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Biomedical subjects

G Zaman

Publications and source records attributed to G Zaman.

18 recordsLinked to original sources
PubMed2004

The adaptive response of bone to mechanical loading in female transgenic mice is deficient in the absence of oestrogen receptor-alpha and -beta.

Postmenopausal osteoporosis represents a failure of the response by which bone cells adapt bone mass and architecture to be sufficiently strong to withstand loading without fracture. To address why this failure should be associated with oestrogen withdrawal, we investigated the ulna's adaptive response to mechanical loading in adult female mice lacking oestrogen receptor-alpha (ERalpha(-/-)), those lacking oestrogen receptor-beta (ERbeta(-/-)) and their wild-type littermates. In wild-type mice, short periods of physiologic cyclic compressive loading of the ulna in vivo over a 2-week period stimulates new bone formation. In ERalpha(-/-) and ERbeta(-/-) mice this osteogenic response was respectively threefold and twofold less (P<0.05). In vitro, primary cultures of osteoblast-like cells derived from these mice were subjected to a single short period of mechanical strain. Twenty-four hours after strain the number of wild-type cells was 61+/-25% higher than in unstrained controls (P<0.05), whereas in ERalpha(-/-) cells there was no strain-related increase in cell number. However, the strain-related response of ERalpha(-/-) cells could be partially rescued by transfection with functional human ERalpha (P<0.05). ERbeta(-/-) cells showed a 125+/-40% increase in cell number following strain. This was significantly greater than in wild types (P<0.05).These data support previous findings that functional ERalpha is required for the full osteogenic response to mechanical loading and particularly the stage of this response, which involves an increase in osteoblast number. ERbeta appears to depress the ERalpha-mediated strain-related increase in osteoblast number in vitro, but in female transgenic mice in vivo the constitutive absence of either ERalpha or ERbeta appears to diminish the osteogenic response to loading.

Adaptation, Physiological↗
PubMed2001

Mechanical strain and estrogen activate estrogen receptor alpha in bone cells.

Bone cells' early responses to estrogen and mechanical strain were investigated in the ROS 17/2.8 cell line. Immunoblotting with antiphosphorylated estrogen receptor a (ER-alpha) antibody showed that when these cells were exposed for 10 minutes to estrogen (10(-8) M) or a single period of cyclic dynamic strain (peak 3400 microepsilon, 1 Hz, 600 cycles), there was an increase in the intensity of a 66-kDa band, indicating phosphorylation of ser122 in the amino terminus of ER-alpha. Increased phosphorylation was detected within 5 minutes of exposure to estrogen and 5 minutes after the end of the period of strain. Estrogen and strain also activated the mitogen-activated protein kinase (MAPK) family member extracellular regulated kinase-1 (ERK-1). Increases in ERK activation coincided with increased ER-alpha phosphorylation. Activation of ERK-1 and the phosphorylation of ER-alpha, by both estrogen and strain, were prevented by the MAP kinase kinase (MEK) inhibitor U0126 and the protein kinase A (PKA) inhibitor (PKI). These data support previous suggestions that resident bone cells' early responses to strain and estrogen share a common pathway, which involves ER-alpha. This pathway also appears to involve PKA and ERK-mediated phosphorylation of ser122 within the amino terminus of ER-alpha. Reduced availability of this pathway when estrogen levels are reduced could explain diminished effectiveness of mechanically related control of bone architecture after the menopause.

Bone and Bones↗
PubMed2000

Mechanical strain activates estrogen response elements in bone cells.

