenesis was carried out to identify the residues that determine interspecies selectivity for nonpeptide antagonist binding. The introduction of rat amino acids in position 224, 310, 324, or 337 of the human V(1)R sequence dramatically altered OPC-21268 affinity for the receptor, whereas binding of AVP, the peptide V(1)R antagonist d(CH(2))(5)Tyr(Me)AVP, and the nonpeptide V(1)R antagonist SR49059 was not altered by these mutations. Computer modeling explained the mutagenesis results. Docking of OPC-21268 onto a homology-built model of the V(1)R receptor yielded a model for the bound ligand in which the hydrophobic part is deeply embedded in the transmembrane region, whereas the polar part is located on the surface of the extracellular side. The increased affinity of the G337A mutant is due to two additional van der Waals contacts of the alanine methyl group with carbon atoms on the antagonist. The I310V mutant reduces the hydrophobicity in the vicinity of the polar oxygen atom of the antagonist. The I224V mutant relieves overcrowding in a hydrophobic binding pocket involving the aromatic residues Trp(175), Phe(179), Phe(307), and Trp(304). Finally, the E324D mutant enables the formation of a hydrogen bond of the carboxylate side chain with the amide side chain of Gln(311), which in turn forms a hydrogen bond with the N57 nitrogen atom of OPC-21268. Thus, a few residues, distinct from those involved in agonist binding, control interspecies selectivity toward OPC-21268 nonpeptide antagonist binding.     

Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK(2) receptor.

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK(2) receptor, either wild-type or mutated, at four aromatic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [(125)I]neurokinin A (NKA), the peptide antagonist [(3)H]MEN 11420, and the nonpeptide antagonist [(3)H]SR 48968 bound to the wild-type receptor with high affinity (K(d) = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K(d) = 4.8 and 11.5 nM, respectively); Y(266)F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K(d) = 2.8 nM and 1.2 nM, respectively); F(270)A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K(d) = 1.6 nM); Y(289)F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K(d) = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H(198)A and Y(266)F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y(289)F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK(2) receptor.     

Antidiuretic effects of a nonpeptide vasopressin V(2)-receptor agonist, OPC-51803, administered orally to rats

OPC-51803 is the first nonpeptide vasopressin (AVP) V(2)-receptor-selective agonist. Its pharmacological profile, including antidiuretic action and receptor binding, was characterized using conscious Brattleboro rats with hereditary diabetes insipidus and Sprague-Dawley rats. In membrane preparations from the liver and kidney, OPC-51803 displaced the [(3)H]AVP binding to V(2)-receptors (K(i) = 49.8 +/- 8.1 nM) more greatly than that to V(1a)-receptors (K(i) = 1061 +/- 60 nM), showing a 21 times higher affinity for V(2)-receptors. At single oral doses of 0.003 to 0.3 mg/kg in female Brattleboro rats, OPC-51803 decreased urine volume (from 10.8 +/- 1.1 to 0.5 +/- 0.2 ml during 0-2 h postdosing) and increased urinary osmolality (from 114 +/- 9 to 432 +/- 114 mOsm/kg) in a dose-dependent manner. During the period of 4-week treatment with OPC-51803, significant and constant antidiuresis was observed. In male Sprague-Dawley rats with normal plasma AVP levels, OPC-51803 at 0.03 to 0.3 mg/kg also produced a dose-dependent antidiuretic action (urine volume: from 2.6 +/- 0.6 to 1.1 +/- 0.2 ml at 0-4 h postdosing). Few changes in urinary parameters, serum parameters, or plasma hormone levels were observed. OPC-51803 did not change blood pressure or heart rate, or inhibit AVP-induced pressor response even at 30 mg/kg p.o. These results demonstrate that OPC-51803 is a V(2)-selective agonist that produces a significant antidiuretic action after single and multiple oral dosing in AVP-deficient and normal AVP states. The data suggest that OPC-51803 is a useful therapeutic drug in the treatment of hypothalamic diabetes insipidus, nocturnal enuresis, and some kinds of urinary incontinence.     

