PTH Antagonist Research Articles

Parathyroid hormone stimulates prostanoid formation in mouse calvarial bones

Bovine parathyroid hormone (bPTH 1-34) caused a time- and dose-dependent enhanced formation of the two prostanoids PGE2 and 6-keto-PGF1 alpha in cultured neonatal mouse calvarial bones, with threshold for action at 0.1 nmol/l. The PGE2 response to PTH was completely blocked by indomethacin, but insensitive to calcitonin. By contrast, indomethacin was without effect on 45Ca release induced by PTH. The PTH analogues (Nle 8, 18, Tyr 34)-bPTH (3-34) amide and (Tyr 34)-bPTH (7-34) amide, which are putative PTH antagonists, did not affect basal production of PGE2, nor did the analogues affect bPTH 1-34 induced PGE2 formation. The data show that PTH stimulates prostanoid formation in mouse bone cells and that this response is not directly linked to PTH-induced bone resorption.

A tumor-secreted protein associated with human hypercalcemia of malignancy. Biology and molecular biology.

This investigation addresses a theoretical concept of tumor pathogenesis proposed over 40 years ago, namely that malignancy-associated hypercalcemia can result from endocrine secretion by tumors of a PTH-like factor. These studies demonstrate that a fragment of hHCF alone, without added or tumor-secreted cofactors or hormones, can produce hypercalcemia and other biochemical abnormalities associated with HHM. The hypercalcemia can be generated by hHCF-(1-34)NH2 action on bone, although kidney and gut could contribute to the HHM syndrome when it occurs naturally. No other tumor-secreted peptide displays this biological profile. These studies establish one (PTH-like) mechanism by which human tumors could produce hypercalcemia. Furthermore, the finding that hHCF-(1-34)NH2 is more potent than PTH in some systems is of considerable interest for the future design of hormone analogs. A broad spectrum of biological properties of hHCF-(1-34)NH2, including production of components of the HHM syndrome, can be inhibited by a PTH antagonist. Because [Tyr-34]bPTH-(7-34)NH2 selectively and competitively occupies PTH receptors, our studies demonstrate formally that hHCF-(1-34)NH2 mediates some (and perhaps all) of its actions via receptors conventionally regarded as intended for interaction with PTH, but which actually may be present to allow for expression of bioactivity of both secreted proteins. Although some structural homology is shared by the two hormones and many contribute to interaction with receptors, the disparity in structure, especially within the 1-34 domains responsible for bioactivity in both hormones, is more pronounced. The similarity in biological profiles despite structural differences between hHCF and PTH is emphasized by the inhibitory action of [Tyr-34]bPTH-(7-34)NH2 against the tumor peptide even in the absence of much of the homologous region in the PTH antagonist. This investigation provides impetus for designing more potent antagonists, which must now be regarded more appropriately as inhibitors of both PTH and hHCF. Such antagonists may best be generated from hybrid structures of the two hormones. In any case, these studies establish a promising new approach to therapy of tumor-associated hypercalcemia.

Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the opossum kidney cell.

Cathepsin-D has been previously reported to cleave intact PTH into PTH-(1-34) and -(35-84) in membranous fractions of rat and bovine kidney. Whether PTH degradation occurs by intact kidney cells, however, has not been examined in detail. We have, therefore, examined this possibility using an opossum kidney (OK) cell line which possesses the characteristics of proximal renal tubules and responds to PTH. PTH radioimmunoreactivity recovered in trichloroacetic acid-soluble products and in fractions eluted from reverse phase HPLC was measured using an antibody directed to the midregion and C-terminus of PTH. In this study, intact OK cells, but not extracellular enzymes, cleaved human (h) PTH-(1-84) into three discrete fragments which were released into the medium in a time- and temperature-dependent fashion. Half-maximal velocity of PTH-degrading activity (PTHDA) was observed at 9 nM hPTH-(1-84). A 1000-fold molar excess of PTH antagonists [hPTH-(3-34) and [Tyr34]hPTH-(7-34)amide] markedly inhibited PTHDA, whereas ACTH, glucagon, or big gastrin did not suppress it, suggesting an involvement of the PTH receptor in PTHDA. This PTHDA was strongly inhibited by phenylmethylsulfonylfluoride and chymostatin, but not by trypsin inhibitor, elastatinal, or inhibitors of aspartic, cysteine, or metalloproteinases, suggesting that it is due to a seryl chymotrypsin-like endopeptidase. Analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by UV absorbance (210 nm), three of which were measurable by PTH RIA, and each corresponded to the three PTH fragments produced by OK cells. All three fragments were predominantly suppressed in the presence of chymostatin, suggesting that chymotrypsin-like activity is solely responsible for PTHDA in intact OK cells. To further explore the cleavage sites of PTH by chymotrypsin, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the conclusion that a chymotrypsin-like enzyme in OK cells cleaved the hormone between residues 23-24, and 34-35 to produce, at least, hPTH-(24-84) and -(35-84). Lysosomal blockers (chloroquine, ammonium chloride, or monensin) did not affect this PTHDA. Our present study indicates that chymotrypsin-like endopeptidase, but not other endopeptidase or lysosomal enzymes, is responsible for the limited hydrolysis of PTH by intact OK cells.

