Treatment with parathyroid hormone hPTH(1-34), hPTH(1-31), and monocyclic hPTH(1-31) enhances fracture strength and callus amount after withdrawal fracture strength and callus mechanical quality continue to increase.

The influence of intermittent hPTH(1-34)NH2, hPTH(1-31)NH2, and monocyclic [Leu27]cyclo (Glu22-Lys26)hPTH(1-31)NH2 treatment on callus formation, mechanical strength, and callus tissue mechanical quality of tibial fractures in rats was investigated after 8 and 16 weeks of healing. In the 8 weeks of healing animals, the PTH-peptides were injected subcutaneously during the entire observation period (15 nmol/kg/day [hPTH(1-34)NH2: 15 nmol = 60 microg]), and control animals with fractures were given vehicle. In the 16 weeks of healing animals, the PTH-peptides were injected only during the first 8 weeks of healing (15 nmol/kg/day), after which the animals were left untreated during the rest of the healing period. After the first 8 weeks of healing, increased fracture strength and callus volume were seen in the PTH-treated rats (ultimate load 66%, ultimate stiffness 58%, callus volume 28%), and the three peptides were equally effective. No difference in callus tissue mechanical quality was found between PTH and vehicle animals. After 16 weeks of healing, no differences in fracture strength, callus volume, or callus tissue mechanical quality were seen between PTH and vehicle. When comparing PTH-treated animals at 8 and 16 weeks, fracture strength and callus tissue mechanical quality continued to increase after the withdrawal of PTH (ultimate load 23%, ultimate stress 88%, elastic modulus 87%) and external callus volume declined during this period (27%).

Structural requirements for conserved arginine of parathyroid hormone.

Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.

Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specific protective immunity against infection with the facultative intracellular pathogen, Listeria monocytogenes.

Protective immunity to intracellular bacterial pathogens usually requires the participation of specific CD8+ T cells. Natural exposure of the host to sublethal infection, or vaccination with attenuated live vaccines are the most effective means of eliciting prolonged protective cell-mediated immunity against this class of pathogens. The ability to replace these immunization strategies with defined sub-unit vaccines would represent a major advance for clinical vaccinology. The present study examines the ability of novel liposomes, termed archaeosomes, made from the polar lipids of various Archaeobacteria to act as self-adjuvanting vaccine delivery vehicles for such defined acellular antigens. Using infection of mice with Listeria monocytogenes as a model system, this study clearly demonstrates the ability of defined, archaeosome-entrapped antigens to elicit rapid and prolonged specific immunity against a prototypical intracellular pathogen. In this regard, all of the tested archaeosomes were superior to conventional liposomes.

Parathyroid hormone: an anabolic treatment for osteoporosis.

Osteoporosis is a disease characterised by low bone mass, structural deterioration of bone and increased risk of fracture. The prevalence, and cost, of osteoporosis is increasing dramatically with our ageing population and the World Health Organization now considers it to be the second-leading healthcare problem. All currently approved therapies for osteoporosis (eg., estrogen, bisphosphonates, calcitonin and selective estrogen receptor modulators) are anti-resorptive agents that act on osteoclasts to prevent further bone loss. A new class of bone anabolic agent capable of building mechanically strong new bone in patients with established osteoporosis is in development. While the parathyroid hormone (PTH) is classically considered to be a bone catabolic agent, when delivered intermittently at low doses PTH potently stimulates cortical and trabecular bone growth in animals humans. The native hPTH-(1-84) and its osteogenic fragment, hPTH-(1-34), have already entered Phase III clinical trials. Understanding the mechanism of PTH's osteogenic actions has led to the development of smaller PTH analogues which can also build mechanically normal bone in osteopenic rats. These new PTH analogues are promising candidates for treating osteoporosis in humans as they are as efficacious as hPTH-(1-84) and hPTH-(1-34), but there is evidence that they may have considerably less ability to induce hypercalcemia, the major side effect of PTH therapy. In addition to treating osteoporosis, PTHs may be used to promote fracture healing, to restore bone loss in immobilized patients, or following excessive glucocorticoid or prolonged spaceflight, and to treat psoriasis.

The effect of monocyclic and bicyclic analogs of human parathyroid hormone (hPTH)-(1-31)NH2 on bone formation and mechanical strength in ovariectomized rats.

