Partially modified retro-inverso-enkephalinamides: topochemical long-acting analogs in vitro and in vivo.

The synthesis of four enkephalinamide analogs is described in which the peptide bond between residues 4 and 5 is reversed with or without simultaneous reversal of the carboxyl-terminal amide bond. These so-called partially modified retro-inverso-isomers are new, potent, topochemical analogs of the enkephalins. Tests, both in vitro and in vivo, have shown that these analogs are considerably longer acting than any previously studied enkephalins. Thus, partial reversal of the peptide bonds of the backbone can result in peptides with enhanced activity compared to a parent compound, provide that the structural complementarity of both the side chains and end groups are conserved.

Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge.

We have recently reported the discovery of a 14-amino-acid osteogenic growth peptide (OGP). In vivo OGP increases bone formation and trabecular bone density. Physiologically it is found in serum complexed to an OGP binding protein (OGPBP). In vitro OGP has a biphasic effect on osteoblastic MC 3T3 E1 and fibroblastic NIH 3T3 cell proliferation; at low concentrations (0.01-1.0 and 1.0-100.0 pM, respectively) it is highly stimulatory with an inhibition at higher doses. To assess possibilities of labeling synthetic OGP to obtain radio- or fluorescent ligands, OGP analogues were extended at the N- or C-termini with Cys or Cys(S-NEtSucc) or the OGP Tyr-10 replaced by 3-I(Tyr). All analogues with N-terminal modifications, as well as the [Cys15]OGP-NH2 retained the OGP-like dose-dependent effect on proliferation of the MC 3T3 E1 and NIH 3T3 cells, although the magnitude of stimulation was lower, approx. 2/3 that of the native-like synthetic OGP. The [Cys15(S-NEtSucc)]OGP-NH2 and [3-I(Tyr10)]OGP shared only the inhibitory activity of OGP. This suppression is further shared by a number of other positively and negatively net charged, but not net neutral, peptides. Both N-terminal-modified analogues displayed a decreased binding activity to the OGPBP. All analogues except reverse OGP, [3-I(Tyr10)]OGP and [Cys15(S-NEtSucc)]OGP-NH2 reacted with anti-OGP antibodies. These data are not only important for labeling purposes but suggest a respective role for the OGP N-and C-terminal regions in binding to the OGPBP and putative OGP receptor. It appears that the OGP proliferative activity represents the net effect of stimulation specific to the OGP structure and nonspecific inhibition associated with the peptide's high positive net charge.

Substance P degrading systems of rat parotid and hypothalamus.

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu6]substance P6-11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K+ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu6]substance P6-11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe8-Gly9 with minor cleavage sites between Phe7-Phe8 and Gly9-Leu10. In the rat parotid slice system the major cleavage occurs between Gly9-Leu10 with a minor cleavage site between Phe7-Phe8. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH2, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.

Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone.

PTH maintains blood calcium concentrations within the physiological range by acting on a G protein-coupled heptahelical receptor (PTH1 Rc) located primarily in cells in bone and kidney. We have undertaken a photoaffinity cross-linking approach to elucidate the nature of the bimolecular interaction of PTH with the human (h) PTH1 Rc. Specifically, we have studied the region of the receptor that interacts with the midregion of PTH-(1-34), position 13, using a benzophenone-containing photoaffinity ligand, 125I-[Nle(8,18),Lys13(epsilon-pBz2),L-2-NaI23,Arg(26,2 7),Tyr34]bPTH-(1-34)NH2 (125I-K13). Using site-directed mutagenesis in combination with biochemical analysis, we have reduced our previously identified contact domain, 17 residues in the extracellular region of the receptor (173-189), to an 8-amino acid domain (182-189). Furthermore, we have found arginine 186 to be of critical importance to the interaction of the hPTH1 Rc with 125I-K13: modification of Arg186 to either lysine or alanine does not modify receptor avidity or signal transduction by the receptor, but eliminates cross-linking to 125I-K13.

The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.

Evaluation in vivo of a potent parathyroid hormone antagonist: [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2.