Evaluation of novel parathyroid hormone analogs using a bovine renal membrane receptor binding assay.

A PTH membrane receptor binding assay based on a stable hormone analog radioligand was refined and used with bovine renal cortical membranes to evaluate PTH reference peptides and novel analogs of the hormone. Systematic studies were performed to optimize several aspects of the receptor binding assay. The sulfur-free agonist analog [Nle8,18,Tyr34]bovine PTH-(1-34)NH2 was iodinated using Iodogen. The monoiodinated derivative was purified and isolated by reverse phase HPLC. Immediate dilution of the purified radioligand in albumin-containing buffer and cold storage of aliquots yielded a tracer that was stable for at least 2 months. When used in the binding assay, the radioligand displayed specific, high affinity, and saturable binding to both bovine and canine renal cortical membranes. The Kd values were 0.47 +/- 0.07 and 0.63 +/- 0.08 nM for bovine and canine membranes, respectively. Maximum binding values were 475 +/- 46 and 395 +/- 48 fmol/mg protein for bovine and canine membranes, respectively. Furthermore, when used on a routine basis, this assay system proved reliable and reproducible. A series of previously characterized PTH agonists and antagonists was tested as reference peptides to validate this assay. Inhibition of binding by agents was dose dependent, parallel concentration-dependent binding curves were observed, and a close correlation between binding affinity and adenylate cyclase activity was obtained. In addition, a series of novel analogs designed to determine the consequences of sequence elongation at the N- and/or C-terminus or incorporation of amino acid substitutions to enhance resistance to enzymatic degradation were examined. With the exception of C-terminal extension from position 34 to position 38, which diminished receptor affinities, all modifications retained full biological activity. This refined receptor binding assay should facilitate future studies of newly designed PTH analogs.

Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells.

PTH receptors on two stable clonal rat osteosarcoma cell lines, ROS 17/2 and ROS 17/2.8, were characterized using an HPLC-purified, synthetic, sulfur-free, radioiodinated analog of bovine PTH, [Nle8,Nle18,Tyr,34]bovine PTH-(1-34)amide. PTH binding is specific for PTH agonists and antagonists and is dependent on the time and temperature of incubation. There is an excellent correlation between binding affinities of PTH agonists and antagonists in these intact cell systems with those in canine renal membranes. Peptides unrelated to PTH do not bind. Both ROS 17/2 and 17/2.8 have a single class of saturable, high affinity PTH binding sites that, by kinetic analysis and Scatchard analysis of saturation and competition studies, has a dissociation constant (Kd) of 0.8-1.4 nM. Bmax is approximately 36,000 and 72,000 sites per cell in ROS 17/2 and 17/2.8, respectively. A close correlation was found between the binding of PTH agonists to their receptors in ROS 17/2 cells with their relative biological potencies as measured by stimulation of adenylate cyclase in plasma membranes prepared from these cells. Prolonged treatment of ROS 17/2 and 17/2.8 cells with PTH agonists results in a dose- and time-dependent decrease of available cell-surface binding sites, without alterations in Kd. PTH antagonists do not regulate PTH receptors. Regulation of PTH receptors by PTH agonists is dependent on the dose and time of exposure to ligand over a dose range of 10(-8) to 10(-11) M. Cells exposed to agonists (greater than or equal to 10(-8) M) for 48 h show maximally decreased receptor number; continued exposure to agonists (greater than or equal to 10(-8) M) does not further decrease PTH receptor number, which remains constant at about 15% of control values. Agonist-induced down-regulation occurs with less than 10(-11) M agonists, a concentration less than 10% of the minimal dose detected by direct ligand competition. Treatment of ROS 17/2 cells with PTH agonists results in a dose- and time-dependent decrease of PTH-stimulated adenylate cyclase. This agonist-induced desensitization correlates closely with the decreased availability of PTH receptors: it is maximal in cells exposed to agonists (greater than 10(-8) M) for 48 h and also does not decrease further with continued exposure of the cells to agonist. Future studies with these stable ROS cell lines should permit detailed analysis of the biochemical mechanisms underlying homologous and heterologous regulation of PTH receptors and desensitization and sensitization of the adenylate cyclase response.

