Inactivation by plasma may be responsible for lack of efficacy of parathyroid hormone antagonists in hypercalcemia of malignancy.

PTH-related protein (PTHrP) has been shown to be a major factor responsible for hypercalcemia of malignancy. PTHrP acts via the PTH/PTHrP receptor, and therefore, PTH antagonists might be expected to reverse the hypercalcemia in malignancy. In the present studies, the PTH antagonists [Tyr34]bovine (b) PTH-(7-34)NH2, [D-Trp12,Tyr34]-bPTH-(7-34)NH2, or PTHrP-(7-34)NH2, were administered to hypercalcemic athymic nude mice bearing a human squamous cell carcinoma of the lung in 60- to 500-fold molar excess of a dose of PTHrP-(1-34) known to produce hypercalcemia. The antagonists had no significant effect on serum calcium levels. In an adenylyl cyclase assay using the ROS 17/2.8 cells, a potent PTH antagonist, [Leu11,D-Trp12]PTHrP-(7-34)NH2 was rapidly inactivated in the presence of rat or human plasma. This inactivation by plasma was not blocked by common inhibitors of proteolysis (aprotinin, soybean trypsin inhibitor, and leupeptin). Preliminary studies demonstrated that inactivation of the PTHrP antagonist was caused by a plasma component with an apparent mol wt of 230,000 daltons. The knowledge of the structure of the PTH/PTHrP receptor combined with the identification of a hormone-inactivating plasma factor should facilitate the design of PTH-antagonists that are effective in vivo.

A novel, mild, specific and indirect maleimido-based radioiodolabeling method. Radiolabeling of analogs derived from parathyroid hormone (PTH) and PTH-related protein (PTHrP).

In an effort to design a mild, non-oxidative and site-specific means of radiolabeling bioactive molecules we have employed maleimido-sulfhydryl chemistry to produce bioactive hormone radioligands. We have prepared two novel radioiodolabeled reagents, 3'-maleimidopropanoyl-3-125I-tyramide and its retro analog, N-maleoyl-N'-3-(4-hydroxy-3-125I-phenyl)propanoyl ethylenediamide, by either oxidative radioiodination of the precursors or radiolabeling of the phenolic component prior to its incorporation into the radiolabeling reagents. These reagents were then used to radiolabel analogs of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) in an efficient way, yielding reaction mixtures which were easily purified. The radioligands obtained are stable upon storage and bind in a reversible manner to a single population of binding sites displaying affinity in the low nanomolar range. The potencies of these analogs are comparable to the non-modified PTH and PTHrP analogs. This study demonstrates the utility of the novel maleimido-based indirect radioiodination approach and highlights some of its advantages over either direct oxidative procedures or acylation using the Bolton-Hunter reagent.

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.

A pharmacological comparison of parathyroid hormone receptors in human bone and kidney.

While abundant information is available characterizing PTH receptor properties in other species, data on human PTH receptors is very limited. We have been interested in the possibility that tissue-specific differences among human PTH receptors (i.e. bone vs. kidney) might exist. We have, therefore, compared pharmacological profiles for a wide array of PTH and PTH-related peptide (PTHrP) analogs in human osteoblast-like cells (SaOS-2) and human renal cortical membranes (RCM) using radioiodinated (Tyr36)hPTHrP(1-36)NH2 as a probe for PTH receptor function. The rank order of receptor affinity for 10 PTH/PTHrP receptor agonists tested was very similar in the bone and kidney assay systems. Binding affinity for these peptides was greater in human (h)RCMs and SaOS-2 membranes than in SaOS-2 intact cells. The relative binding affinities for (Tyr36)hPTHrP(1-36)amide, hPTH(1-34), bovine (b)PTH(1-34), and rat PTH(1-34) were similar in human RCMs, SaOS-2 membranes, and SaOS-2 cells. bPTH(1-84) and hPTHrP(1-74) both manifested lower receptor affinity than the amino-terminal analogs. Seven PTH/PTHrP receptor antagonists were also studied in this homologous human assay system. The binding affinity for hPTHrP(7-34)NH2 was 2- to 3-fold greater than that for (Tyr34)bPTH(7-34)NH2 in RCM and SaOS-2 membranes. However, in SaOS-2 cells, a striking reversal in the relative binding affinities was observed; the PTHrP(7-34) analog was nearly 3-fold less potent than (Tyr34)bPTH(7-34)NH2, underscoring a difference between intact and broken cell preparations. Two hybrid PTH-PTHrP receptor antagonists demonstrated similar relative affinity to each other in the human bone and kidney assay systems. Affinity cross-linking of receptors in human renal and skeletal tissues demonstrated an indistinguishable dominant 85-kilodalton receptor protein. We conclude that the binding and bioactivity profiles of a broad array of PTH and PTHrP peptides are very similar or identical in human renal and skeletal tissues. Differences relating to intact vs. broken cell preparations accounted for some variation in potency. These studies emphasize the importance of employing homologous assay systems to study PTH receptor function and the existence of interspecies differences among PTH receptors. The results support the possibility that PTH receptors in human bone and human kidney are very similar if not identical.

