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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.

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 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 [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.

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.

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.

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 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.

A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents., (Copyright 2000 Academic Press.)

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.

To examine the importance of the N- or C-termini of PTH(1-34) the effects of truncated fragments of PTH on human receptors in osteoblast-like SaOS-2 cells and rat receptors in rats ROS 17/2 cells were examined. Fura-2-loaded cells were used to monitor cytosolic free Ca(2+) concentration ([Ca2+]i), and the Cytosensor microphysiometer was used to monitor extracellular acidification rate (ECAR). C-terminally truncated fragments (1-31) and (1-28) of hPTH(1-34)NH(2) stimulated an increase in [Ca(2+)](i) and ECAR in both cell lines. hPTH(3-34)NH(2) and other N-terminally truncated fragments did not stimulate [Ca(2+)](i) or ECAR in either cell type. The signal transduction pathway of PTH-induced ECAR in ROS 17/2 cells was investigated to compare with previous results in SaOS-2 cells. Potentiation by IBMX, attenuation by 8Br-cAMP and lack of effect of the PKC inhibitor chelerythrine chloride support a cAMP/PKA-mediated signal transduction pathway in ROS 17/2, while the protein kinase C pathway was predominant in SaOS-2 cells. We conclude that the intact N-terminus of PTH is essential in PTH signaling mediated via either the cAMP/PKA or inositol lipid/Ca(2+)/PKC pathways in osteoblast-like cells., (Copyright 1999 Academic Press.)

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 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.

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.

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.

Protein kinase C-delta (PKC-delta) appears to be variously involved in proliferation and apoptosis. To compare the changes of this enzyme in these two processes, we have determined the levels and activities of the 79-kDa PKC-delta holoenzyme and its catalytically active 47- and 40-kDa C-terminal fragments in the nuclei of proliferating untreated polyomavirus-transformed pyF111 rat fibroblasts and pyF111 cells treated with the apoptogenic topoisomerase-II inhibitors VP-16 (etoposide), VM-26 (teniposide), and doxorubicin. PyF111 cells were chosen because they hyperexpress PKC-delta and they are hypersusceptible to apoptosis because they do not express the antiapoptotic proteins Bcl-2 and Bcl-XL. The highest PKC-delta activity in cells before they started proliferating or were exposed to one of the inhibitors was in the NM (nuclear envelope-containing) fraction, which contained the holoenzyme and both C-terminal fragments, while only the two fragments were in the nucleoplasmic (NP) fraction where they were tightly associated with chromatin. When the cells began proliferating the amounts of the PKC-delta holoenzyme and the two fragments increased in the NM and the NP fractions and the already high PKC-delta activity either increased or stayed the same in these fractions until the end of the 72-h incubation. And there was no leakage of cytochrome c from the mitochondria into the cytoplasm. VP-16 exposure caused a prompt release of cytochrome c from the mitochondria into the cytosol and at the same time triggered a sharp drop (35% by 3 h and 60% by 6 h) in the PKC-delta activity in the NM fraction without changing the actual amounts of the holoenzyme or its fragments. This prompt inactivation of PKC-delta and its fragments during the first 6 h of exposure to the drug was not due to their dephosphorylation and could not be reversed by phosphatidylserine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). Between 6 and 24 h the PKC-delta activity in the NM fraction dropped a further 20%, the kinase's activity transiently surged in the NP fraction, and cytoplasmic CPP-32-like (DEVD-specific caspase) activity increased without an increase in the proteolysis of nuclear PKC-delta or PARP. Between 24 and 72 h nuclear CPP-32-like activity increased along with a massive proteolysis of PKC-delta, an accumulation of various PKC-delta fragments, and the cleavage of PARP. But despite this proteolysis, the cells were still able to maintain or even increase the amounts of holoenzyme and 40- and 47-kDa fragments in the NM and NP fractions before dying. VM-26 and doxorubicin caused the same prompt release of cytochrome c from the mitochondria and dramatic drop of NM PKC-delta activity as did VP-16. Thus, high levels of activity of nuclear PKC-delta, particularly PKC-delta in the nuclear membrane, might have a role driving the cell cycle of pyF111 cells. On the other hand, the prompt and sustained large drop in the activity of PKC-delta at this site that precedes the onset of the caspase-mediated proteolysis of the isoform may be involved in starting and driving apoptogenesis in pyF111 fibroblasts exposed to topoisomerase-II inhibitors., (Copyright 1999 Academic Press.)