Evaluation of a parathyroid hormone antagonist in an in vivo multiparameter bioassay.

The antagonist properties of a bovine parathyroid hormone analogue ([Tyr34]bPTH-(7-34] amide were quantitatively assessed in vivo in a multiparameter assay to estimate the potency of the antagonist against the major actions of PTH. The analogue inhibited PTH-stimulated urinary excretion of phosphate and adenosine 3',5'-cyclic monophosphate in vitamin D-deficient thyroparathyroidectomized rats in a dose-dependent manner. At a molar dose ratio as low as 5:1 of antagonist to PTH, partial inhibition occurred. PTH stimulates the activity of 25-hydroxyvitamin D3-1 alpha-hydroxylase in renal proximal tubules. When coinfused with PTH, this analogue completely inhibited PTH-stimulated 1 alpha-hydroxylase activity at a molar dose ratio of 25:1 of antagonist to PTH and partially inhibited the activity at a molar dose ratio of 10:1. The analogue revealed no PTH-like agonist activity for stimulation of the 1 alpha-hydroxylase. Taken together, these studies indicate that [Tyr34]bPTH-(7-34) amide is a potent antagonist of several of the parameters of PTH action in vivo and demonstrate the feasibility of designing a PTH antagonist that can interact simultaneously with all the PTH receptors responsible for the hormone's major actions in vivo.

Structure-activity relationship of parathyroid hormone: relative sensitivity of rabbit renal microvessel and tubule adenylate cyclases to oxidized PTH and PTH inhibitors

It has been shown previously that secondary structural changes of bPTH-(1-34) (synthetic amino-terminal (1–34) fragment of bovine parathyroid hormone), obtained by oxidation of the methionines 8 and 18, abolished its hypotensive but not its hypercalcemic action. Hence, it has been postulated that the various physiological effects of the hormone are mediated by different receptors that require different regions or configurations of the peptide. To further examine this hypothesis the relative sensitivity of the PTH-responsive adenylate cyclase of microvessels and tubules isolated from rabbit kidney cortex, to oxidized PTH and PTH inhibitors, was examined. In the presence of GTP, bPTH-(1-34) stimulated both microvessel and tubule adenylate cyclase in a dose-dependent fashion and with analogous affinities (ED 50 = 52 nM in the microvessels and 85 nM in the tubules). Hydrogen peroxide treatment of bPTH-(1-34) resulted in the loss of the adenylate cyclase stimulating potency in the microvessels while there was substantial enzyme activation (ED 50 = 900 nM) in the tubules. Oxidized PTH inhibited the untreated PTH-stimulated adenylate cyclase, suggesting that oxidized PTH still retains an affinity for vascular receptor sites. Similar treatment of the sulfur-free PTH analog [Nle 8,18, Tyr 34]bPTH-(1-34)NH 2, where methionines have been replaced by norleucine, had little or no effect in both fractions. In the microvessels the synthetic PTH antagonist analogs [Nle 8,18, Tyr 34]bPTH-(3-34)NH 2 and [Tyr 34]bPTH-(7-34)NH 2, strongly inhibited the adenylate cyclase responses to bPTH-(1-34). No inhibition was seen in the tubules with the same molar ratios of inhibitor to native PTH. Together, these results suggest strongly that the differences in the adenylate cyclase response to various PTH fragments most likely represent a difference in the structural requirements for PTH actions between microvessels and tubules.

Skin-derived fibroblasts respond to human parathyroid hormone-like adenylate cyclase-stimulating proteins.