Expression of the parathyroid hormone-related protein gene in the avian oviduct: potential role as a local modulator of vascular smooth muscle tension and shell gland motility during the egg-laying cycle.

The phylogenetic conservation of the primary structure of PTH-related protein (PTHrP) supports an important, yet undetermined, role(s) for this molecule in the biology of birds and mammals. As an initial step toward understanding the function of PTHrP in birds, we investigated the expression of PTHrP mRNA in tissues of the egg-laying hen. This analysis revealed that PTHrP mRNA is expressed at various levels in lung, brain, heart, and tissues of the digestive tract, including the proventriculus (secretory stomach), gizzard, and small intestine. In the oviduct tissues of adult birds, PTHrP mRNA was detected in the isthmus (membrane-secreting) and shell gland (calcium-secreting) portions, but not in magnum (albumin secreting) tissue. During oviduct development, high levels of PTHrP mRNA present in the oviducts of the 12-week-old bird suggest a role for PTHrP in oviduct development. Interestingly, as the oviduct matures, relatively high levels of PTHrP mRNA segregate with the distal tissues that ultimately differentiate into the isthmus and shell gland (uterus). To address a possible role for PTHrP in the differentiated function of the shell gland, we followed the expression of PTHrP in the shell gland at different times in the laying cycle and found levels of PTHrP to transiently increase as the egg moves through the oviduct, gradually returning to basal levels in the 15-h calcification period. We localized the cycle-associated fluctuations in PTHrP mRNA levels to the shell gland serosa and smooth muscle layer. Immunoreactive PTHrP was localized to the serosal membrane as well as the smooth muscle layer of serosal arterioles, suggesting that PTHrP may modulate vascular smooth muscle activity. In support of this hypothesis, synthetic chicken PTHrP (1-34)NH2 was found to relax the resting tension of isolated shell gland blood vessels in a dose-dependent manner. Together, these data indicate that the expression of the PTHrP gene in the avian oviduct is both temporally and spatially regulated during the egg-laying cycle and that PTHrP may function as an autocrine/paracrine modulator of shell gland smooth muscle activity of both ductal and vascular origins. The vasorelaxant property of N-terminal fragments of PTHrP supports a role for this molecule in the temporal increase in blood flow to the shell gland during egg calcification.

Solid-phase synthesis and biologic activity of human parathyroid hormone (1-84).

We have chemically synthesized the full-length, 84 amino acid, human parathyroid hormone (hPTH) on a greater than 100 mg scale by the Merrifield solid-phase technique of stepwise peptide synthesis using a benzhydrylamine support. The peptide was purified by high-performance liquid chromatography and found to be greater than 96% pure. The authenticity or the sequence of the synthetic peptide was confirmed by repetitive Edman degradation. Furthermore, tryptic digestion of hPTH generated the predicted fragments. The synthetic full-length hormone was evaluated for biologic activity in assays of PTH receptor binding and stimulation of adenylate cyclase activity (using bovine renal cortical membranes and rat and human bone cells). Synthetic hPTH (1-84) was found to be highly potent in binding to PTH receptors (Kb = 1-25 nM) and stimulating adenylate cyclase (Km = 1-14 nM). The availability of significant quantities of synthetic full-length hPTH and future analogs will permit widespread use in multiple in vitro and in vivo assays to delineate their spectrum of biologic properties. Available supplies of the synthetic hormone will also enable evaluation of the effectiveness of PTH antagonists at inhibiting the action of native sequence hormone at its receptors.

