Effect of parathyroid hormone and antagonist on aortic cAMP levels

Experiments were designed to further investigate the vasoactive mechanisms of parathyroid hormone (PTH) on vascular smooth muscle cells. Time courses of the cAMP responses to the fragment (1-34) of bovine PTH (bPTH(1-34)) on cAMP levels have been studied in rat isolated aorta and in aortic myocytes in primary culture. In both aorta and myocytes bPTH (1-34) induced an increase in cAMP levels that was maximal and reached, respectively, 1.6- and 1.9-fold the basal level after 2 min of contact with bPTH (1-34). The effect of bPTH (1-34) on aortic cAMP content was concentration dependent in the range of 30-300 nM. (Nle8,18, Tyr34)-bPTH (3-34)amide, an antagonist of bPTH (1-34) with a stimulant effect on renal and vascular adenylate cyclase activity, inhibited the cAMP-increasing effect of bPTH (1-34). These results are in favour of a role for cAMP in the vasodilating effect of PTH.     

Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.     

Age, strain and species differences in circulating parathyroid hormone

A new, sensitive parathyroid hormone (PTH) radioimmunoassay that appears specific for the intact hormone, and its validation for measuring rat PTH are described. The assay is based on antibody C2-7 from chicken immunized with bovine PTH; it has a detection limit of 6 pg of bPTH per assay tube and measures basal PTH in most rats; it is responsive to provoked changes in endogenous PTH concentration, and the intra-assay and inter-assay coefficients of variation are 6.0% and 7.2%, respectively. Multiple dilutions of rat serum and parathyroid gland extract, result in competitive inhibition curves that are parallel to that of highly purified bPTH. Under our assay conditions the C2-7 antibody cross-reacts well with intact PTH but synthetic fragments of the hormone (1-34bPTH, 1-34hPTH, 28-48hPTH, 44-68hPTH, 53-84hPTH) do not depress tracer (125l-bPTH) binding to the antibody. Studies designed to validate the assay gave predictable results such as enhanced secretion of the hormone in response to EDTA infusion, and failure to detect the hormone in serum following thyroparathyroidectomy. In addition, we made the novel observation that in F344 rats circulating immunoreactive PTH increases progressively with aging.     

Potentiation of antigen-induced mast cell activation by 1-34 bovine parathyroid hormone

Peptides such as parathyroid hormone (PTH), somatostatin, and gastrin have been reported to stimulate mast cell mediator release. Preincubation of rat serosal mast cells with synthetic 1-34 bovine parathyroid hormone (1-34bPTH) significantly enhanced antigen-induced 5-hydroxytryptamine (5-HT) release. Enhancement of 5-HT release by 1-34bPTH was dose dependent between 5 and 2000 nM. In the absence of antigen, mean net 5-HT release was less than 1% when naive or passively sensitized mast cells were incubated with 1000 nM 1-34bPTH for time intervals up to 90 min. These findings indicate that 1-34bPTH, at relatively low concentration, potentiates antigen-induced 5-HT release from mast cells.     

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)     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

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.     

Relaxation of bovine, porcine and human brain arteries by parathyroid hormone

We have studied the relaxant effect of bovine parathyroid hormone (bPTH) on helical strips of branches of bovine and human middle cerebral arteries and bovine and porcine basilar arteries. All arteries were studied after contraction with prostaglandin (PG) F2 alpha or KCl. In the case of all arteries contracted with PGF2 alpha, the ED50 of PTH vasorelaxation related to maximal vasorelaxation induced by papaverine ranged from 9 to 14 nM for bPTH-(1-34) and 100 to 220 ng/ml for native bPTH-(1-84). The PTH inhibitor, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, attenuated the vasorelaxant effect of both bPTH-(1-34) and bPTH-(1-84). The vasorelaxant effects of PTH which we have observed in this study are consistent with the stimulatory effects of PTH on vascular adenylate cyclase which we had previously reported.     

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.     