Human tumors and keratinocyte-conditioned medium contain PTH-like adenylate cyclase-stimulating proteins. Human dermal fibroblasts have receptors that recognize PTH and a factor associated with humoral hypercalcemia of malignancy in rats. We examined 10 human dermal fibroblast lines for an adenylate cyclase response to PTH. Six of 10 lines tested displayed a definite response (2.4- to 3.8-fold over basal) to 10(-6) M bovine PTH-(1-34). This response was inhibited by the PTH analog and antagonist Nle8,18,Tyr34-bPTH-(3-34). We also examined whether human dermal fibroblasts are capable of responding to either a human PTH-like tumor-derived factor or the PTH-like factor contained in human keratinocyte-conditioned medium. Both human humoral hypercalcemia of malignancy-associated tumor extract (2.5 X 10(-10) M) and keratinocyte-conditioned medium (8 X 10(-10) M) stimulated human dermal fibroblast adenylate cyclase. These concentrations are markedly lower than those required for PTH-induced adenylate cyclase stimulation. This activity was also inhibited by the PTH analog. The high prevalence of PTH-responsive adenylate cyclase in dermal fibroblast lines and the apparent potency of tumor-derived and keratinocyte-derived PTH-like factors in dermal fibroblasts suggest that these factors may play a role in normal dermal physiology.

Parathyroid hormone-like adenylate cyclase-stimulating activity from a human carcinoma is associated with bone-resorbing activity.

We found previously that a human renal carcinoma cell line derived from a hypercalcemic patient induces humoral hypercalcemia when grown as allografts in the nude mouse and secretes a protein that activates adenylate cyclase via the PTH receptor. The purpose of this study was to examine the conditioned medium of this cell line for bone-resorbing activity in vitro. Processed conditioned medium produced dose-dependent stimulation of bone resorption in cultured fetal rat limb bone explants. Two PTH antagonists were used to assess the PTH receptor dependence of this bone-resorbing activity. Neither [8Nle,18Nle,34Tyr]bovine (b) PTH-(3-34) amide nor [34Tyr]bPTH-(7-34)amide inhibited bone resorption or limb bone cAMP accumulation induced by either processed conditioned medium or equivalent concentrations of bPTH-(1-34). As an alternate means to assess whether this tumor-derived PTH-like protein had intrinsic bone-resorbing activity, the latter was measured during partial purification of PTH-like adenylate cyclase-stimulating activity (ACSA) from conditioned medium by consecutive gel filtration and reverse phase HPLC. The bone-resorbing activity in conditioned medium could not be resolved from PTH-like ACSA by these two separation techniques, indicating that the activities may be intrinsic to the same protein. These results are consistent with the view that a tumor-derived protein with PTH-like ACSA and bone-resorbing activity may be responsible for hypercalcemia in vivo.

Endothelium-Independent Linkage of Parathyroid Hormone Receptors of Rat Vascular Tissue with Increased Adenosine 3′,5′-Monophosphate and Relaxation of Vascular Smooth Muscle*

We examined the mechanisms involved in the relaxation of rat vascular smooth muscle by PTH. PTH increased intracellular cAMP 10-fold in cultured vascular smooth muscle cells from rat aorta. Forskolin, methylisobutylxanthine, and papaverine all potentiated PTH action. The cAMP responses to PTH were not altered by concurrent addition of propranolol, phentolamine, atropine, or [Sar1,Ile8]angiotensin II. Only the synthetic PTH antagonist analog [Nle8,Nle18,Tyr34] bovine PTH-(3-34) inhibited the cAMP and vascular relaxation responses to PTH. Isoproterenol produced increases in intracellular cAMP and adenylate cyclase activity which were additive to those produced by PTH. In contracted rat aortic strips, PTH caused a dose-dependent relaxation which was not altered by removal of the vessel intima or treatment with nordihydroguaiaretic acid. Also, membrane preparations from intact aortas or aortas with the endothelium or adventitia removed displayed identical PTH-stimulated adenylate cyclase activities. These findings indicate that the relaxant action of PTH in rat aorta does not require an intact endothelium and results from a direct effect on the vessel medial layer. Relaxation appears to be mediated by a receptor unique for PTH which is linked to the adenylate cyclase of vascular smooth muscle cells.

Two species of adenylate cyclase-stimulating activity in a murine squamous carcinoma model of humoral hypercalcemia of malignancy.