Cyclic parathyroid hormone related protein antagonists: lysine 13 to aspartic acid 17 [i to (i + 4)] side chain to side chain lactamization.

Cyclization of parathyroid hormone related protein (7-34)amide [PTHrP(7-34)NH2] via covalent bond formation between the epsilon-amino of Lys13 and the beta-carboxyl of Asp17 yielded a 20-membered ring lactam. This analogue, [Lys13,Asp17]PTHrP(7-34)NH2, was 5-10-fold more potent than the linear parent peptide (Kb = 15 and 18 nM in PTH receptor binding assays, and Ki = 130 and 17 nM in PTH-stimulated adenylate cyclase assays in bovine renal cortical membrane and in human bone derived B10 cells, respectively). In contrast, a linear analogue in which charges in positions 13 and 17 were eliminated and other stereoisomers of the above-mentioned lactam in which either Lys13 and/or Asp17 were replaced by the corresponding D-amino acids were much less potent with regard to antagonist bioactivity than the parent peptide. The rationale for the design of the lactam as well as the conformational implications for the PTHrP sequence in light of reported models suggested for the 1-34 peptide are described. The potential use of conformationally constrained analogues for elucidating the "bioactive conformation" of antagonists and for the design of substantially simplified molecular structures for antagonists is discussed.

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.

Effects of hydrophobic substitutions at position 18 on the potency of parathyroid hormone antagonists.

Position 18 in a parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2 (ii), was shown to tolerate substitutions by a range of amino acids with retention of inhibitory activity. The effects of hydrophobic substitutions at this position as a means of enhancing binding interactions with the receptor were evaluated. Substitution of Nle at position 18 with either D-Ala, D-Trp, or L-Trp in analog ii or with Trp (D or L) in the recently reported, highly potent antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 (in vitro activities; Kb = 15 nM and Ki = 125 nM), was performed. In terms of activity on renal receptors, one antagonist, [Nle8,D-Trp12,18,Tyr34]bPTH(7-34)NH2, is the most active in vitro PTH antagonist yet reported (Kb = 4 nM; Ki = 30 nM). The rationale for design of this antagonist and the conclusions regarding PTH-receptor interactions are discussed.

Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH2 by substitution of PTH amino acids at positions 10 and 11.

PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2 have previously been shown to be shown to be more potent antagonists than the corresponding PTH peptide, [Tyr34]bPTH(7-34)NH2. However, these peptides also display partial agonism for adenylate cyclase activity in ROS 17/2.8 cells. In this study, design of a pure potent antagonist of PTH and PTHrP by removal of agonism from PTHrP(7-34)NH2 with retention of antagonist potency was accomplished. Since [Tyr34]bPTH(7-34)NH2 lacks agonist activity, we introduced two amino acids native to the PTH sequence into their respective positions in PTHrP and the potent D-Trp12 analog. [Asn10Leu11]- and [Asn10,leu11,D-Trp12]-PTHrP(7-34)NH2 were found to be 23- and 26-fold more potent as antagonists in ROS cells than PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2, respectively. In addition, these peptides did not display partial agonism, even in an assay based on highly responsive cells pretreated with dexamethasone and pertussis toxin. In contrast, when the PTHrP sequence Asp10,Lys11 was inserted into [Tyr34]hPTH(7-34)NH2, antagonist potency declined by more than 6-fold and PTH-like agonist activity was installed. These results demonstrate that the activation domain of both PTH and PTHrP can be extended to include the 1-12 region and that the 10-12 region, in addition to the N-terminal hexapeptide, is important not only for receptor binding but also for hormonal signal transduction.

Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists.

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.

The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: the receptor binding domains of PTH and PTH-related protein are located within the 14-34 region.