The involvement of the estrogen receptor in the early responses of bone cells to mechanical strain was investigated by subjecting subconfluent monolayer cultures of ROS.SMER #14 cells (ROS 17/2.8 cells stably transfected with additional ER alpha) to 17 beta-estradiol or a single short period of dynamic mechanical strain (600 cycles, 1 Hz). The basal proliferation rate of ROS.SMER #14 cells was similar to ROS 17/2.8 cells, whose proliferative responsiveness to strain and estrogen is similar to that of primary cultures of rat long bone-derived osteoblasts. At peak strains of 3400 mu epsilon, strain-related proliferation in ROS.SMER #14 cells was 1.4 times that of ROS 17/2.8 cells. At 10(-8) mol/L, 17 beta-estradiol-related proliferation was nearly twice greater. The ROS.SMER #14 cells were transiently transfected with an estrogen-responsive reporter, 2ERE-pS2-CAT, containing two consensus estrogen response elements (ERE) linked to a chloroamphenicol acetyl transferase gene. Strain increased normalized ERE-CAT activity threefold and estradiol (10(-8) mol/L) sixfold. Both strain-related and estradiol-related increases in proliferation and ERE-CAT activity were blocked by the estrogen antagonist ICI 182,780 (10(-6) mol/L). These data show that strain as well as estrogen stimulates increased proliferation in ROS 17/2.8 cells and increased ER alpha-related ERE activity in ROS cells transfected with ER alpha. Proliferation is greater in the cells with more estrogen receptors. Both strain- and estrogen-related proliferation and ERE activity are blocked by the estrogen antagonist ICI 182,780. This indicates that ROS cells' early responses to mechanical strain involve ER alpha and estrogen-responsive genes.

Animals↗
PubMed1999

Mechanical strain stimulates ROS cell proliferation through IGF-II and estrogen through IGF-I.

The mechanism by which mechanical strain stimulates bone cell proliferation was investigated and compared with that of estrogen in ROS 17/2.8 cells. Similarity of strain-related responses between ROS cells and osteoblasts was established by demonstrating that ROS cells respond to a short single period of strain in their substrate (1000-3500 microepsilon, 600 cycles, 1 Hz) by a similar strain magnitude-related increase in glucose 6-phosphate dehydrogenase activity as rat osteoblasts and osteocytes in explants in situ. ROS17/2.8 cells also showed similar proliferative responses to strain and 17beta-estradiol, as assessed by [3H]thymidine incorporation and cell counting, as primary cultures of long bone-derived osteoblast-like cells. Strain-related increase in proliferation in ROS cells was accompanied by a 4-fold increase in levels of insulin-like growth factor-II (IGF-II) in conditioned medium. Neither strain nor estrogen had an effect on the conditioned medium levels of IGF-I. Exogenous truncated IGFs tIGF-I and tIGF-II both increased proliferation in a dose-dependent manner. The neutralizing monoclonal antibody (nMAb) to IGF-I blocked proliferation stimulated by tIGF-I but not that due to tIGF-II and vice versa. IGF-I receptor blocking antibody (IGF-IRBAb) blocked the proliferative effect of tIGF-I but not that to tIGF-II. The proliferative effect of estrogen was abolished by IGF-I nMAb and IGF-IRBAb, but these antibodies had no effect on the proliferative response to strain. In contrast IGF-II nMAb abolished the proliferative effect of strain but had no effect on that of estrogen. These data show that ROS17/2.8 cells have similar responses to strain and estrogen qualitatively and quantitatively as rat osteoblasts in situ and rat long bone-derived osteoblast-like cells in primary culture. Estrogen-related proliferation in ROS17/2.8 cells appears to be mediated by IGF-I acting through the IGF-I receptor and does not involve IGF-II. In contrast, strain-related proliferation appears to be mediated by IGF-II and does not involve either IGF-I or the IGF-I receptor.

Animals↗
PubMed1999

Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes.

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non-load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.

Animals↗
PubMed1998

RoBo-1, a novel member of the urokinase plasminogen activator receptor/CD59/Ly-6/snake toxin family selectively expressed in rat bone and growth plate cartilage.

Using differential display polymerase chain reaction, we cloned a novel cDNA named RoBo-1 from rat tibia. RoBo-1 is abundantly expressed in bone, including the hypertrophic chondrocytes of the growth plate where cartilage is remodeled into bone. RoBo-1 mRNA expression increased in response to two modulators of bone metabolism, estradiol and intermittent mechanical loading, suggesting a role in bone homeostasis. The 1.6-kilobase cDNA encodes a 240-amino acid protein with a cysteine spacing pattern, suggesting that RoBo-1 is a novel member of the urokinase plasminogen activator receptor/CD59/Ly-6/snake toxin family. Furthermore, the C-terminal contains a glycosyl-phosphatidylinositol attachment site, suggesting that it is a cell surface protein similar to other mammalian members of this family. The strongest homology of RoBo-1 is to the snake serum-derived phospholipase A2 inhibitors, which uniquely contain two of the cysteine domains but are secreted proteins. Interestingly, RoBo-1 is likely the first membrane-anchored member of this family containing two cysteine domains. Thus, the tissue specificity, responsiveness to bone protective mediators, along with its relationship to the multifunctional urokinase plasminogen activator receptor/CD59/Ly-6/snake toxin family suggests that RoBo-1 may play a novel role in the growth or remodeling of bone.