A nonpeptide vasopressin V(1a) receptor antagonist, SR 49059, does not prevent cisplatin-induced emesis in piglets

We determined the pharmacological and the antiemetic properties of SR 49059, a selective nonpeptide V(1a) receptor antagonist, on cisplatin-induced emesis in the piglet. Firstly, we clearly demonstrate that SR 49059 is a potent V(1a) receptor antagonist in vitro and in vivo in the piglet. In binding studies, [3H]-SR 49059 exhibited high affinity for V(1a) receptors in piglet liver membranes (K(d) of 0.76 +/- 0.12 nM and B(max) of 138 +/- 22 fmol/mg prot.). In vivo, in decerebrate piglets, SR 49059 (1 mg/kg iv) antagonized AVP (500 ng/kg iv)-induced hypertension for at least 150 min and also blocked, for at least 270 min at 3 mg/kg iv, the pressor responses to exogenous LVP. After single and repeated iv or icv administration, we studied the antiemetic properties of SR 49059 on cisplatin-induced emesis in piglets. Animals receiving an emetic dose of cisplatin (5.5 mg/kg, iv) were observed continuously for 60 h. Piglets acting as controls were iv administered with vehicle 15 min prior to cisplatin infusion (T0(-15min)), while experimental animals received a single iv administration of SR 49059 at the dose of 1 or 3 mg/kg. In additional piglets, we administered SR 49059 (3 mg/kg) every 12 h from T0(-15min) to T48(-15min) (cumulative dose, 15 mg/kg). Another set of animals - observed only during the acute phase - was administered with SR 49059 (10 mg/kg) every 3 h from T0(-15min) to T15(-15min) (cumulative dose, 60 mg/kg). Lastly, 10 piglets were given a bilateral icv injection of SR 49059 (500 microg and 1500 microg/side) 1 h prior to cisplatin infusion. In all groups treated with SR 49059, the latency of the first emetic episode and the incidence of vomiting during the acute, the delayed and the cumulative phases remained statistically similar to that observed in controls, suggesting that V(1a) receptors are not involved in the onset and completion of nausea and vomiting.     

Receptor binding, behavioral, and electrophysiological profiles of nonpeptide corticotropin-releasing factor subtype 1 receptor antagonists CRA1000 and CRA1001

Receptor binding, behavioral, and electrophysiological profiles of 2-[N-(2-methylthio-4-isopropylphenyl)-N-ethylamino]-4-[4-(3-flu orophe nyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine (CRA1000) and 2-[N-(2-bromo-4-isopropylphenyl)-N-ethylamino]-4-[4-(3-fluoropheny l)- 1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine (CRA1001), putative novel and selective antagonists for corticotropin-releasing factor1 (CRF1) receptor were examined. Both CRA1000 and CRA1001 inhibited 125I-ovine CRF binding to membranes of rat frontal cortex with IC50 values of 20.6 and 22.3 nM, respectively. Likewise, CRA1000 and CRA1001 inhibited 125I-ovine CRF binding to membranes of rat pituitary. In contrast, both CRA1000 and CRA1001 were without affinity for the CRF2beta receptor when examined using rat heart. In mice orally administered CRA1000 and CRA1001 reversed the swim stress-induced reduction of the time spent in the light area in the light/dark exploration task. In nonstress conditions, CRA1000 and CRA1001 were without effect on the time spent in the light area in the same task in mice. Orally administered CRA1000 and CRA1001 dose dependently reversed the effects of i.c.v. infusion of CRF on time spent in the open arms in the elevated plus-maze in rats. Lesioning of olfactory bulbs induced hyperemotionality, and this effect was inhibited by either acute or chronic oral administration of CRA1000 and CRA1001 in rats. The firing rate of locus coeruleus neurons was increased by i.c.v.-infused CRF. This excitation of locus coeruleus neurons was significantly blocked by pretreatment with i.v. administration of CRA1000 and CRA1001. CRA1000 and CRA1001 had no effects on the hexobarbital-induced anesthesia in mice, the rotarod test in mice, the spontaneous locomotor activity in mice, and a passive avoidance task in rats. These observations indicate that both CRA1000 and CRA1001 are selective and competitive CRF1 receptor antagonists with potent anxiolytic- and antidepressant-like properties in various experimental animal models, perhaps through inhibition of CRF1 receptors. CRA1000 and CRA1001 may prove effective for treating subjects with depression- and/or anxiety-related disorders without the side effects seen in the related currently prescribed medications.     

