Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues.

Two synthetic analogues of bovine parathyroid hormone (PTH) with NH2-terminal modifications, PTH-(3-34) and [desamino-Ala-1]PTH-(1-34), were found to lack agonist activity but to demonstrate antagonist properties when tested in the rat renal cortical adenylyl cyclase assay in vitro against the native hormone or against PTH-(1-34), the active synthetic NH2-terminal tetratriacontapeptide. The inhibition exhibited by these analogues was proportional in degree to the dose of inhibitor, abolished by oxidation of the analogue, reversible by addition of an excess of active hormone, and specific for parathyroid hormone-stimulated renal adenylyl cyclase. No inhibition of basal or sodium fluoride-stimulated renal adenylyl cyclase could be demonstrated. Two other synthetic bovine analogues, PTH-(13-34) and PTH-(1-26), were devoid of agonist and antagonist properties. The over-all results suggest that the requirements for receptor binding of parathyroid hormone are rather broad. Conformational factors or binding interactions involving specific residues, or both seem to require the entire sequence from residue 3 to residue 27 for receptor binding to occur. A dichotomy between receptor binding and adenylyl cyclase activation was demonstrated only by alterations or deletions involving the first 2 NH2-terminal residues of the hormone and emphasizes the importance of these residues in eliciting the biological activity of parathyroid hormone. The two antagonists, [desamino-Ala-1]PTH-(1-34) and PTH-(3-34), should be useful in further analysis of the initial steps in hormone action.     

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.     

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.     

Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.     

Synthesis of fully active biotinylated analogues of parathyroid hormone and parathyroid hormone-related protein as tools for the characterization of parathyroid hormone receptors.

The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys13 or Lys26 residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)NH2, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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)     

Cerebral Vascular Adenylate Cyclase: Evidence for Coupling to Receptors for Vasoactive Intestinal Peptide and Parathyroid Hormone

We have studied the responsiveness of vascular adenylate cyclase to vasoactive intestinal peptide (VIP) and parathyroid hormone (PTH) using preparations of cerebral microvessels and arteries. Cerebral microvessels obtained from rats, guinea-pigs, cattle, and pigs all responded potently to bovine (b) PTH-(1-34), whereas considerable between-species variability was observed in the responsiveness to VIP. The homologous peptide to VIP, PHI (porcine heptacosapeptide), stimulated adenylate cyclase in both rat microvessels and a broken-cell preparation of bovine arteries. The ED50 values for activation of bovine arterial adenylate cyclase by VIP, PHI, and bPTH-(1-34) were 6.9 nM, 10 nM, and 100 nM, respectively, with the following order of efficacy: VIP = PHI greater than bPTH-(1-34). The other related peptides, hpGRF (human pancreatic growth hormone releasing factor), secretin, and glucagon, and the fragment VIP-(10-28) were inactive. The PTH antagonist, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, inhibited bPTH-(1-34) activation of vascular adenylate cyclase but did not affect activation by VIP using either microvessels or arteries. VIP or PHI demonstrated an additive effect with bPTH-(1-34) on vascular adenylate cyclase activity. However, the effects of VIP and PHI were nonadditive with each other. These data suggest that VIP and bPTH-(1-34) activate cerebral vascular adenylate cyclase by interacting with pharmacologically distinct receptors, whereas PHI and VIP likely interact with a common receptor.     

Taming psoriatic keratinocytes--PTHs' uses go up another notch

The native parathyroid hormone (PTH) and several of its N-terminal adenylyl cyclase-activating fragments and their analogs have become the star stimulators of bone growth for treating osteoporosis, accelerating fracture healing, and strengthening the anchorage of prosthetic bone implants and one of them (Lilly's Forteo--recombinant hPTH-(1-34) has recently arrived in the clinic. But something entirely different has been lurking in the background-the ability of the adenylyl cyclase stimulating hPTH-(1-34) to calm hyperproliferating keratinocytes and reduce psoriatic lesions. By contrast PTH-(7-34) which cannot stimulate adenylyl cyclase actually stimulates keratinocyte proliferation. Normal keratinocytes make PTHrP after they lift off the basal lamina and have stopped cycling. But they have an unconventional PTH/PTHrP receptor which is not coupled to adenylyl cyclase. Psoriatic keratinocytes do not make PTHrP and have only a broken-down, proliferation-limiting terminal differentiation-driving Notch-Notch ligand mechanism. Putting these and other facts together produces a possible picture of an exogenously applied adenylyl cyclase-activating PTH pinch hitting for the missing PTHrP and restoring normal keratinocyte proliferative activity epidermal structure by stimulating dermal fibroblasts which do have the conventional adenylyl cyclase-linked PTHR1 and in response directly or indirectly restore the overlying basal keratinocytes' Notch-Notch ligand terminal differentiation-driving mechanism and consequently a normal epidermal structure.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

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)     

Stimulation of membrane-associated protein kinase-C activity in spleen lymphocytes by hPTH-(1-31)NH2, its lactam derivative, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

Human parathyroid hormone, hPTH-(1-34), stimulates adenylyl cyclase and phosphatidylinositol-bisphosphate-specific phospholipase-C (PIP2-PLC), as indicated by increased membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. The C-terminally truncated hPTH-(1-31)NH2 stimulates adenylyl cyclase as strongly as hPTH-(1-34) in these cells, but it does not stimulate PKC activity. Even [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, a 6-fold stronger adenylyl cyclase stimulator than hPTH-(1-34), cannot stimulate PKC activity in ROS cells. Therefore PTH required its 32-34 region to stimulate PIP2-PLC/PKCs in this osteosarcoma line. In contrast, hPTH-(1-31)NH2 [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 and even hPTH-(1-30)NH2 can stimulate PKC activity in freshly isolated rat spleen lymphocytes as strongly as hPTH-(1-34)NH2. The difference in the ability of membrane-associated PKC activity in spleen lymphocytes, but not in ROS cells, to be stimulated by C-terminally truncated PTH fragments might be due to different receptor densities or to the lymphocyte's atypical PTH/PTHrP receptor.     

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.     

Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: role of Na+/H+ exchange.

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.     

Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor

The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.     

Amino-terminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: implications for design of signal-specific PTH ligands.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.     

The hydrophobic residues phenylalanine 184 and leucine 187 in the type-1 parathyroid hormone (PTH) receptor functionally interact with the amino-terminal portion of PTH-(1-34).

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH(2)-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182-190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe(184), Arg(186), Leu(187), and Ile(190) as important determinants of maximum binding of (125)I-labeled bovine PTH-(1-34) and (125)I-labeled bovine PTH-(3-34) and determinants of responsiveness to the NH(2)-terminal analog, PTH-(1-14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu(22), Trp(23)]PTHrP-(15-36) to inhibit the cAMP response induced by PTH-(1-34). At Phe(184) and Leu(187), hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1-34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe(184) and Leu(187) in the PTH-1 receptor plays an important role in determining functional interaction with the 3-14 portion of PTH.     

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.     

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 ED50 values of 11.5 nM and 64 nM respectively, in the absence of GTP. 10(-4)M 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 ED50 were 190 nM and 64 nM respectively. [Nle8, Nle18, Tyr34] -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.