Amino Acid Sequence↗
PubMed1998

Heme oxygenase isozymes in bone: induction of HO-1 mRNA following physiological levels of mechanical loading in vivo.

Heme oxygenases (HO) are responsible for the production of carbon monoxide, which has been suggested to act similarly to nitric oxide as a signaling molecule. Inducible HO-1 and constitutive HO-2 were located in sections of weight-bearing ulnae of the rat by immunocytochemistry. Intense HO-1 localization was restricted to peri- and endosteal sites, whereas HO-2 staining occurred in osteoblasts and osteocytes throughout the cortex. Northern blot hybridization of mRNA levels for HO-1 and HO-2 extracted from bones was also performed. Six hours after a single 10 min period of noninvasive mechanical loading of the ulna in vivo, generating physiological levels of strain sufficient to initiate an osteogenic response, the level of mRNA for the inducible HO-1 isoform was increased, but that of HO-2 was unchanged. The presence of a constitutive and strain-related upregulation of an inducible enzyme capable of producing carbon monoxide suggests that carbon monoxide may participate not only in bone cells' basal metabolism but also in their adaptive response to mechanical load.

Animals↗
PubMed1997

Expression of tenascin-C in bones responding to mechanical load.

A number of early biochemical responses of bone cells to mechanical loading have been identified, but the full sequence of events from the sensing of strain to the formation of new bone is poorly characterized. Extracellular matrix proteins can modulate cell behavior and would be ideal molecules to amplify the early response to loading. The extracellular matrix protein, tenascin-C, supports differentiation of cultured osteoblast-like cells. The current study was carried out to investigate whether expression patterns of tenascin-C in loaded bones support a role for this protein as a mediator of the osteoregulatory response to loading. Tenascin-C expression was investigated by Northern blot analysis in rat ulnae subjected to an established noninvasive loading regimen engendering physiological strain levels. RNA extracted from loaded compared with contralateral control bones 6 h after loading showed a significant increase in tenascin-C transcript expression. The presence of tenascin-C was investigated by immunohistochemistry in bones of animals killed 3, 5, or 15 days after the initiation of daily loading. In animals killed at 3 or 5 days, periosteal surfaces undergoing load-induced reversal from resorption to formation showed enhanced tenascin-C staining. In animals killed at 15 days, the bone formed in response to loading was clearly demarcated from old bone by strong tenascin-C staining of reversal lines. Within this new bone, tenascin-C staining was seen in the lacunae of older but not more recently embedded osteocytes. The results presented here indicate that tenascin-C expression by bone cells is enhanced in the early osteogenic response to loading. This may indicate that tenascin-C acts as a mediator of the mechanically adaptive response.

Animals↗
PubMed1997

Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture.

Mechanical loading of bone explants stimulates prostaglandin E2 (PGE2) and prostacyclin (PGI2) release and increases glucose 6-phosphate dehydrogenase (G6PD) activity. This response is blocked by indomethacin and imitated by exogenous PGs. In the experiments reported here, primary cultures of rat long bone-derived osteoblast-like cells were exposed to a dynamic strain and exogenous PGs in the culture dish. Strain (3400 mu epsilon, 600 cycles, 1 Hz) caused an immediate release of PGI2 into the culture medium but had no effect on PGE2. Strain also caused an increase in G6PD activity per cell and an increase in the smallest transcript of insulin-like growth factor II (IGF-II) (IGF-II T3) but had no effect on the expression of transforming growth factor-beta1 (TGF-beta1). Indomethacin inhibited strain-induced release of PGI2 and suppressed strain-induced stimulation of IGF-II T3 transcript. PGI2 (1 microM) increased G6PD activity and mRNA levels of all three transcripts of IGF-II but had no effect on the mRNA levels of IGF-I or TGF-beta1. PGE2 (1 microM) stimulated G6PD activity and caused a marked increase in IGF-I and the largest transcript of IGF-II (IGF-II T1) but had no effect on the IGF-II transcripts T2 and T3 or on TGF-beta1 mRNA levels. These findings show similarities in response between osteoblast-like cells strained in monolayer culture and bone cells in loaded bone explants in situ. They provide support for a role for IGF-II and PGI2 in the early strain-related response of osteoblasts in loading-related bone modeling/remodeling.