Characterization of the rabbit renal receptor for native parathyroid hormone employing a radioligand purified by reversed-phase liquid chromatography

Iodinated native bovine parathyroid hormone (bPTH(1-84)) was separated from uniodinated hormone by reversed-phase liquid chromatography techniques after lactoperoxidase labeling. Analysis of iodinated residues after enzymatic digestion indicated that the major labeled product was largely monoiodinated on the sole tyrosine residue. This material retained full bioactivity in an in vitro renal adenylate cyclase assay. Binding of 125I-bPTH(1-84) to rabbit renal membranes at 4 degrees C was proportional to membrane protein concentration and was saturable and dissociable. Radioligand binding was inhibited by concentrations of unlabeled bPTH(1-84) required to stimulate adenylate cyclase in the same membrane preparation but was not inhibited by non-PTH peptides other than adrenocorticotropin at high concentrations (greater than 10 microM). Synthetic NH2-terminal analogues of bPTH(1-84) all elicited approximately equivalent inhibition of radioligand binding which was, however, less potent than unlabeled bPTH(1-84), suggesting a role for the carboxyl region of the molecule in the interaction of bPTH(1-84) with its receptor. Activity of the NH2-terminal agonists was similar to bPTH(1-84) in stimulating adenylate cyclase. Although substitution in sequence position one, of serine in human PTH(1-34) for alanine in bPTH(1-34), reduced activity in the adenylate cyclase assay, inhibition of 125I-bPTH(1-84) binding by both peptides and by an analogue of bPTH(3-34) was equivalent, consistent with a minimal contribution of the first 2 residues for receptor binding of the NH2-terminal region of PTH. The results illustrate the utility of the radiolabeled preparation of native bPTH we have developed and emphasize the importance of probing the PTH receptor with an intact hormone to maximize information concerning the mechanism of PTH action.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Radioimmunoassay specific for amino (N) and carboxyl (C) terminal portion of parathyroid hormone.

A radioimmunoassay specific for the amino (N) terminal portion of the parathyroid hormone (PTH) molecule (N-PTH radioimmunoassay) has been developed by iodinating synthetic 1-34bovine PTH (1-34bPTH) and using commercially available bPTH antiserum. A radioimmunoassay specific for the carboxyl (C) terminal (C-PTH radioimmunoassay) has been carried out by adding enough amount of 1-34bPTH to the PTH radioimmunoassay system. The data obtained from N- and C-PTH radioimmunoassay were compared with those obtained from the PTH radioimmunoassay. It was observed that plasma levels of N-PTH, indicating biologically active PTH, were only one 8th to 32th to those of PTH and those of C-PTH were almost equal to those of PTH. These data corresponded well with those reported previously by using the antiserum specific for each terminal of the PTH molecule from the other laboratory. The half life of plasma N-PTH and C-PTH determined following the removal of parathyroid adenoma was less than 10 min and about 45 min respectively. These data indicate that the N-PTH radioimmunoassay can be done by iodinating 1-34bPTH and using commercially available antiserum.     

Dissociation of cAMP accumulation and phosphate uptake in opossum kidney (OK) cells with parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP)