In an effort to design and select potent parathyroid hormone (PTH) antagonists suitable for clinical utility, a PTH analog was evaluated in vivo in an animal model to assess its properties in preparation for human studies. The previously described PTH antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2, which is highly active in vitro, was documented in these studies to be an effective antagonist of the PTH-stimulated calcemic response in vivo. In thyroparathyroidectomized (TPTX) rats, the efficacy of the antagonist was demonstrated to be dose-dependent. Inhibition was demonstrated when intravenous administration of antagonist started 1 h prior to coinfusion with the PTH agonist [Nle8,18,Tyr34]bPTH(1-34)NH2. Maximal inhibition by antagonist (an 84% decline in serum calcium levels compared with agonist alone) of the calcemic response was observed when a 200-fold molar excess of antagonist (12 nmol/h) was administered. At dose ratios of antagonist:agonist as low as 10:1, a 40-50% inhibition of PTH-stimulated calcemic response is evident, provided a longer (2 h) lead time for antagonist infusion is allowed. Based on these and related studies, the antagonist [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 has displayed sufficient potency to obtain approval from the appropriate institutional and regulatory agencies for clinical trials in hypercalcemic states of parathyroid and tumor origin.

Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice.

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

A role for N-cadherin in the development of the differentiated osteoblastic phenotype.

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADdeltaC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADdeltaC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation.

Recent developments in retro peptides and proteins--an ongoing topochemical exploration.

Main-chain peptidomimetics based on peptide-bond reversal and inversion of chirality represent important structural alterations for peptides and proteins, and are highly significant for biotechnology; these modifications have been widely applied: the D-HIV-protease dimer cleaves only all-D substrate; an all-D-hexapeptide opioid is able to produce analgesia following intraperitoneal administration. Antigenicity and immunogenicity can be achieved by metabolically stable antigens such as all-D- and retro-inverso-isomers of natural antigenic peptides. Isomers, including the retro- and retro-inverso- forms, of hybrid peptides derived from cercropin A and melittin, maintain antimicrobial activity. Therefore, an insight is provided into structure-activity relationships and the rational design of biologically important isomeric peptides.

Direct identification of two contact sites for parathyroid hormone (PTH) in the novel PTH-2 receptor using photoaffinity cross-linking.

Direct examination of the interacting sites between PTH and the human PTH2 receptor (PTH2R) was conducted by photoaffinity cross-linking followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. Photoreactive analogs of PTH, individually substituted with an L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in cells stably expressing recombinant PTH2R. One highly bioactive analog, [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]PTH-(1-34)NH 2 (Bpa1-PTH), was chosen for cross-linking studies. In addition, a PTH analog in which the photoreacive moiety is at the mid-region position 13 (K13) was demonstrated to be bioactive, then cross-linked to PTH2R. The minimal digestion-restricted domain containing the contact site ("contact domain") for 125I-Bpa1-PTH is in the sixth transmembrane domain and part of the third extracellular loop, spanning residues Ser364-Met395 of the receptor. This domain was further confirmed and refined by cross-linking 125I-Bpa1-PTH to two receptor mutants, PTH2R[V380M]- and PTH2R[V380M,M395L]-receptors. Treatment of the cross-linked conjugates with cyanogen bromide identified a single amino acid (position 380) as the putative contact point. The contact domain for 125I-K13 is located in the N-terminal extracellular tail of the receptor (in the C-terminal portion) and spans Gln138-Met147. Further validation of this contact domain was accomplished by photocross-linking to point-mutated PTH2R[K137R] receptor. Previous studies in which PTH analogs were cross-linked to human PTH/PTHrP receptor (PTH1R) identified Met425 and Phe173-Met189 as the contact sites for Bpa1-PTH and K13, respectively. These studies demonstrate that both receptor subtypes, PTH1- and PTH2-receptors, use analogous sites for interaction with positions 1 and 13 in PTH.

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

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

Histidine at position 5 is the specificity "switch" between two parathyroid hormone receptor subtypes.