The [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 lactam is a stronger stimulator of adenylyl cyclase activity and a better stimulator of trabecular bone in the ovariectomized (OVX) rat model of osteopenia than hPTH-(1-31)NH2. This enhanced activity is due in large part to the stabilization of the amphiphilic receptor-binding alpha-helix in the Ser17-Gln29 region. The goal of the present study was to determine whether further cyclization could produce a more active hPTH analog. To this end, we compared the relative bioactivities of the bicyclic hPTH analog [Glu17,Leu27]cyclo(Lys13-Glu17,Glu22-Lys26)-hPTH-(1-31)NH2, made by replacing Ser17 with Glu17 and introducing a second lactam linkage between Lys13 and Glu17. The relative EC50 for adenylyl cyclase stimulation by the bicyclic hPTH analog was similar to the EC50 of the monocyclic [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, but the bicyclic analog was still more active than hPTH-(1-31)NH2. As expected from adenylyl cyclase stimulation being responsible for PTH's anabolic action, the bicyclic hPTH analog [Glu17, Leu27]cyclo(Lys13-Glu17, Glu22-Lys26)-hPTH-(1-31)NH2 was able to increase femoral trabecular volume and thickness and mechanical strength in OVX rats, but it was no more effective than [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 when injected once daily in a dose of 0.8 nmol/100 g body weight. Thus, further constraint of the conformation of hPTH-(1-31)NH2 by introducing two lactam link-ages between Lys13-Glu17 and Glu22-Lys26 did not raise the osteogenicity above that of the monocyclic analog.

Stimulation of protein kinase C activity in cells expressing human parathyroid hormone receptors by C- and N-terminally truncated fragments of parathyroid hormone 1-34.

The parathyroid hormone (PTH) fragment PTH(1-34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1-34), such as human PTH(1-31) [hPTH-(1-31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1-31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3-34) [bPTH(3-34)], hPTH(3-34)NH2, and hPTH(13-34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3-34)NH2 and hPTH(13-34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1-34)NH2 and hPTH(1-31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1-34)NH2 and hPTH(1-31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1-34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.

The parathyroid hormones: bone-forming agents for treatment of osteoporosis.

This is a 3-part article. Part 1 is an overview of bone formation and resorption and the consequences of estrogen loss on bone. Part 2 comprehensively reviews the most current data on the ability of a family of potent osteoblast-stimulating bone-builders, the native 84-amino-acid parathyroid hormone (PTH), and certain of its 31- to 38- amino-acid fragments to stimulate the growth of animal and human bones. These PTHs are currently in, or about to enter, clinical trial as treatment for postmenopausal osteoporosis. Part 3 provides a detailed consideration of how these PTHs might stimulate bone growth via PTH receptor signaling.

The parathyroid hormone, its fragments and analogues--potent bone-builders for treating osteoporosis.

As populations age a rising number of men and women, but especially women during the first decade after menopause, become victims of a severe, accelerated loss of bone with crippling fractures known as osteoporosis. This often results in costly, prolonged hospitalisation and perhaps indirectly, death. Osteoporosis in women is caused by the menopausal oestrogen decline, which removes several key restraints on the generation, longevity and activity of bone-resorbing osteoclasts. Although there are many antiresorptive drugs on or coming onto the market (calcitonin, bisphosphonates, oestrogen and SERMS) that can slow or stop further bone loss, there are none that can restore lost bone mechanical strength by directly stimulating osteoblast activity and bone growth. However, there is a family of potent bone-building peptides, namely the 84 amino acid parathyroid hormone (PTH). Its 31 to 38 amino acid N-terminal fragments are currently in or about to enter clinical trials. We can predict that these peptides will be effective therapeutics for osteoporosis especially when supplemented with bisphosphonates or SERMs to protect the new bone from osteoclasts. These peptides should also accelerate the healing of fractures in persons of all ages and restore lost bone mass and mechanical strength to astronauts following their return to earth after long voyages in space.

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones.

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone.

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.

Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2.

Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I-[Nle8,18,Tyr34]bovine PTH(1-34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH-related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the alpha-helix in the residue 17-28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 analog dramatically reduced the IC50 for displacing 125I-[Nle8,18,Tyr34]bPTH(1-34)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1-28)NH2. Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs.

The stimulation of vertebral and tibial bone growth by the parathyroid hormone fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27] cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10-25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1-L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal.

High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli.

A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.

Design and applications of parathyroid hormone analogues.