Animals↗
PubMed1997

Enhancement by sex hormones of the osteoregulatory effects of mechanical loading and prostaglandins in explants of rat ulnae.

Explants of ulnae from 5-week-old male and female rats were cleaned of marrow and soft tissue and, in the presence and absence of 10(-8) M 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT), mechanically loaded or treated with exogenous prostanoids previously shown to be produced during loading. Over an 18-h period, mechanical loading (peak strain 1300 mu epsilon, 1 Hz, 8 minutes, maximum strain rate 25,000 mu epsilon/s), prostaglandin E2 (PGE2) and prostacyclin (PGI2) (10(-6) M), each separately produced quantitatively similar increases in cell proliferation and matrix production in bones from males and females, as indicated by incorporation of [3H]thymidine into DNA and [3H]proline into collagen. E2 and DHT both increased [3H]thymidine and [3H]proline incorporations, E2 producing greater increases in females than in males. Indomethacin abrogated the effects of loading, but had no effects on those of sex hormones. Loading, or prostanoids, together with sex hormones, produced responses generally equal to or greater than the addition of the individual influences acting independently. In females there was a synergistic response in [3H]thymidine incorporation between loading and E2, which was quantitatively similar to the interaction between E2 and PGE2 or PGI2. The interaction between loading and E2 for [3H]proline incorporation was not mimicked by these prostanoids. In males the synergism in [3H]proline incorporation seen between loading and DHT was mimicked by that between PGI2 and DHT. We conclude that loading stimulates increased bone cell proliferation and matrix production in situ through a prostanoid-dependent mechanism. This response is equal in size in males and females. Estrogen and testosterone increase proliferation and matrix production through a mechanism independent of prostanoid production. The interactions between loading and hormones are reproduced in some but not all cases by E2 and prostaglandins. E2 with loading and prostaglandins has greater effects in female bones, while DHT with loading and prostaglandins has greater effects in males.

Animals↗
PubMed1996

Mechanical loading and sex hormone interactions in organ cultures of rat ulna.

The separate and combined effects of loading and 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) on [3H]thymidine and [3H]proline incorporation were investigated in cultured ulna shafts from male and female rats. Ulnae were cultured and loaded to produce physiological strains in the presence or absence of 10(-8) M E2 or DHT. Loading engendered similar increases in incorporation of [3H]thymidine and [3H]proline in male and female bones. E2 engendered greater increases in incorporation in females than in males, and DHT greater increases in males than in females. In males E2 with loading produced increases in both [3H]thymidine and [3H]proline incorporation, which approximated to the arithmetic addition of the increases due to E2 and loading separately. In females E2 with loading produced increases greater than those in males, and substantially greater than the addition of the effects of E2 and loading separately. Loading with DHT in males also showed additional [3H]thymidine and [3H]proline incorporation. In females there was additional incorporation of [3H]proline, but not [3H]thymidine. The location of incorporation of [3H]thymidine and [3H] proline was consistent with their level of incorporation reflecting periosteal osteogenesis, in which case the early osteogenic effects of sex hormones are gender-specific when acting alone and in combination with loading. In males the effects of estrogen and testosterone add to, but do not enhance, the osteogenic responses to loading. In females testosterone with loading produces an additional effect on [3H]proline incorporation but no greater effect than loading alone on that of [3H]thymidine. In contrast, estrogen and loading together produce a greater effect than the sum of the two influences separately. Because premenopausal bone mass will have been achieved under the influence of loading and estrogen acting together, these findings suggest that the bone loss which follows estrogen withdrawal may result, at least in part, from reduction in the effectiveness of the loading-related stimulus on bone cell activity. This stimulus is normally responsible for maintaining bone mass and architecture.

Animals↗
PubMed1995

Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?