The clinical effects of vasopressin receptor antagonists in heart failure

The neurohormone arginine vasopressin (AVP) is a promising target in the treatment of heart failure because AVP promotes congestion and hyponatremia, each of which is associated with poor outcomes. Diuretics are standard therapy for heart failure, but they have several limitations, including worsening renal function and hyponatremia. Blocking AVP leads to effective aquaresis, improvements in hemodynamics and renal function parameters, weight loss, and normalization of serum sodium, without changes in blood pressure or heart rate. In placebo-controlled trials in the inpatient and outpatient setting, the AVP receptor antagonist tolvaptan reduced body weight and edema and normalized serum sodium in patients with heart failure.     

Hyponatremia and the use of vasopressin receptor antagonists in critically ill patients

Hyponatremia in critically ill patients is a common and challenging problem. Increased levels of arginine vasopressin almost always contribute to the etiology. Inhibition of the vasopressin receptor with a vasopressin receptor antagonist (vaptan) is a novel approach to the treatment of hyponatremia. Vaptans are well suited to the treatment of chronic hyponatremia associated with syndrome of inappropriate anti-diuretic hormone secretion (SIADH) and hypervolemic states like cirrhosis or congestive heart failure. No data are available on the use of vaptans in acute hyponatremia, and they are not indicated in hypovolemic hyponatremia. The focus of this review is the treatment of critically ill patients with hyponatremia with vaptans and other measures.     

Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts.

We investigated immunohistochemical localization of V2 vasopressin receptor along the nephron using a specific polyclonal antibody. Staining was observed in some of thick ascending limbs and all of principal and inner medullary collecting duct (IMCD) cells. Not only basolateral but also luminal membrane was stained in collecting ducts, especially in terminal IMCD (tIMCD). To learn the functional role of luminal V2 receptor in tIMCD, we studied the luminal effects of arginine vasopressin (AVP) on osmotic water permeability (Pf), urea permeability (Pu), and cAMP accumulation using isolated perfused rat tIMCD. In the absence of bath AVP, luminal AVP caused a small increase in cAMP accumulation, Pf and Pu, confirming the presence of V2 receptor in the lumen of tIMCD. In contrast, luminal AVP inhibited Pf and Pu by 30-65% in the presence of bath AVP by decreasing cAMP accumulation via V1a or oxytocin receptors and by an unknown mechanism via V2 receptors in the luminal membrane of tIMCD. These data show that V2 receptors are localized not only in the basolateral membrane but also in the luminal membrane of the distal nephron. Luminal AVP acts as a negative feedback system upon the basolateral action of AVP in tIMCD.     

Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.     

Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene

The V2 vasopressin receptor (V2R) plays a key role in the maintenance of a normal body water balance. To generate an in vivo model that allows the physiological and molecular analysis of the role of V2Rs in kidney function, we have created mouse lines that lack functional V2Rs by using targeted mutagenesis in mouse embryonic stem cells. Specifically, we introduced a nonsense mutation known to cause X-linked nephrogenic diabetes insipidus (XNDI) in humans (Glu242stop) into the mouse genome. V2R-deficient hemizygous male pups showed a decrease in basal urine osmolalities and were unable to concentrate their urine. These pups also exhibited an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Interestingly, female mice heterozygous for the V2R mutation showed normal growth but displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis and immunoelectron microscopic studies showed that the loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1). The V2R mutant mice described here should serve as highly useful tools for the development of novel therapeutic strategies for the treatment of XNDI.     

Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.

The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.     

125I-8-L-arginine vasopressin binding to human mononuclear phagocytes.