Previous studies examining the interaction of PTH and PTH-related protein (PTHrP) with target tissue have for the most part emphasized the similarity between the two hormones in binding to and activating receptors. This observation that two peptides with limited homology have equal affinities for the same receptor is unusual. In this report we investigated two aspects of PTH/PTHrP-receptor interactions. First, the nonhomologous 14-34 regions of PTH and PTHrP were synthesized and evaluated. Second, hybrid peptides containing the 7-18 fragment of one hormone combined with the 19-34 region of the other hormone were studied to determine whether interactions between these two regions are required for receptor recognition. All four peptides were examined in bovine renal cortical membrane and rat osteosarcoma (ROS 17/2.8) cell PTH-binding and PTH-stimulated adenylate cyclase assays. The results indicate that the receptor-binding domains of PTH and PTHrP lie outside of the 1-13 region, the region containing sequence homology shared by the two hormones, and that two peptides of different amino acid sequence bind with equal affinity to the bovine renal PTH receptor. However, in the absence of the N-terminal region, the rat bone PTH receptor displays a preference for the C-terminal (19-34 sequence) region of PTHrP.

Modifications of position 12 in parathyroid hormone and parathyroid hormone related protein: toward the design of highly potent antagonists.

Truncated N-terminal fragments of parathyroid hormone (PTH), [Tyr34]bovine PTH(7-34)NH2, and parathyroid hormone related protein (PTHrP), PTHrP(7-34)NH2, inhibit [Nle8,18,[125I]iodo-Tyr34]-bPTH(1-34)NH2 binding and PTH-stimulated adenylate cyclase in bone and kidney assays. However, the receptor interactions of these peptides are 2-3 orders of magnitude weaker than those of their agonist counterparts. To produce an antagonist with increased receptor-binding affinity but lacking agonist-like properties, structure-function studies were undertaken. Glycine at position 12 (present in all homologues of PTH and in PTHrP), which is predicted in both hormones to participate in a beta-turn, was examined by substituting conformational reporters, such as D- or L-Ala, Pro, and alpha-aminoisobutyric acid (Aib), in both agonist and antagonist analogues. Except for N-substituted amino acids, which substantially diminished potency, substitutions were well tolerated, indicating that this site can accept a wide latitude of modifications. To augment receptor avidity, hydrophobic residues compatible with helical secondary structure were introduced. Incorporation of the nonnatural amino acids D-Trp, D-alpha-naphthylalanine (D-alpha-Nal), or D-beta-Nal into either [Tyr34]bPTH(7-34)NH2 or [Nle8,18,Tyr34]bPTH(7-34)NH2 resulted in antagonists that were about 10-fold more active than their respective 7-34 parent compound. Similarly, [D-Trp12]PTHrP(7-34)NH2 was 6 times more potent than the unsubstituted peptide but retained partial agonistic properties, although markedly reduced, similar to PTHrP(7-34)NH2. The antagonistic potentiating effect was configurationally specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian (chicken) parathyroid hormone: synthesis and comparative biological evaluation of the 1-34 fragment.

The full-length amino acid sequence of the avian (chicken) form of parathyroid hormone (cPTH) has recently been elucidated. We have chemically synthesized, purified to a high degree, and analytically and biologically characterized the N-terminal 1-34 fragment of the avian hormone. The biological properties of cPTH-(1-34)NH2 were evaluated and compared to the bovine fragment bPTH-(1-34) in several assays. The potency of cPTH-(1-34)NH2 in binding to PTH receptors, in stimulating adenylate cyclase activity and in relaxing smooth muscle tissue was approximately one-tenth that of bPTH-(1-34). Comparison of the avian sequence to other native PTH related sequences suggests that changes in the binding domain of the 1-34 active fragment may account for the decline in potency.

A new highly potent parathyroid hormone antagonist: [D-Trp12,Tyr34]bPTH-(7-34)NH2.