The PTH and PTH-related protein (PTHrP) system consists of two hormones, at least two G protein-coupled seven-transmembrane domain receptors, and at least two intracellular signal transduction pathways for each receptor. The PTH/PTHrP receptor is present in the conventional target tissues of PTH action, namely kidney and bone. Both PTH and PTHrP bind to and activate the PTH/PTHrP receptor with equal affinity and efficacy. The newly discovered receptor, termed the human (h) PTH2 receptor, has 70% homology with the PTH/PTHrP receptor, but is found predominantly in brain and pancreas. It interacts selectively with PTH and not with PTHrP. PTH and PTHrP differ in several positions, including position 5 (Ile in PTH; His in PTHrP). To define the role of position 5 in receptor selectivity, we designed and synthesized a series of hybrid analogs containing specific elements of both the PTH and PTHrP sequences. Using human cell lines stably expressing either human receptor subtype, we evaluated the biological profile of the hybrids in assays of receptor binding and action. Both point-mutated hybrids, [Ile5]PTH-(1-34) and [His5]PTH-(1-34), bind to and stimulate cAMP accumulation and the release of cytosolic free calcium in HEK293/C-21, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH/PTHrP receptor. However, only [Ile5]PTHrP-(1-34), and not [His5]PTH-(1-34), binds to and stimulates cAMP accumulation and the release of cytosolic free calcium in HEK293/BP-16, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH2 receptor. The segmental hybrid PTHrP-(1-14)-PTH-(15-34) binds to and activates the hPTH/PTHrP receptor, but not the hPTH2 receptor, similar to the biological profile of His5-containing ligands: PTHrP-(1-34) and [His5]PTH-(1-34). Exchanging Ile5 for His5 in the segmental hybrid produces the analog [Ile5]PTHrP-(1-14)-PTH-(15-34), which interacts with both receptor subtypes. We conclude that His5 in PTHrP is the major structural determinant of receptor subtype specificity in the hPTH/PTHrP and hPTH2 two-receptor system. The mechanism of the specificity "switch" remains to be elucidated, but may result from a subtle perturbation of the bioactive conformation and/or from a direct steric hindrance at the hPTH2 receptor-ligand interface created by histidine at position 5. The hPTH2, but not the hPTH/PTHrP, receptor can discriminate between the two hormones based on the structural differences generated at position 5.

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.

The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.

We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.

Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.

Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK-293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (> 400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.

Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals, OGP increases osteogenesis. Immunoreactive OGP (irOGP) in high abundance was demonstrated in normal animal serum mainly as an OGP-OGP-binding protein (OGPBP) complex. Here we show the presence of an OGP-OGPBP system in normal human serum. The total irOGP content, of which the bound peptide comprises at least 80-90%, ranged from 480-4460 mumol/L, several orders of magnitude higher than that of other regulatory polypeptides. The steady state/total irOGP ratio declined between 23 and 49 yr of age. The bound irOGP, purified by boiling, ultrafiltration, and hydrophobic high pressure liquid chromatography, was identical to OGP obtained previously from rat regenerating marrow and mouse stromal cell cultures in terms of its amino acid sequence, immunoreactivity, and mitogenicity. These data demonstrate the usefulness of our immunoassay to measure circulating OGP. More importantly, the identity of the human OGP with that of other species indicates the peptide's evolutionary conservation and, thus, its biological importance. The natural occurrence of OGP in man signifies its potential role in the prevention of bone loss and rescue of bone mass, especially in osteoporosis.

Stereospecific antibodies to propranolol.

The beta-adrenergic antagonist propranolol was activated through its side chain, coupled to bovine serum albumin, and injected into BALB/c mice. After fusion of the splenocytes from these immunized mice with the NS-1 myeloma cell line, two hybridomas, producing monoclonal anti-propranolol antibodies, were isolated. Clone P-49 was monospecific for propranolol, with a significant preference for the 1-stereoisomer, as compared to the d form. On the other hand, clone P-28 cross-reacted with alprenolol as well as some other beta-antagonists. Both classes of antibodies competed with A431 epidermoid carcinoma beta 2-adrenoceptors for the binding of [3H]propranolol. When ascites cells from clone P-28 were fixed with glutaraldehyde, the anti-propranolol monoclonal antibody became cell bound. These cell-bound P-28 antibodies bind propranolol and other beta-adrenergic ligands with a similar ranking order to the soluble monoclonal antibody. The cell-bound antibody displayed a 5-fold higher affinity towards 1-propranolol than the soluble monoclonal antibody. The practical implications of these findings are discussed.

GTP analogue hydrolysis by the Gs protein: implication for the role of catalytic glutamine in the GTPase reaction.