Bovine parathyroid hormone (PTH) 1-34 [bPTH(1-34)] and human PTH related protein [hPTHrP(1-34)] stimulated cAMP accumulation in opossum kidney (OK) cells with Km of 5 x 10(-9) M, but inhibition of phosphate uptake was obtained with 17-fold lower Km of 3 x 10(-10) M. Phosphate uptake was partially inhibited with [Nle8.18Tyr34]bPTH(3-34)NH2 without concomitant cAMP stimulation. With hPTHrP(7-34)NH2, cAMP accumulation was increased in parallel to inhibition of phosphate uptake. [D-Trp12Tyr34]bPTH(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 had no agonist activity on cellular cAMP and inhibition of phosphate uptake. bPTH(1-34)-stimulated cAMP accumulation was antagonized by [Nle8.18Tyr34]bPTH(3-34)NH2, [D-Trp12Tyr34]bPTH(7-34)NH2, hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 with Ki of 1.4 x 10(-7), 2 x 10(-7), 4.7 x 10(-7) and 3.7 x 10(-6) M, respectively. But [Nle8.18Tyr34]bPTH(3-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2 reversed the inhibition of phosphate uptake only marginally, and hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 were inactive. With hPTHrP(1-34) the Ki for cAMP accumulation of [Nle8,18Tyr34]bPTH(3-34)NH2 and hPTHrP(7-34)NH2 were 1.9 x 10(-7) and 7.2 x 10(-7) M, and inhibition of phosphate uptake was partially reversed with [Nle8,18Tyr34]bPTH(3-34)NH2, but not with hPTHrP(7-34)NH2. The present results indicate that truncated hPTHrP(7-34)NH2, unlike [Tyr34]hPTH(7-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2, elevates cellular cAMP and inhibits phosphate uptake. bPTH(1-34)- and hPTHrP(1-34)-evoked cAMP accumulation is suppressed by PTH and PTHrP fragments while inhibition of phosphate uptake remains largely unaltered.     

Effects of bPTH fragments (1-34), (3-34) and (7-34) on uterine contraction

Synthetic bovine parathyroid hormone containing the NH2 terminal 34 amino acids [bPTH-(1-34)] was recently demonstrated to inhibit oxytocin stimulated uterine contraction in vitro. The parathyroid hormone analogues [Nle8, Nle18, Tyr34]bPTH-(3-34)amide [NTA-(3-34)] and [Tyr34]bPTH-(7-34)amide [NTA-(7-34)] have been reported to act as inhibitors of antagonists of parathyroid hormone (PTH) in numerous assays. In the present study the effects of these PTH analogues on uterine contraction and the ability of these analogues to act as antagonists to the uterine inhibitory action of bPTH-(1-34) in vitro were investigated. The NTA-(3-34) fragment had no effect on oxytocin stimulated uterine contractions. However, the NTA-(3-34) fragment was able to alter the ability of bPTH (1-34) to reduce oxytocin stimulated uterine contraction in a dose-related manner. Bovine PTH(1-34) (0.3 microgram/ml) reduced the contractile response obtained with oxytocin (0.5 mU/ml) by 20%. A dose of 15 micrograms/ml) of NTA-(3-34) abolished this inhibitory action of bPTH-(1-34) on oxytocin stimulated uterine contraction. In contrast the NTA-(7-34) caused a change in itself, stimulated contraction of resting uterine horns in a dose-related manner; 3.0 micrograms/ml of NTA-(7-34) caused a change in gram tension of + 1.5 grams. Bovine PTH-(1-34) was able to reduce the uterine contraction stimulated by NTA-(7-34) and 0.3 microgram/ml of bPTH-(1-34) reduced the contractile response obtained with 3.0 micrograms/ml of NTA-(7-34) by as much as 70%.(ABSTRACT TRUNCATED AT 250 WORDS)     

The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes

The stimulation of DNA synthesis in primary cell cultures of chicken chondrocytes by parathyroid hormone was studied by assaying [3H]thymidine incorporation into DNA. Optimal assay conditions were determined by varying cell age, plating density, and incubation time. Under these conditions DNA synthesis was significantly stimulated by parathyroid hormone (PTH) and some of its fragments: cells treated with human (h)PTH(1-84), bovine (b)PTH(1-34) and [Nle8,18,Tyr34]bPTH(3-34)amide and hPTH(13-34) displayed 2.6-fold enhanced [3H]thymidine incorporation in a dose-dependent manner. The fragment hPTH(28-48) led to a similar stimulation, whereas [Tyr43]hPTH(43-68) and [Tyr52,Asp76]hPTH(52-84) had no effect. Using a series of synthetic hPTH peptides covering the central region of the hormone molecule (residues 25-47), we could delimitate further this putative mitogenic functional domain to a core region between amino acid residues 30 and 34. The effect of PTH on [3H]thymidine incorporation could not be mimicked by forskolin, indicating that the corresponding signal is not mediated by cAMP. It is, however, inhibited by EGTA and cannot be provoked in the absence of calcium ions in the medium. Therefore, the results presented indicate a hitherto unidentified functional domain of PTH in the central part of the molecule which exerts its mitogenic effect on chondrocytes in a cAMP-independent manner but seems to involve calcium ions for signal transduction.     

Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line

In the present study we investigated the characteristics of the transport of inorganic phosphate (Pi) in an opossum kidney cell line endowed with parathyroid hormone (PTH) receptors. In confluent epithelial cell culture, a Na-dependent Pi transport (NaPiT) was identified. Preincubation for 1 h with bovine (b)PTH(1-34) at 10(-7) M inhibited the NaPiT from 2.76 +/- 0.11 to 1.08 +/- 0.10 nmol/mg protein X 2 min-1 (p less than 0.001). This inhibition was already expressed 5 min after exposure to 10(-7) M bPTH. It was associated with a 4-fold increase in cellular cyclic AMP. The NaPiT was significantly inhibited at 10(-9) M bPTH, a hormonal concentration which stimulated the cellular cyclic AMP by only 30%. Kinetic analysis of the NaPiT inhibition by 10(-7) M bPTH revealed a decrease in Vmax (from 4.14 +/- 0.32 to 2.41 +/- 0.14 nmol/mg protein X 2 min-1) with no change in Km (0.093 +/- 0.016 versus 0.094 +/- 0.012 mM). The effect of bPTH on NaPiT was not associated with a change in the Na-dependent glucose methylglucopyranoside transport also present in the opossum kidney cell line. The inhibitory influence of bPTH on NaPiT was not affected by blockage of new protein synthesis by cycloheximide. Stimulation of cyclic AMP production by 10(-5) M forskolin, 10 micrograms/ml cholera toxin, 10(-5) M prostaglandin E2 or addition of 10(-5) M dibutyryl cyclic AMP mimicked the PTH-induced reduction in NaPiT. In conclusion, the present study indicates that the opossum epithelial cell line is endowed with a Na-dependent Pi transport system which is selectively inhibited by PTH and agents which increase cyclic AMP production.     

Characterization of canine renal receptors for the parathyroid hormone-like protein associated with humoral hypercalcemia of malignancy

Parathyroid hormone-like proteins (PTHLP) display actions in the kidney which are similar to those of parathyroid hormone (PTH). We compared the binding properties of PTHLP and PTH in canine renal cortical membranes to determine if they interacted with the same or different receptors. Radioiodination to high specific activity (greater than 400 microCi/micrograms) of [Nle8,18,Tyr34]human PTH-(1-34)amide and [Tyr36]PTHLP-(1-36)amide was performed using the lactoperoxidase method. Complete enzymatic digestion of both radioligands demonstrated that the peptides were monoiodinated. Both radioligands retained full biological activity in the renal adenylate cyclase assay, and neither was significantly degraded during incubation with highly purified canine renal membranes under binding assays conditions. Specific binding reached equilibrium by 20 min at 20 degrees C. Competition binding studies using unlabeled [Nle8,18,Tyr34]human PTH-(1-34)amide, [Tyr36] PTHLP-(1-36)amide, and bovine PTH-(1-34) with either radioligand revealed similar binding affinities for all three peptides. Biologically inactive PTHLP fragments did not show significant displacement. In contrast to its similar binding affinity, [Tyr36]PTHLP-(1-36)amide was 6-15-fold less potent than bovine PTH-(1-34) in the renal adenylate cyclase assay, suggesting less efficient receptor-effector coupling. Photoaffinity cross-linking using either radioligand in canine renal membrane labeled indistinguishable 70,000-dalton proteins. In the presence of multiple protease inhibitors, binding to an 85-kDa component was observed. Labeling of both receptor forms was specifically abolished by an excess of either cold peptide and dose-response curves using affinity cross-linked membranes corroborated the apparent binding affinities determined by conventional radioligand binding assays. We conclude that PTHLP-(1-36) and amino-terminal PTH analogues bind to indistinguishable receptors in canine renal cortical membranes, but display differential coupling to post-receptor events.     