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.

PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cbeta (PLCbeta). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCbeta. We further tested the hypothesis that phospholipase D is responsible for PKC activation in distal tubule cells. PTH-(1-31) increased [Ca2+]i in distal tubule but not in proximal tubule cells, whereas PTH-(3-34) caused a partial increase in [Ca2+]i in proximal cells, but had no effect in distal cells. PTH-(7-34) blocked increases in [Ca2+]i in distal tubule cells stimulated by PTH-(1-34) and PTH-(1-31). The PLC inhibitor U73122 abolished the PTH-induced rise in [Ca2+]i and inositol trisphosphate formation by proximal tubule cells, but had no effect on PTH-stimulated Ca2+ uptake by distal tubule cells. These results support the view that activation of PKC by PTH in distal tubule cells does not involve PLCbeta. PTH did, however, activate phospholipase D with attendant formation of diacylglycerol in distal cells. As activation of PKC is required for induction of calcium transport by PTH, we conclude that PTH receptors are capable of activating multiple phospholipases and that the structural requirements for such activation differ in proximal and distal tubule cells.

The immunological cross-reactivity of several peptides with specific pattern-property characteristics related to the epitopes of human immunodeficiency virus type 1 (HIV-1) gp160/ 120 envelope proteins has been investigated. Proteins with similar primary structures can be expected to show functional or topographic similarities, such as specific epitopes which may cross-react with antibodies derived from the immunisation of animals with other members of the same protein family. These structure-function characteristics may be revealed as periodicities derived from presentations based on the discrete Fourier transformation of the distributions of various physico-chemical amino acid descriptors, constituting the polypeptide backbone and amino acid side-chains of the protein molecule. Such approaches, for example, have permitted prediction of periodicities corresponding to secondary structural motifs, including amphipathic alpha-helices and beta-sheets, within protein sequences, and have helped to clarify potential binding sites for ligands, substrates or cofactors with interacting macromolecules. Based on this approach, characteristic periodicities have been identified which represent common Fourier transform spectral properties of the envelope (ENV) gp160/120 glycoproteins from a range of HIV-1 isolates. In addition, similar periodicities have been detected as components of the discrete Fourier transform representation of the corresponding amino acid descriptors of the CD4 binding domain of gp120. Accordingly, we have synthesised several peptides having periodic characteristics in their discrete Fourier transform representations similar to these HIV-1 proteins. These nonhomologous synthetic peptides induced cross-reactive antibodies in New Zealand White rabbits. Polyclonal antibodies raised to one of these peptides reacted with HIV-1 ENV gp120-related proteins, as determined by enzyme-linked immunosorbent assay and Western blotting techniques. These findings provide further evidence for a role of immunological cross-reactivity and molecular biomimicry in the development of peptide-based vaccines directed against viral or bacterial pathogens.

hPTH-(1-31)NH2, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH2. We have now shown that [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight.

N-terminal fragments of PTH and PTHrP, such as hPTH-(1-34) and hPTHrP-(1-34), are sufficiently similar with respect to amino acid sequence, location of functional domains, and higher order configuration to activate the same PTH/PTHrP receptor and the same two signal enzymes, adenylyl cyclase and phospholipase-Cbeta. Therefore, it was expected that hPTHrP-(1-31)NH2 would stimulate bone growth in ovariectomized rats as strongly as hPTH-(1-31)NH2. Like hPTH-(1-31)NH2, hPTHrP-(1-31)NH2 stimulated adenyly cyclase in ROS 17/2 osteosarcoma cells as strongly as the standard hPTH-(1-34) and like hPTH-(1-31)NH2, triggered a large drop in mean blood pressure when injected intravenously. Unlike hPTH-(1-31)NH2, however, hPTHrP-(1-31)NH2 could not stimulate trabecular growth in the distal femurs of young, sexually mature, ovariectomized rats.