The endocrine parathyroid hormone (PTH) is the major regulator of serum calcium levels. In contrast, the autocrine/paracrine parathyroid hormone-related peptide (PTHrP) has been associated with organism development. Both are secreted as much larger molecules but have their major functions associated with their N-terminal 34 residues. They share a common receptor expressed in organs critical to PTH function - bone, kidney, and intestine. PTH and PTHrP receptor activation stimulates adenylyl cyclase (AC), phospholipase C (PLC), and phospholipase D (PLD) in target cells. It has been possible to separate the AC-stimulation from that of PLC. AC-stimulation requires at least the N-terminal 28 residues of PTH and PLC-stimulation requires a minimum of residues 29-32-NH2. Intermittent administration of PTH stimulates bone growth and requires AC-stimulation. The shortest linear sequence of hPTH with essentially full anabolic activity for bone growth-stimulation is hPTH(1-31)NH2. Two applications are postulated for PTH and PTHrP-based pharmaceuticals - treatment of bone loss due to osteoporosis and reversal of the hypercalcemic effect of malignancy. PTHrP analogues which strongly inhibit PTHrP AC-stimulation showed promise for the treatment of malignancy-associated hypercalcemia in animal trials but failed in human ones. However, both animal and human trials of hPTH have shown significant bone growth-stimulating effects. New deletion, substitution and cyclized analogues of PTH show great promise both for greater in vitro activity and possibly for improved delivery and greater specificity as agents for restoration of bone loss in osteoporosis.

The bone-building action of the parathyroid hormone: implications for the treatment of osteoporosis.

As populations age in the world's 7 major pharmaceutical markets, an increasing number of men and women, but especially women during their first postmenopausal decade, are falling victim to a bone disease that can result in hospitalisation, disability and death. This disease is osteoporosis. Indeed, by the year 2007, as many as 153,000,000 people will have experienced at least some significant osteopenia, if not osteoporosis. Although there are many different drugs, such as calcitonin, the bisphosphonates, estrogen and selective estrogen receptor modulators, that can slow or even stop further bone loss, there are currently few that can replace already lost bone by directly stimulating bone growth. However, a family of potent bone-building (or bone-anabolic) peptides is currently undergoing clinical development. These are the first-generation 84-amino acid native parathyroid hormone (PTH) and its 34- to 38-amino acid N terminal fragments, and the potent second-generation mini-PTHs. In this article, we briefly summarise what has so far been learned about how these molecules stimulate the production of new biomechanically strong bone in animals and humans.

Comparison of the biochemical responses to human parathyroid hormone-(1-31)NH2 and hPTH-(1-34) in healthy humans.

The 1-31 fragment of human PTH [hPTH-(1-31)NH2] has been shown, like hPTH-(1-34), to have anabolic effects on the skeletons of ovariectomized rats when given intermittently, but, unlike hPTH-(1-34), it does so without affecting serum calcium concentrations and does not activate the protein kinase C second messenger pathway in some target cells. To investigate the biochemical responses to hPTH-(1-31) in humans, we have directly compared it to hPTH-(1-34) during the course of slow infusions of each. Ten healthy adults, five men and five women, aged 26+/-5 yr (range, 22-37), each received 8-h continuous infusions of 8 pmol/kg.h hPTH-(1-34) and hPTH-(1-31) given in random order at least 2 weeks apart. During the infusions there were significant increases in both plasma and urinary cAMP (P < 0.05), but there were no differences in the responses between the two peptides (P = 0.362 for plasma; P = 0.987 for urine). There were also significant phosphaturic and natriuretic responses to the two peptides, which again were not different between peptides. During the infusion of hPTH-(1-34) serum ionized calcium (Ca2+) increased from 1.21+/-0.033 to 1.29+/-0.046 mmol/L (P < 0.01), and endogenous hPTH-(1-84) decreased from 29.6+/-9 to 15.0+/-5.7 pg/mL (P < 0.01), such that there was a negative correlation between them (r2 = 0.45). However, when hPTH-(1-31) was infused, neither serum Ca2+ (1.24+/-0.03 vs. 1.25+/-0.03) nor hPTH-(1-84) (26.8+/-5 vs. 30.7+/-12 pg/mL) was affected. Circulating concentrations of 1,25-dihydroxyvitamin D3 increased from 92+/-42 to 131+/-63 pmol/L (P < 0.05) during infusion of hPTH-(1-34) and from 92+/-27 to 110+/-42 pmol/L (P = NS) during hPTH-(1-31) infusion. There was also a significant increase in the urinary measure of type I collagen degradation of aminoterminal telopeptides from 78+/-45 to 101+/-51 nmol/mmol creatinine (P < 0.05) when hPTH-(1-34) was infused, but it was not affected (68+/-30 vs. 66+/-24 nmol/mmol creatinine) by hPTH-(1-31). Therefore, hPTH-(1-31) appears to be equivalent and equipotent to hPTH-(1-34) in the release of cAMP from target tissues and the renal handling of phosphate and sodium. However, at the doses employed, it does not increase serum calcium, is a weaker stimulator of the 25-hydroxyvitamin D-1alpha-hydroxylase, and does not induce rapid bone resorption.