Based upon N-terminal parathyroid hormone (PTH) analog structure-activity relationship studies, position 12 was found to possess a wide structural latitude and was chosen as a site for single amino acid substitutions. Replacement of the naturally-occurring Gly with D-Trp at position 12 in the PTH antagonists [Tyr34]bPTH-(7-34)NH2 and [Nle8,18,Tyr34]bPTH-(7-34)NH2 increased in vitro receptor affinity. The D-Trp12 containing analogs were 12-fold more potent than their unsubstituted counterparts as inhibitors of PTH binding to renal and bone PTH receptors and 13-27-fold more potent as inhibitors of PTH-stimulated renal and bone adenylate cyclase activity. Based upon Scatchard analyses of saturation binding experiments and Schild analyses of adenylate cyclase experiments, [D-Trp12,Tyr34]bPTH-(7-34)NH2 was shown to interact with PTH receptors in a competitive manner. These studies demonstrate, therefore, that D-Trp12 substitution in PTH antagonists improves inhibitory properties in vitro and is compatible with a helical conformation at this position as a new direction for the design of PTH antagonists.

Perifused precision-cut liver slice system for the study of hormone-regulated hepatic glucose metabolism.

A nonrecirculatory perfusion system for precision-cut rat liver slices has been developed and utilized for investigating hormone-regulated hepatic glucose metabolism. In this system, slices are cultured in a highly controlled environment and exhibit excellent retention of viability as judged by their maintenance of intracellular potassium and glycogen contents. Using this system, the complex physiological phenomenon of hormone-regulated glycogenolysis was investigated at both extra- and intracellular sites. Specifically, the sensitive responses of intracellular cyclic AMP (cAMP) production, activation of cyclic AMP-dependent protein kinase, and production of glucose upon glucagon stimulation have been measured. The maximal responses observed for these parameters were either equal to or greater than those previously reported for either isolated hepatocytes or perfused livers, demonstrating the sensitivity of this technique. Upon dose-response examination of glucagon challenge, it was observed that high doses of glucagon (greater than 16 nM) stimulate glucose production by activating the cAMP-second messenger cascade. In contrast, low doses (less than 4 nM) stimulate this process without production of intracellular cAMP or activation of cAMP-dependent protein kinase, suggesting the operation of cAMP-independent messenger. Since this system permits measurements of parameters common to many cellular processes, this methodology is suitable for addressing both pharmacological and toxicological questions.

The 7-34-fragment of human hypercalcemia factor is a partial agonist/antagonist for parathyroid hormone-stimulated cAMP production.

The N-terminal fragment of human hypercalcemia factor (hHCF), hHCF-(1-34)NH2, has bioactivities similar to PTH in vitro and in vivo. Because it interacts with PTH receptors and is more potent than PTH in some systems, the hHCF sequence may provide interesting leads for the design of potent and selective PTH and hHCF antagonists. Based on the antagonist activity of [Tyr34]bovine PTH-(7-34)NH2 [( Tyr34]bPTH-(7-34)NH2), we synthesized the corresponding fragment of hHCF, hHCF-(7-34)NH2 and examined its properties in vitro. In the bone-derived rat osteosarcoma cell line ROS 17/2.8, hHCF-(7-34)NH2 and [Tyr34]bPTH-(7-34)NH2 were equipotent for inhibition of radiolabeled PTH-binding. In contrast, hHCF-(7-34)NH2 was 8-fold more potent that [Tyr34]bPTH-(7-34)NH2 for inhibiting PTH-stimulated cAMP production. hHCF-(7-34)NH2 also inhibited PTH-binding and PTH-stimulated adenylate cyclase activity in bovine renal cortical membranes: hHCF-(7-34)NH2 and [Tyr34]bPTH-(7-34)NH2 were equipotent in this system. In addition, hHCF-(7-34)NH2 antagonized hHCF-(1-34)NH2 action in both systems with similar inhibition constants. However, unlike the PTH analogue, hHCF-(7-34)NH2 (8 microM) was a weak partial agonist, producing a 2.4-fold increase in cAMP (5% of the maximal response) in ROS cells. This same system also detects agonism for [Nle8, 18Tyr34]bPTH-(3-34)NH2, another PTH partial agonist/antagonist. These results demonstrate that hHCF-(7-34)NH2 interacts with PTH receptors based in large part on the region which is not homologous to PTH, and suggest the utility of the ROS 17/2.8 cell system for identifying weak agonism of PTH and hHCF analogues in vitro.

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.