Parathyroid hormone (PTH) is classically considered to be a bone catabolic agent; however, when delivered intermittently at low doses, PTH potently stimulates cortical and/or trabecular bone growth in animal and clinical studies. Understanding the mechanism of PTH's osteopenic actions has led to the making of small, novel, and possibly noninjectable PTH analogues that can build biomechanically normal bone in osteopenic rats and are promising candidates for treating osteoporosis in humans.

Human parathyroid hormone (hPTH)-(1-31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase-C as do hPTH(1-84) and hPTH(1-34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side-chain lactams have been introduced in the hydrophilic face of the receptor-binding region of the fragment's Ser17-Val31 amphiphilic alpha-helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this alpha-helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2, which had six times the AC-stimulating ability of hPTH(1-31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix-stabilizing lactam derivative [Leu27]-cyclo(Lys26-Arg30)-hPTH(1-31)NH2 did not have a greater AC-stimulating ability than hPTH(1-31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTH's anabolic action, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1-31)NH2 or [Leu27]-cyclo(Lys26-Arg30)-hPTH(1-31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1-31)NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.

In a search for analogues of human parathyroid hormone (hPTH) with improved activities and bioavailabilities, we have prepared the following three lactam analogues of hPTH-(1-31)-NH2 (1) or [Leu27]hPTH-(1-31)-NH2 (2): [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 (3), [Leu27]cyclo(Lys26-Asp30)-hPTH-(1-31)-NH2 (4), and cyclo(Lys27-Asp30)-hPTH-(1-31)-NH2 (5). Analogues 1, 2, and 5 had seven or eight residues of alpha-helix, as estimated from their circular dichroism (CD) spectra, in contrast to 12 residues in cyclic analogues 3 and 4. Thus, lactams 3 and 4 stabilized a helix previously shown to exist within residues 17-29. The adenylyl cyclase activity (EC50), measured in rat osteosarcoma 17/2 cells, of 5 (40.3 +/- 2.3 nM) was half that of its linear form 1 (19.9 +/- 3.9 nM). The linear Leu27 mutant 2 was twice as active (11.5 +/- 5.2) as analogue 1, and lactam analogue 3 was 6-fold more active (3.3 +/- 0.3 nM). Lactam analogue 4 had less activity (16.9 +/- 3.3 nM) than 2, its linear form. Peptides hPTH-(1-30)-NH2 (6), [Leu27]hPTH-(1-30)-NH2 (7), and [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-30)-NH2 (8) all had AC-stimulating activities similar to that of 1. When injected intravenously, with a dose of 0.8 nmol/100 g of analogue in acid saline, hypotensive effects paralleled their adenylyl cyclase activities. They behaved quite differently when applied subcutaneously. Analogues 1, 5, and 6, the weakest, showed about half the drop in blood pressure observed with 3 and 4, the most active. In contrast, the time required to reach a maximum drop in blood pressure of 4-8, after subcutaneous administration, was 2-4 times that of the other analogues. Thus, the bioavailabilities of the lactam analogues, unlike their adenylyl cyclase-stimulating activities, were highly dependent on the presence or conformation of Val31.

Parathyroid hormone (PTH), hPTH-(1-84), and its hPTH-(1-34), hPTH-(1-31)NH2, and hPTH-(1-30)NH2 fragments reduced the tail artery pressure in anesthetized female Sprague-Dawley rats by 42.4-67.1% within about 1 minute after injection into a femoral vein, but reduced the pressure by only 8.5-36.2% 2-19 minutes after subcutaneous injection. hPTH-(1-84) and hPTH-(1-34) stimulate both adenylyl cyclase and phospholipase-C in their target cells, but the hypotensive action must have been stimulated specifically by adenylyl cyclase activation, because hPTH-(1-30)NH2 and hPTH-(1-31)NH2, which can only stimulate adenylyl cyclase, were potently hypotensive when injected intravenously whereas hPTH-(7-84), which can only stimulate phospholipase-C, was not significantly hypotensive when injected intravenously. Since PTH's osteogenic action is also mediated by adenylyl cyclase stimulation, it was expected that the hypotensive response might be used to screen new PTH constructs for possible osteogenicity. Indeed, the osteogenic activities of subcutaneously injected hPTH-(1-31)NH2, hPTH-(1-34), and hPTH-(1-84) correlated closely to their hypotensive activities, with hPTH-(1-34) being much more hypotensive and significantly more osteogenic than the other two molecules. hPTH-(1-31)NH2 and hPTH-(1-84) were equally osteogenic and hypotensive. However, this correlation broke down with hPTH-(1-30)NH2 which does not stimulate bone formation, but in the present study it stimulated adenylyl cyclase and reduced tail artery pressure almost as much as hPTH-(1-31)NH2 and hPTH-(1-34). Nevertheless, the ability to significantly reduce arterial pressure is a common property of osteogenic PTH and PTH fragments and is thus a rapidly determinable preliminary indicator of in vivo bioactivity of PTH fragments.