Cardiac actions of bovine parathyroid hormone (1-34), isoproterenol, and propranolol in turtles.

1. In isolated turtle hearts bovine parathyroid hormone (b-PTH) (1-34) stimulated both force of contraction and rate (beats/min) apparently stimulating beats/min to a larger extent. 2. Isoproterenol stimulated both rate and force, perhaps affecting contractile force more. 3. Propranolol alone clearly decreased contractile force and beats/min. 4. Pre-treatment with propranolol removed stimulatory effects of both bPTH (1-34) and isoproterenol. Propranolol's effect on isoproterenol was expected. Its effect on bPTH (1-34) may be related to beta-stimulation by PTH or to some other non-specific inhibitory effect of propranolol.     

甲状旁腺素、表皮生长因子及维生素A酸对UMR106细胞EGF受体的调节作用

本文研究了EGF、PTH和RA对UMR106细胞EGF受体的调节作用。结果显示PTH能上调EGF的受体,UMR106细胞经bPTH(1-34)处理3天,EGF受体的相对结合率与对照比较提高了40.3％,每个细胞的EGF受体数目从7.22×10~3增加到1.44×10~4,Kd从2.02×10~(-11)增加到3.68×10~(-11)mol/L。而RA则能下调EGF受体,以RA处理3天,EGF受体数目从7.22×10~3下降到4.28×10~3,Kd则从2.02×10~(-11)增加到4.17×10~(-11)mol/L。提示PTH和RA可能通过调变其EGF受体而分别起到正性和负性生长调节作用。     

Characterization of the interaction of parathyroid hormone with the mitochondrial ATPase

Parathyroid hormone (PTH) has been shown to bind specifically to the beta subunit of the mitochondrial ATPase on nitrocellulose blots. We have now examined this interaction further, using intact mitochondria, submitochondrial particles, and the purified F1 ATPase. With intact mitochondria, 1 microM concentrations of PTH and its biologically active 1-34 fragment activate the ATPase about 3-fold. This effect was reduced to a 1.4-fold activation with 3-34 and 7-34 fragments of the hormone, and oxidized PTH gave no detectable activity. Activation could only be observed below pH 7. PTH had no significant effect on the activity of the purified enzyme or on submitochondrial particles. However, specific binding of an iodinated PTH analog, [Nle 8,18-Tyr 34] bPTH (1-34) amide, was found with submitochondrial particles and the purified ATPase. Binding affinity with the purified enzyme was about 10(-3) that of the plasma membrane receptor, and the molar stoichiometry was close to 1:1 (PTH:intact enzyme). With submitochondrial particles the affinity was about 10-fold higher than with the purified enzyme. This binding was further examined with PTH derivatives and fragments, and compared to that seen in the plasma membrane receptor. Oxidation of methionine 18 in PTH reduced the affinity about 50%, oxidation of methionine 8 reduced the affinity 95%, and oxidation of both methionines further decreased affinity in both membranes and submitochondrial particles. However, when compared to the native hormone, the 3-34 and 7-34 PTH fragments had much higher affinity for the submitochondrial particles than for the plasma membranes. PTH also reduced chemical crosslinking of the ATP analog, p-fluorosulfonyl benzoyl 5'-adenosine, to the alpha subunit of this enzyme, but did not alter labeling of the enzyme with 3'-O-(4'-benzoyl) benzoyl ATP, suggesting that the hormone binds near a regulatory nucleotide binding site. Direct chemical crosslinking of PTH to the beta-subunit of the enzyme was attained with a cleavable, photoactivate crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3-dithiopropionate. The crosslinked protein was cleaved with cyanogen bromide and the labeled fragments were sequenced. The labeled fragments were found to be segments of the protein which have previously been implicated as being close to the noncatalytic ATP binding sites.