Hydrolysis of GTP, bound to members of the G-protein superfamily, terminates their downstream signaling activity. A conserved glutamine serves a critical role in this pivotal guanosine triphosphatase (GTPase) reaction. However, the role of the catalytic glutamine in GTP hydrolysis is still not well understood. We have employed substrate-assisted catalysis to probe the catalytic mechanism of Gs alpha using GTP analogues. These GTP analogues, each having different functional groups, were designed to support or refute particular putative GTPase mechanisms. We have found that a hydrogen donor group, in close proximity to the gamma-phosphate of GTP, is necessary and sufficient to substitute for the function of the catalytic glutamine in the GTPase reaction.

Inositol 1-,4-,5-trisphosphate-dependent Ca2+ signaling by the recombinant human PTH/PTHrP receptor stably expressed in a human kidney cell line.

We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expressing various numbers of the recombinant human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (Rc). Using this expression system we examined ligand (PTH or PTHrP) binding characteristics and cyclic AMP responsiveness. We have now extended these studies to investigate the calcium signal transduction pathways activated by the hPTH/PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP Rc, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ concentration [Ca2+]i. In HEK-293 clone C-21, stably expressing approximately 400,000 Rc/cell, PTH stimulated an increase in [Ca2+]i by Ca2+ release from intracellular stores; PTH released Ca2+ exclusively from the IP3 sensitive Ca2+ pool. Unlike previous studies, the ability of PTH to elicit both cAMP responses and [Ca2+]i transients occurred over a wide range of Rc numbers (between 400,000 and 3000 Rc/cell); both responses were always observed at PTH concentrations in the same dose range although the magnitude of the responses decrease with Rc number. Pretreatment of C-21 cells with pertussis toxin for 24 h, which significantly enhanced PTH-stimulated cAMP accumulation, did not modulate PTH-stimulated [Ca2+]i transients. At each PTH concentration tested which resulted in increased cAMP levels, there was also an increase in [Ca2+]i transients. Treatment of C-21 cells with a battery of midregion and C-terminal PTH or PTHrP peptides showed no effect on either [Ca2+]i transients or cAMP accumulation, indicating a lack of functional interactions between these peptides and the form of the hPTH/PTHrP Rc stably expressed in these cells. Immunological analysis of G-protein expression demonstrated the presence of Gs, Gi, and Gq in all parental and transfected cells lines examined. Taken together, these data demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cells, elicits responses in both the cAMP and IP3-dependent [Ca2+]i pathways and is responsive only to N-terminal PTH/PTHrP peptides.

Antagonism of morphine-induced respiratory depression by novel anticholinesterase agents.

This study compared the effects of 3 novel antiAChE agents (derivatives of dimethylaminoethyl-phenyl carbamate) with that of physostigmine on the respiratory depression induced by morphine in rabbits. Each drug, RA6, (1 mg i.v., 2 mg s.c.) RA7 (1 or 2 mg i.v.); RA15 (0.25 or 0.5 mg i.v.), physostigmine (0.05 or 0.1 mg i.v.) or saline (1 ml), was injected simultaneously with morphine (8 mg i.v.) to groups of 6-10 rabbits. Respiration rate, blood gases and pH were monitored for 3 hr. Plasma ChE was measured before and at 15 min intervals after injection. The 4 antiAChE's were given to 40 other rabbits, which were sacrificed at the time of maximal antagonism of the respiratory depressant effect of morphine, in order to measure the activity of AChE in the medulla, cortex and hippocampus. Physostigmine (0.1 mg) only antagonized the increase in paCO2 induced by morphine at 15 and 30 min. The drugs RA15 (0.5 mg), RA6 (2.5 mg) and RA7 (2 mg) almost completely prevented the respiratory depression, without obvious signs of peripheral cholinergic hyperactivity, for at least 3 hr. There was no relationship between the degree of antagonism of the effects of morphine with any drug and that of inhibition of ChE in plasma. In contrast, a highly significant correlation (P less than 0.01) was found between the former and the amount of inhibition of AChE in the medulla. It is suggested that the novel carbamates may have potential therapeutic application in reducing the respiratory depression of opiates, without impairing analgesia.