A recombinant human parathyroid hormone, rhPTH-(1-84), which is currently in Phase II clinical trial, and hPTH-(1-31)NH2 (Ostabolin) are promising anabolic agents for treating osteoporosis because they can stimulate cortical and trabecular bone growth in osteopenic, ovariectomized (OVX) rats and in osteoporotic, postmenopausal women when injected subcutaneously and intermittently at low doses. We have now found that, despite their different sizes and signaling properties (rhPTH-(1-84) stimulates adenylyl cyclase and phospholipase C; hPTH-(1-31)NH2 only stimulates adenylyl cyclase), they are equally osteogenic in OVX rats. Thus daily subcutaneous injections of 0.6 nmol/100 g of body weight of rhPTH-(1-84) or hPTH-(1-31)NH2 into 3-month-old OVX rats for 6 weeks starting 2 weeks after OVX equally reduced the otherwise large OVX-triggered loss of femoral trabecular bone. Daily subcutaneous injections of 0. 4 or 0.8 nmol/100 g of body weight of the two agents for 6 weeks also equally increased the mean thickness of the remaining femoral trabeculae in 3-month-old and 1-year-old OVX rats to 20 to 80% above the value in normal animals when started 9 weeks after ovariectomy.

One of the effects of transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). To investigate the role of the Ras oncogene product in gap junction closure, and to incorporate GJIC into the gamut of transformation-related properties which can be regulated by increasing levels of oncogene expression, a panel of murine C3H10T1/2 and 3T3L1 fibroblasts was constructed in which graded increments of Ras(leu61) could reliably be obtained. The inducibly activated Ras(leu61) protein substantially reduced or eliminated GJIC at levels much lower than those needed for neoplastic transformation, as indicated by acquisition of the ability to proliferate in the absence of anchorage. These results indicate that disruption of GJIC, although necessary, is not sufficient for neoplastic transformation in this system.

A decrease in gap junctional, intercellular communication (GJIC) has been associated with cells neoplastically transformed by a variety of factors. To investigate the role of the Ras oncogene product in gap junction function, a panel of murine C3H10T1/2 (10T1/2) fibroblasts was constructed in which the levels of ras gene expression could be effectively up- or down-regulated. Intercellular communication was measured using a novel technique of in situ electroporation of adherent cells on a partly conductive slide. The introduction of increasing amounts of activated Ras(leu61) in mouse 10T1/2 fibroblasts proportionally reduced GJIC, while the downregulation of endogenous c-ras gene expression increased junctional permeability. These results indicate that Ras plays an important role in the junction closure pathway leading to the proliferation of normal cells. However, differentiation of c-Ras-deficient preadipocytes entirely abolished their initially extensive GJIC, indicating that junction closure in response to adipocytic differentiation is independent of Ras.