The binding of vasopressin to human circulating blood cells was examined. Direct binding studies with preparations of single cell types indicated that the mononuclear phagocyte system is almost entirely responsible for binding of the hormone. Binding of 125I-8-L-arginine vasopressin (AVP) (40 pM) in the presence of excess unlabeled hormone was saturable (2.8 +/- 0.4 fmol/2 x 10(6) cells per ml), was linear with cell number, was dependent upon the concentration of the radioligand used, and was reversible. Binding equilibrium was achieved in 30--40 min at 22 degrees C. Scatchard analysis of binding at this time showed an apparent dissociation constant of 25 +/- 0.21 pM, providing an estimate of 640 +/- 80 sites/cell. Pretreatment of the cells with cytochalasin B, an agent that can block phagocytosis, did not modify radioligand binding, which indicates that 125I-AVP uptake by the cells is due to binding and not to endocytosis. Specificity of vasopressin-sensitive sites on mononuclear phagocytes was demonstrated with a series of vasopressin analogues with various degrees of antidiuretic potency, and with peptide hormones that bind to specific receptors on circulating blood cells but that lack antidiuretic activity. AVP (40 pM) elevated the intracellular level of cyclic AMP from 137 +/- 8.6 to 350 +/- 20.5 pmol/mg cell protein. The binding affinities of the various analogues were correlated with their ability to stimulate intracellular cyclic AMP synthesis (Lys8-vasopressin less than deamino(8-D-Arg)-vasopressin less than oxytocin).     

Study of V1a vasopressin receptor gene single nucleotide polymorphisms in platelet vasopressin responsiveness

There is a significant heterogeneity among individuals in terms of platelet aggregation response to arginine vasopressin (AVP). The aim of this study was to evaluate whether four single nucleotide polymorphisms (SNPs) in the promoter region of vasopressin V1a receptor gene (V1aR) could be used as genetic markers for divergent platelet aggregation response to AVP. Seventeen of 33 subjects showed more than 60% of maximum platelet aggregation and were classified as responders. Sixteen were classified as nonresponders because they had less than 30% aggregation. In a preliminary study, V1aR gene sequences were determined in two responders and two nonresponders. We found four SNPs in the promoter region of the V1aR gene: -6951G/A, -4112A/T, -3860T/C, and -242C/T. In all 33 subjects the genotypes of four SNPs were determined using either polymerase chain reaction (PCR) with allele-specific primers or PCR followed by restriction-fragment length polymorphism (RFLP). There were no differences in the AVP-induced aggregation between the subjects with and without variant alleles of each four SNPs. The genotype frequencies of four SNPs of V1aR were almost identical between AVP responders and nonresponders. These results suggest that the four SNPs in the promoter region of the V1aR gene may not be useful as genetic markers for platelet aggregation heterogeneity.     

Biochemical and pharmacological properties of SR 49059, a new, potent, nonpeptide antagonist of rat and human vasopressin V1a receptors.

SR 49059, a new potent and selective orally active, nonpeptide vasopressin (AVP) antagonist has been characterized in several in vitro and in vivo models. SR 49059 showed high affinity for V1a receptors from rat liver (Ki = 1.6 +/- 0.2) and human platelets, adrenals, and myometrium (Ki ranging from 1.1 to 6.3 nM). The previously described nonpeptide V1 antagonist, OPC-21268, was almost inactive in human tissues at concentrations up to 100 microM. SR 49059 exhibited much lower affinity (two orders of magnitude or more) for AVP V2 (bovine and human), V1b (human), and oxytocin (rat and human) receptors and had no measurable affinity for a great number of other receptors. In vitro, AVP-induced contraction of rat caudal artery was competitively antagonized by SR 49059 (pA2 = 9.42). Furthermore, SR 49059 inhibited AVP-induced human platelet aggregation with an IC50 value of 3.7 +/- 0.4 nM, while OPC-21268 was inactive up to 20 microM. In vivo, SR 49059 inhibited the pressor response to exogenous AVP in pithed rats (intravenous) and in conscious normotensive rats (intravenous and per os) with a long duration of action (> 8 h at 10 mg/kg p.o). In all the biological assays used, SR 49059 was devoid of any intrinsic agonistic activity. Thus, SR 49059 is the most potent and selective nonpeptide AVP V1a antagonist described so far, with marked affinity, selectivity, and efficacy toward both animal and human receptors. With this original profile, SR 49059 constitutes a powerful tool for exploring the therapeutical usefulness of a selective V1a antagonist.     