Stimulation of femoral trabecular bone growth in ovariectomized rats by human parathyroid hormone (hPTH)-(1-30)NH(2).

It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTH's bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH(2), which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH(2) to stimulate bone growth despite its having hPTH-(1-31)NH(2)'s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH(2)'s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragment's ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2-10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10-50 nmol of hPTH-(1-30)NH(2)/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH(2)/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH(2), [Leu(27)]cyclo(Glu(22)-Lys(26))-hPTH-(1-31)NH(2), hPTH-(1-34)NH(2) and [Leu(27)]cyclo(Glu(22)-Lys(26))-hPTH-(1-34)NH(2). These results have removed a major objection to the proposal that PTH's osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause.

Prolonged low-dose infusion of human parathyroid hormone does not increase femoral cancellous bone volume in ovariectomized rats.

OBJECTIVE: Daily injections of human parathyroid hormone (hPTH) increase bone volume in various animal species and in osteoporotic women. For hPTH to be widely accepted as an anabolic therapy for treating postmenopausal osteoporosis alternative delivery options need to be explored to replace the need for daily patient subcutaneous self-injection. Among these are inhalation, oral delivery and the use of programmable implanted minipumps to deliver the peptide. While infusion of high doses of PTH causes bone loss and hypercalcemia, no studies have assessed the effects of prolonged infusion of low doses of PTH on bone growth. DESIGN AND METHODS: [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2) was delivered by Alzet minipumps to ovariectomized rats for 6 weeks after which histomorphometric indices (cancellous bone volume, trabecular thickness, mean trabecular number) of bone formation were measured in distal femurs. RESULTS: Infusing low doses (0.05 and 0.1 nmole/100g body weight/day) of the hPTH analog, [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2), for 6 weeks does not prevent the ovariectomy-induced loss of rat femoral cancellous bone volume, trabecular thickness or trabecular number. CONCLUSION: These results support the absolute requirement of daily injections for the osteogenic action of hPTH on bone.

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.

Differential expression of alpha1, alpha2, alpha3, and alpha5 GABAA receptor subunits in seizure-prone and seizure-resistant rat models of temporal lobe epilepsy.

Temporal lobe epilepsy remains one of the most widespread seizure disorders in man, the etiology of which is controversial. Using new rat models of temporal lobe epilepsy that are either prone or resistant to develop complex partial seizures, we provide evidence that this seizure susceptibility may arise from arrested development of the GABAA receptor system. In seizure-prone (Fast kindling) and seizure-resistant (Slow kindling) rat models, both the mRNA and protein levels of the major alpha subunit expressed in adult brain (alpha1), as well as those highly expressed during development (alpha2, alpha3, and alpha5), were differentially expressed in both models compared with normal controls. We found that alpha1 subunit mRNA expression in the Fast kindling strain was approximately half the abundance of control rats, whereas in the Slow kindling strain, it was approximately 70% greater than that of controls. However, Fast rats overexpressed the alpha2, alpha3, and alpha5 ("embryonic") subunits, having a density 50-70% greater than controls depending on brain area, whereas the converse was true of Slow rats. Using subunit-specific antibodies to alpha1 and alpha5 subunits, quantitative immunoblots and immunocytochemistry revealed a concordance with the mRNA levels. alpha1 protein expression was approximately 50% less than controls in the Fast strain, whereas it was 200% greater in the Slow strain. In contrast, alpha5 subunit protein expression was greater in the Fast strain than either the control or Slow strain. These data suggest that a major predispositional factor in the development of temporal lobe epilepsy could be a failure to complete the normal switch from the GABAA receptor alpha subunits highly expressed during development (alpha2, alpha3, and alpha5) to those highly expressed in adulthood (alpha1).