These studies were designed to investigate the role of protein kinase C (PKC) in the regulation of ATP-triggered intracellular Ca2+ ([Ca2+]i) oscillations in chicken granulosa cells. Granulosa cells were obtained from the two largest preovulatory follicles (F1 and F2) of hens and [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. Adenosine triphosphate (100 mumol/l) triggered an immediate, large [Ca2+]i spike that was followed by oscillations that returned to the resting level between spikes. The ATP (100 mumols/l) also stimulated a 1.70 +/- 0.1-fold increase in membrane-associated PKC activity over control levels. The frequency of the ATP-triggered [Ca2+]i oscillations was reduced in a concentration-dependent (1-10 nmol/l) manner by treating the cells for 2 min with a PKC activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA). A higher TPA concentration (100 nmol/l) completely prevented ATP from triggering the initial [Ca2+]i spike and oscillations. Adding TPA during the ATP-triggered [Ca2+]i oscillations immediately stopped the oscillatory activity. Interestingly, PKC inhibitors failed to amplify the ATP-triggered [Ca2+]i oscillations. Instead, adding the PKC inhibitors staurosporine (20 nmol/l), calphostin C (200 nmol/l) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7; 100 mumols/l), either before or during the ATP (100 mumols/l)-triggered [Ca2+]i response, also completely blocked the [Ca2+]i oscillations. Therefore, ATP-triggered [Ca2+]i oscillations in chicken granulosa cells appear to be regulated by a negative feedback loop requiring PKC, because the [Ca2+]i oscillations were prevented by either full activation or inhibition of PKC activity.

The effects of glucose, K+, and cholecystokinin octapeptide (CCK-8) on intracellular free Ca2+ concentration ([Ca2+]i) and membrane-associated protein kinase C (PKC) activity were examined in cultured islet cells from neonatal rats. Raising the glucose concentration from 2.8 to 22.2 mM or external K+ (from 5 to 45 mM), or adding CCK-8 (200 nM) all triggered a [Ca2+]i surge that peaked between 3 and 10 min afterward, depending on the stimulus, and then declined, either to a suprabasal plateau (glucose and K+) or to basal levels (CCK-8). These same manipulations triggered a burst of membrane-associated PKC activity that peaked between 5 and 10 min and then variously declined. Incubation in Ca(2+)-free medium abolished both the effects of glucose and K+ on [Ca2+]i and the stimulation of membrane-associated PKC activity. The K(+)-triggered stimulation of PKC activity was also inhibited by pretreating the cells with the general Ca2+ entry blocker lanthanum (1 mM). However, incubation in Ca(2+)-free medium did not affect the CCK-8-induced release Ca2+ from internal stores, although it abolished the burst of membrane-associated PKC activity, which showed the importance of Ca2+ influx as opposed to internal release for PKC activation. Thus, glucose, the principal stimulator of insulin secretion, rapidly stimulates Ca2+ influx into islet cells from neonatal rats, and it is probably this influx that stimulates membrane-associated PKC activity.

Endogenous, cellular Ras proteins (c-Ras) mediate the transforming action of the polyoma virus middle Tumor antigen (mT), which is accompanied by elimination of gap junctional, intercellular communication (GJIC). In this report we show that reducing the c-Ras content of murine C3H10T1/2 fibroblasts (10T1/2) through the expression of an anti-sense ras gene, increased GJIC by 60-80% mT totally eliminated GJIC in normal 10T1/2 cells but it reduced GJIC no more than 50% in the c-Ras deficient lines. These results indicate that endogenous c-Ras is at least partly responsible for the mT-induced gap junction closure.

The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR) a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 microM) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5-20 microM), but not its remacemide precursor (10-100 microM), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5-20 microM) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 microM NMDA and reduced the NMDA-triggered increases in intracellular free Ca2+ concentration ([Ca2+]i) by up to 70%. By contrast, remacemide (50 and 100 microM) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca2+]i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca2+]i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca2+]i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.

The human parathyroid hormone, hPTH-(1-84), and its hPTH-(1-34) fragment are promising anabolic agents for treating osteoporosis because they can strongly stimulate the production of biomechanically effective cortical and trabecular bone in osteopenic ovariectomized (OVX) rats and trabecular bone in osteoporotic postmenopausal humans. The ideal PTH fragment for treating osteoporosis would be the smallest and functionally simplest fragment that activates only one signal mechanism and still strongly stimulates trabecular bone growth. A new PTH fragment, hPTH-(1-31)NH2, which only stimulates adenylyl cyclase instead of stimulating both adenylyl cyclase and phospholipase-C as do hPTH-(1-84) and hPTH-(1-34), is this minimum, high-potency anabolic fragment. hPTH-(1-31)NH2 (which we have named Ostabolin) can greatly thicken trabeculae and increase the dry weight and calcium content of trabecular bone in the distal femurs of osteopenic, young, sexually mature OVX Sprague-Dawley rats when injected subcutaneously each day for 6 weeks at doses between 0.4 and 1.6 nmole/100 g of body weight.