Differentiation of arginine vasopressin antagonistic effects by selective V2 versus dual V2/V1a receptor blockade in a preclinical heart failure model

Arginine vasopressin (AVP) is increased in patients with heart failure (HF). Its actions are linked to free water reabsorption (V2-) and arteriolar vasoconstriction (V1a receptor). AVP can exacerbate the cardiorenal syndrome with excess fluid retention and afterload increase. Tolvaptan (TOL; selective V2 antagonist) and Conivaptan (CON; dual V1a/V2 antagonist) are two AVP antagonists that counteract the action of AVP with distinct profiles. We investigated the therapeutic effects of CON and TOL in an acute HF model. Mongrel dogs were paced continuously at 220 beats/min. After 14 days, the animals underwent acute testing. Dogs were instrumented to measure cardiac output, blood pressure, pulmonary artery pressure, and left ventricular dP/dtmax. Additionally, during the acute experiments, vasopressin was infused intravenously (4 mU/kg/min) to achieve constant and controlled pathophysiological levels of AVP. Subsequently, animals received either CON or TOL (n = 6; 0.1-mg/kg bolus). There were no significant differences in effect on mean arterial pressure, dP/dtmax, central venous pressure, and urine output between CON and TOL. In contrast, cardiac output increased by 0.15 l/min after CON and decreased by 0.6 l/min after TOL (P < 0.01). Accordingly, the total peripheral resistance increased after TOL by 250 dyn*s/cm and decreased after CON by 125 dyn*s/cm (P < 0.01). In conclusion, it was demonstrated that in an acute HF model, CON lowered, whereas TOL increased afterload. The results suggest that dual V1a/V2 blockade in the acute HF setting could be beneficial compared with selective V2 blockade. Chronic experiments are needed to determine whether this finding can translate into a sustained clinical advantage.     

Blocking effects of V1 (OPC-21268) and V2 (OPC-31260) antagonists on the negative inotropic response to vasopressin in isolated dog heart preparations.

We investigated the effects of V1 IOPC-21268, 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2( 1H)- quinolinone) and V2 (OPC-31260, 5-dimethylamino-1[4-(2-methylbensoylamino)benzoyl]-2,3,4,5-tetrahy dro-1H- benzazepine) vasopressin receptor antagonists on the negative inotropic response to arginine vasopressin (AVP) in the isolated perfused heart preparations of the dog. AVP (7.5-750 pmol) decreased the atrial and ventricular contractile force when the preparation was perfused with constant pressure or constant flow. AVP induced a small increase in sinus rate. Desmopressin, a selective vasopressin V2 agonist, did not change the sinus rate and atrial or ventricular contractile force. OPC-21268 (0.01-3 mumol) and OPC-31260 (0.01-1 mumol) induced a small negative inotropic effect. Both OPC-21268 and OPC-31260 inhibited the negative inotropic response to AVP in a dose-dependent manner. The doses of 50% inhibition (ID50) for OPC-21268 and OPC-31260 on the inotropic effect were 0.30 +/- 0.16 mumol and 0.084 +/- 0.034 mumol, respectively. Neither OPC-21268 nor OPC-31260 affected the acetylcholine-, adenosine- or norepinephrine-induced inotropic and chronotropic effects. It has been reported that the concentration of OPC-21268 that displaced 50% of specific AVP binding is 0.4 microM for V1 receptors and > 100 microM for V2 receptors and the concentration of OPC-31260 is 0.01 microM for V2 receptors and 1 microM for V1 receptors. We, therefore, suggest that AVP directly causes negative inotropic effects mediated at least in part by V1 receptors in the dog heart.     

Detection of a high-affinity prostaglandin I2 binding site in the human thyroid

This study is concerned with the identification and the pharmacological properties of PGI2 receptor binding sites on human thyroid membrane fractions. Scatchard analysis is not linear, revealing a high- and a low-affinity receptor binding site. (3 H)Iloprost binding experiments were performed under various clinical conditions: in thyroid cancer the low-affinity binding sites disappear totally and the specific high-affinity binding sites are diminished according to the grade of differentiation of the cancer. An alteration in Bmax and Kd is also observed in cold nodules, in Hashimoto's and Riedl's thyroiditis and in hyperthyroidism, whereas hot nodules exhibit an increase in both the receptor subpopulations. The data provide evidence for specific PGI2 binding sites and support the suggestion of a direct regulatory key-role of PGI2 in thyroid intermediary metabolism.