PTH receptor-stimulating proteins may be a common mediator of humoral hypercalcemia of malignancy (HHM). Such proteins exhibit adenylate cyclase-stimulating activity (ACSA) in PTH-sensitive assays, and ACSA has been used to follow their purification. Acid/urea tumor extracts from a murine squamous carcinoma model of HHM were previously shown to have very high ACSA, which was partially, but incompletely, inhibited by the PTH antagonist Nle8,18,Tyr34-bovine PTH-(3-34) amide. ACSA from murine tumor extracts has now been further purified using solvent fractionation and reverse phase HPLC. Approximately half of the ACSA is attributable to a family of three proteins (peaks IA, IB, and IC) with properties characteristic of the PTH receptor-stimulating protein extracted from rat Leydig cell and human HHM tumors. The ACSA in these three peaks of murine tumor extract elutes in the same region as human tumor ACSA on reverse phase HPLC, has a dose-response curve parallel to that of PTH, and is fully inhibited by the PTH-(3-34) antagonist in both the renal cortical and rat osteosarcoma (ROS) adenylate cyclase assays. The remaining half of the ACSA from murine tumor extracts elutes as a single peak (peak II) at a higher acetonitrile concentration on reverse phase HPLC. In the renal cortical assay, its dose-response curve differs from that of PTH, its ACSA is not affected by the PTH-(3-34) antagonist, and it potentiates PTH- or peak I-stimulated adenylate cyclase activity. In the PTH-sensitive intact cell ROS assay, peak II exhibits no ACSA. We conclude that the potent ACSA of murine tumor acid/urea extract results in large part from amplification of the PTH-specific ACSA (peak I) by peak II. Peak II is a distinct protein, not previously reported in tumor extracts, that may act as a postreceptor step in the adenylate cyclase system.

Purification of Peptides with Parathyroid Hormone-Like Bioactivity from Human and Rat Malignancies Associated with Hypercalcemia*

We have purified peptides with PTH-like bioactivity from a rat Leydig cell tumor (H-500) and a human squamous cell carcinoma, both associated with a syndrome of humor-induced hypercalcemia. Tumor extracts were shown to be active in an in vitro renal cytochemical bioassay and in an in vitro osteosarcoma cell (UMR 108) adenylate cyclase assay; activity in both assays could be reduced by the PTH antagonist [norleucine-8,18,tyrosine-34]bovine PTH-(3-34)-amide. Partially purified extracts of both tumors and of rat tumor-conditioned culture medium were active in vivo in thyroparathyroidectomized rats in preventing hypocalcemia and increasing fractional phosphorus excretion and cAMP excretion. Ion exchange chromatography demonstrated that active peptides were basic in character. Employing reverse phase HPLC and gel permeation HPLC, active peptides of approximately 9,000 and 9,500 daltons were purified from extracts of the human and rat tumors, respectively, which had similar but not identical compositions. Two additional bioactive peptides were detected in rat tumor extract, and the more active had a mol wt of approximately 28,000. The results demonstrate that peptides that mimic PTH in a variety of in vivo and in vitro bioassays can be extracted from malignancies associated with hypercalcemia, that multiple molecular species may be detected in tumors that demonstrate PTH-like activity, and that at least one of these peptides may be similar in two tumors of highly divergent cell and species origin.

Formation of parathormone 8–34 by cathepsin-D digestion of parathormone and its efficacy as a hormone antagonist

It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe 34 and Val 35 yielding PTH(1–34) and PTH(35–84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8–34) and PTH(1–7). Time course studies demonstrated that these 2 fragments are formed from the (1–34) peptide generated through the initial cleavage at Phe 34-Val 35 of PTH. The identification of the bPTH(8–34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8–34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8–34) did not affect the PTH-stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response.

A factor produced by cultured rat Leydig tumor (Rice 500) cells associated with humoral hypercalcemia stimulates adenosine 3',5'-monophosphate production via the parathyroid hormone receptor in human skin fibroblasts.

The Rice-500 Leydig cell tumor of Fischer rats is associated with humoral hypercalcemia in vivo and produces a factor that stimulates cAMP formation in cultured rat osteosarcoma cells. We found that cultured human skin fibroblasts respond to both human PTH-(1-34) and the factor produced by cultured rat Leydig tumor cells with a dose-dependent rise in cAMP formation. The time courses for stimulation of the two agents were similar, and stimulation by both was blocked by the competitive PTH antagonist [8,18-norleucine,34-tyrosine]bovine PTH-(3-34) amide. These data suggest that PTH-like factors secreted by a murine tumor are capable of interacting with the human PTH receptor.