Low concentrations of the C-terminal parathyroid hormone-related protein (PTHrP) fragments, PTHrP-(107-111) and PTHrP-(107-139), stimulated membrane-associated protein kinase Cs (PKCs), but not adenylyl cyclase or an internal Ca2+ surge, in early passage human skin keratinocytes and BALB/MK-2 murine skin keratinocytes. The fragment maximally stimulated membrane-associated PKCs in BALB/MK-2 cells at 5 x 10(-9) to 10(-8) M. The maximally PKC-stimulating concentrations of PTHrP-(107-111) also stopped or stimulated BALB/MK-2 keratinocyte proliferation depending on whether the cells were, respectively, cycling or quiescent at the time of exposure. Thus, just one brief (30-minute) pulse of 10(-8) M PTHrP-(107-111) stopped the proliferation of BALB/MK-2 keratinocytes for at least 5 days. On the other hand, daily 30-minute pulses of 10(-8) M PTHrP-(107-111) started and then maintained the proliferation of initially quiescent BALB/MK-2 cells. Similarly PTHrP-(107-111) inhibited DNA synthesis by cycling primary adult human keratinocytes, but it stimulated DNA synthesis by quiescent human keratinocytes.

The brittle, fracture-prone bones of an osteoporotic postmenopausal woman are the products of an excessive uncompensated resorption of trabecular bone by osteoclasts. Osteoporosis is currently treated with the osteoclast suppressors calcitonin, bisphosphonates, or oestrogen, which stop further bone resorption without stimulating new bone growth. Here, James Whitfield and Paul Morley review the growing evidence that small adenylate cyclase-stimulating fragments of the parathyroid hormone are promising therapeutic agents for osteoporosis that potently stimulate osteoblasts to make mechanically strong or supranormally strong bone.

The neuroprotective action of (S)-alpha-phenyl-2-pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 microM) or glutamate (50 microM) for 15 min resulted in the death of 85-95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 microM) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 microM) prevented the loss of membrane-associated protein kinase C activity that developed by 4 h after transient exposure to 50 microM NMDA or 50 microM glutamate. FPL 15896AR also reduced by approximately 35% the magnitude of NMDA-triggered increases in intracellular free Ca2+ concentration in the cortical cultures. These data indicate that NMDA-mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.

Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium. Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J. Biol. Chem. 270, 1362-1368), we proposed that Ca2+ x calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ x calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs from phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ x calmodulin can profoundly affect PKCs' signaling at the substrate level.

The effects of extracellular divalent cations on the intracellular Ca2+ concentration ([Ca2+]i) in neonatal rat islet cells were investigated to determine whether these cells, like several others, have signal-generating surface cation sensors. Raising the external Ca2+ concentration by 1 mM increments triggered either sustained increases in [Ca2+]i or large sharp [Ca2+]i spikes followed by return to a suprabasal level. The external Ca(2+)-triggered [Ca2+]i responses were abolished by treating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), but not by another phospholipase C inhibitor, U-73,122 (2.5 microM), or the voltage-sensitive Ca2+ channel blockers nifedipine (20 microM) and methoxyverapamil (D600; 50 microM). [Ca2+]i responses were also triggered by barium (Ba2+; 1 mM) and cobalt (Co2+; 1 mM). The Ba2+ responses were also inhibited by neomycin and unaffected by nifedipine or D600 and the Co2+ response required external Ca2+. Therefore, neonatal rat pancreatic islet cells may display divalent cation receptors/sensors on their surfaces. Activation of these putative receptors, which are coupled to neomycin-sensitive, voltage-independent, dihydropyridine-insensitive channels, by Ca2+, Ba2+ or Co2+ would trigger [Ca2+]i responses by opening these channels to admit external Ca2+ into the cell. The physiological function(s) of such cell-surface divalent cation receptors/sensors and the [Ca2+]i surges they generate in pancreatic islet cells is not known.

Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca(2+)-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca2+]i). Glutamate (1 mM) induced either sustained or transient increases in [Ca2+]i. The sustained glutamate-induced increase in [Ca2+]i was mimicked by NMDA (40 microM). The NMDA-triggered [Ca2+]i response was abolished by incubating the cells in Ca(2+)-free medium or by pretreating them with Mg2+ (2 mM) or MK-801 (0.1 microM). These responses were unaffected by the non-NMDA antagonist CNQX (10 microM), but they required glycine (3-30 microM). Kainate (40 microM) and AMPA (40 microM) did not affect [Ca2+]i. Without external Ca2+, glutamate triggered transient, sometimes oscillating, increases in [Ca2+]i. These responses were mimicked by the metabotropic agonist trans-(1S, 3R)-1-amino-1,3-cyclopentanedicarboxylic acid (300 microM). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca(2+)-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship of human (h) PTH in the regulation of the vitamin D receptor (VDR) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M caused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2.8 cells with 0.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] led to up-regulation of VDR content by 340-370% of the control value. hPTH-(1-34) decreased the VDR up-regulatory effect of 1,25-(OH)2D3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA by reverse transcription-polymerase chain reaction showed that PTH-(1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)

Analogues of human parathyroid hormone (hPTH) truncated at the C-terminal end have been studied for adenylyl cyclase (AC) activity and for solution conformation by circular dichroism (CD) spectroscopy. Analogues of hPTH-(1-34)-NH2, containing the first 28-31 residues, had only a slightly diminished ability to stimulate AC in rat osteosarcoma (ROS) cells as compared to that of the parent analogue. CD data on hPTH-(16-34)-NH2 and C-terminal deletion mutants of hPTH-(1-34)-NH2 supported the presence of a partially stable alpha-helix over residues 17-28. A carboxyl-terminal mutant, hPTH-(1-30)-OH, showed both reduced helix and greatly reduced AC-stimulating activity as compared to the corresponding amide analogue. In contrast, both of these analogues, in the presence of palmitoyloleoylphosphatidylserine (POPS) vesicles, showed an equal stabilization of alpha-helix. All other analogues showed at least some enhancement of alpha-helix in the presence of POPS. However, both in neutral, aqueous buffer and in POPS, the relative amount of alpha-helix decreased greatly as the peptide was shortened below the 1-28 sequence. These data provide additional support for an amphiphilic alpha-helix over residues 21-28 being the conformation for receptor binding of hPTH for stimulation of AC activity. Modeling human parathyroid hormone-related peptide as an alpha-helix over this same region, and comparison to hPTH, suggests that both may bind via the hydrophobic face to the receptor.

A reduction of gap-junctional intercellular communication (GJIC) often accompanies neoplastic transformation. The present work demonstrates that transformation by the oncogenic human DNA virus, human papilloma virus 16(HPV16), also reduces GJIC between L6 rat myoblasts. HPVs are associated with anogenital cancers, the incidence of which is increasing in HIV positive patients of both sexes. Using videofluorescence imaging of Fura-2 loaded cells a lack of GJIC between transformed HPV16-L6 cells was first indicated by uncoordinated brief [Ca2+]i spikes in clusters of DMSO-treated HPV16-L6 cells instead of the synchronous, sustained [Ca2+]i surges in clusters of DMSO-treated L6 cells. Reduced GJIC between HPV16-L6 cells was demonstrated directly by a much reduced transfer of lucifer yellow dye from HPV16-L6 cells, which had been loaded with the dye through electroporation with an EPIZAP II in situ electroporator, to neighbouring nonelectroporated HPV16-L6 cells. One reason for this reduced GJIC between HPV16-L6 cells could have been their dramatically enhanced activity of membrane-associated PKC which is known to phosphorylate connexins and down-regulate gap junctions. However, the main reason was the viral-induced inhibition of the expression of a major gap junction component, Cx43 (Connexin 43), in the transformed myoblasts.

It is commonly believed that the parathyroid hormone's (PTH's) main function in bone is to stimulate osteoclastic resorption. However, intermittent injections of small doses of PTH holoprotein, but more often its bioactive hPTH-(1-34) fragment, have been shown to stimulate bone growth in animals and humans through their ability to stimulate adenylyl cyclase and not their ability to independently activate a protein kinases-C stimulating mechanism. This anabolic action suggests that PTH might be an effective therapeutic for osteoporosis. If so, the hormone must be able to restore severely depleted trabecular bone, and the goal of this study was to find out if it can. To do this, we started a multiweek program of daily subcutaneous injections of 0.8 nmoles of hPTH-(1-34)/100 g body weight into rats at 4, 8, or 16 weeks after ovariectomy (OVX) and the increasingly severe selective loss of trabecular bone. These injections strongly stimulated femoral trabecular bone to grow and mineralize at the same rate regardless of how much of it had been lost before the injections were started. Thus, the progressively depleting trabecular bone in the femurs of OVX rats does not lose its anabolic responsiveness to PTH. This finding is another indication of the likelihood of small, adenylyl cyclase-stimulating PTH fragments being effective therapeutics for osteoporosis.

Raising the external Ca2+ concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca(2+)- or TPA-stimulated keratinocytes. Raising the external Ca2+ concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca2+ concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. Lopez-Barahona, M., and Aaronson, S. A. (1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca2+ did not. Furthermore, Ca(2+)-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca2+.CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca2+.Ca2+.calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca2+ to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca2+.calmodulin complexes, formed by the internal Ca2+ surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.

Ca2+ and Ca(2+)-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis). However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signals from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis ("diffpoptosis") when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis ("diffpoptosis") mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa. Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding of how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their responsiveness to Ca2+ are needed to assess the drawbacks and advantages of using Ca2+ as a chemopreventor.

The activation of the multifunctional cell signalling enzymes, the protein kinase Cs (PKCs), is generally thought to result from the translocation of inactive cytosolic enzymes to activation sites in cell membranes. However, recent studies suggest that PKCs may also be stimulated in cells by processes independent of translocation. One possible mechanism is the modulation of the activity of PKCs already resident in membranes. A PKC assay that measures enzyme activity directly in isolated native membranes has revealed the presence of an activatable pool of PKCs resident in native membranes of various cells and tissues. In 3T3-L1 cells, some or all of this pool of membrane PKCs was stimulated within 10 min of exposing the cells to 10 ng/ml epidermal growth factor or 100 ng/ml fibroblast growth factor. Similar increases in PKC activity were observed in native membranes isolated from CTLL-2, WEHI-231 and S49 lymphoma cells that had been exposed to interleukin-2. These growth factors all stimulated membrane PKC activity without detectably translocating cytosolic enzymes to the membranes. In intact WEHI cells, low concentrations (5-10 microM) of a diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), or low concentrations (2-10 nM) of phorbol 12-myristate 13-acetate sufficed to activate PKCs already resident in membranes, but much higher concentrations (50-100 microM and 50-100 nM respectively) were needed to detectably stimulate the translocation of cytosolic PKCs. A phosphatidylcholine-specific phospholipase C also selectively stimulated membrane PKCs in WEHI cells at concentrations that were much less than those needed to induce the translocation of cytosolic enzymes. Furthermore, interleukin-2 and low concentrations of OAG both stimulated the phosphorylation of the 85 kDa PKC-selective substrate protein in intact WEHI cells in which translocation of PKCs was not evident. These results suggest that the membranes of some cells maintain a pool of activatable PKCs that respond to lower levels of extracellular stimuli than cytosolic PKCs, and that can be stimulated by signals which produce diacylglycerols through the hydrolysis of phospholipids other than polyphosphoinositides.

Glucose-induced insulin release, glucose-induced rises in intracellular free Ca2+ concentration ([Ca2+]i), and voltage-dependent Ca2+ channel activity were assessed in monolayer cultures of beta-cells from 3-5-day-old rats. The glucose-stimulated insulin secretory responses and [Ca2+]i rises were like those in adult rat beta-cells rather than fetal rat beta-cells. Voltage-dependent Ca2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca2+]i rise and, like deprivation of extracellular Ca2+, prevented the glucose-induced rise in [Ca2+]i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca2+ channels was demonstrated directly by measuring Ca2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca2+]i after membrane depolarization by 45 mM K+ or 200 microM tolbutamide. Thus, in cultured beta-cells of 3-5-day-old rats the coupling of glucose stimulation to Ca2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.