Distribution of parathyroid hormone-sensitive adenylate cyclase in isolated rabbit renal cortex microvessels and glomeruli

The effect of the synthetic amino-terminal fragment of bovine parathyroid hormone, bPTH-(1–34), on the adenylate cyclase of microvessels and glomeruli isolated from rabbit kidney cortex was studied in the presence and absence of guanosine triphosphate (GTP). bPTH-(1–34) stimulated the vascular and glomerular adenylate cyclase in a dose-dependent manner with apparent ED 50 values of 11.5 nM and 64 nM respectively, in the absence of GTP. 10 −4M GTP greatly amplified the vascular response to bPTH-(1–34) while, in the glomeruli, both GTP, and bPTH-(1–34) had only additive effects. In the presence of GTP, vascular and glomerular apparent ED 50 were 190 nM and 64 nM respectively. [Nle 8, Nle 18, Tyr 34] -bPTH-(3–34) amide, described as a PTH antagonist, inhibited the action of bPTH-(1–34) in the microvessels and to a lesser extent in the glomeruli. PTH is therefore a potent stimulator of adenylate cyclase in rabbit renal microvessels and glomeruli, and may play a role in the regulation of renal blood flow and glomerulo-tubular feedback control.

Parathyroid Hormone Receptors Coupled to Cyclic Adenosine Monophosphate Formation in an Established Renal Cell Line*

We examined the relationship between PTH binding and stimulation of cAMP formation in a cell line derived from opossum kidney (OK). In the presence of isobutylmethylxanthine (1 mM) bovine PTH(1-34) [bPTH(1-34)] (244 nM) stimulated cAMP accumulation in confluent cultures up to 40-fold over basal; this response to PTH was stable for 35 passages. The concentration of bPTH(1-34) required to raise cell cAMP levels half-maximally was 5-12 nM. Binding of [125I]bPTH(1-34) to OK cells was saturable; Scatchard analysis of competitive binding data yielded a dissociation constant (KD) = 6 +/- 2 nM, with 1.0 pmol binding sites/mg cell protein. Under steady state binding conditions 89% of labeled PTH remained precipitable by 10% trichloroacetic acid, suggesting minimal metabolism of the hormone. The PTH antagonist (8Nle, 18Nle, 34Tyr)bPTH(3-34)amide competed for [125I]bPTH(1-34) binding sites and inhibited the action of bPTH(1-34) to raise cAMP levels. The intact PTH molecule, bPTH(1-84), and the weak agonist hPTH(1-34) synthesized by Brewer were both less potent than bPTH(1-34) (6 times and 30 times, respectively) with regard to binding and cAMP production. Calcitonin and arginine vasopressin did not bind to PTH receptors but raised cAMP levels in OK cell cultures 3- and 10-fold, respectively; neither glucagon nor ACTH(1-24) influenced PTH binding of cAMP in OK cells. Varying the extracellular calcium concentration in the medium bathing cells did not influence basal or PTH-stimulated cAMP generation. These data suggest that PTH receptors in OK cells are of high affinity, are selective for PTH, and are coupled to adenylate cyclase. This established epithelial cell line provides a model in which to study the mechanism of action of PTH in the kidney.

Coupling of the canine renal parathyroid hormone receptor to adenylate cyclase: modulation by guanyl nucleotides and N-ethylmaleimide.

Guanyl nucleotides greatly enhance PTH stimulation of renal adenylate cyclase activity. As part of an investigation of the mechanism of this potentiation, we evaluated the effect of guanosine triphosphate (GTP) and a GTP analog, guanylimidodiphosphate [Gpp(NH)p] on the specific binding of l25I-labeled bovine (b) PTH-(l–34) to canine renal cortical plasma membranes. Dissociation of [l25I]bPTH-(l–34) from receptors was slow (t½ 200 min) in the absence of added guanyl nucleotide, whereas in the presence of GTP or Gpp(NH)p (100 μM), dissociation was rapid (t½ 1 min). The increase in dissociation rate prompted by Gpp(NH)p was reflected in a 3- to 4-fold decrease in PTH receptor affinity, from 5 nM to 17 nM. In contrast to the slow dissociation rate of the l25I-labeled agonists [Nle8,Nle18,Tyr34]bPTH-(l–34)amide and bPTH(l–34), the dissociation rate of the l25I-labeled antagonist analog of PTH [Nle8,Nle18,Tyr34]bPTH-(3–34)amide was rapid (t½ 2 min). Moreover, binding of the labeled PTH antagonist analog was not...