The structural competence of the skeleton is maintained by an adaptive mechanism in which resident bone cells respond to load-induced strains. To investigate the possible role of the messenger molecule nitric oxide (NO) in this response, we studied NO production in well-characterized organ culture systems, rat long bone-derived osteoblast-like (LOBs) cells, and embryonic chick osteocytes (LOCYs) in monolayer culture. In superfused cancellous bone cores, loading (for 15 min) produces increases in NO2- (stable NO metabolite) release during the loading period, which paralleled those in PGI2 and PGE2. Loading of rat vertebrae and ulnae produces increases in NO2- release, and in ulnae NO synthase inhibitors diminish these responses. Transient rapid increases in NO release are stimulated by strain in both LOBs and LOCYs. Polymerase chain reaction amplification of extracted mRNA shows that rat ulnae, LOBs, and LOCYs express both the inducible and neuronal (constitutive) isoforms of NO synthase. Adaptability to mechanical strain relies on assessment of the strain environment followed by modification of bone architecture. Immediate increases in NO production induced by loading suggest the involvement of NO in strain measurement and cellular communication to establish strain distribution, as well as potentially in adaptive changes in bone cell behavior.

Adaptation, Physiological↗
PubMed1994

Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae.

The shafts of ulnae from 110 g male rats were cultured, and after a period of 5 h preincubation one of each pair of bones was either loaded cyclically (500 g, 1 Hz, 8 minutes) to produce physiologic strains (-1300 mu epsilon) or treated with exogenous prostacyclin (PGI2) or prostaglandin E2 (10(-6) M, 8 minutes) in the presence or absence of 17 beta-estradiol (10(-8) M). PGI2, PGE2, and loading stimulated almost immediate increases in glucose 6-phosphate dehydrogenase (G6PD) activity in osteocytes and osteoblasts. This increase was uniform throughout the section with exogenous PGs in the medium but was related to local strain magnitude in loading. Elevated G6PD levels in response to loading and PGI2 persisted for 18 h, by which time, ALP activity in surface osteoblasts was elevated and [3H]proline incorporation into collagen increased. PGE2 produced similar immediate and sustained increases in G6PD activity and [3H]proline incorporation after 18 h but no change in ALP activity. Bones cultured for 18 h with 17 beta-estradiol increased their [3H]proline incorporation, as did those loaded, and treated with PGI2 and PGE2. Loading and PGI2 but not PGE2 produced similar proportional increases in [3H]proline incorporation above the increased baseline of estradiol alone. These results suggest that estrogen and loading together produce a greater osteogenic response than either separately. If so, estrogen withdrawal would result in a rapid fall in bone mass to establish a new equilibrium appropriate to the reduced effectiveness of the loading-related stimulus. Such a fall in bone mass is a characteristic feature of estrogen withdrawal at the menopause.

Alkaline Phosphatase↗
PubMed1993

Early strain-related changes in cultured embryonic chick tibiotarsi parallel those associated with adaptive modeling in vivo.

A model was developed for the application of cyclic mechanical loads to 17 day embryonic chick tibiotarsi in culture. A single 20 minute period of intermittent loading at 0.4 Hz, producing physiologic peak strains and strain rates, resulted in two peak strain magnitude-related responses that were previously reported in vivo: (1) a rapid increase in glucose 6-phosphate dehydrogenase activity in osteoblasts and osteocytes and (2) increased RNA synthesis, as shown by increased incorporation of [3H]uridine into extracted RNA. The RNA response was detectable 8 h following loading but was more pronounced by 24 h. Both responses were blocked by indomethacin (10(-6) M). These results demonstrate that embryonic chick bones in organ culture exhibit cellular responses to loading similar to those previously identified in adult canine cancellous bone cultures in vitro and adult avian cortical bone in vivo. These findings are consistent with a sequence of events between loading and new bone formation that includes an immediate strain magnitude-related, prostanoid-dependent increase in activity of the pentose monophosphate shunt in osteoblasts and osteocytes, followed by a similarly strain magnitude-related increase in RNA synthesis over the subsequent 24 h.

Adaptation, Physiological↗
PubMed1992

Cultured embryonic bone shafts show osteogenic responses to mechanical loading.

Pairs of 17-day embryonic chick tibiotarsi were removed and maintained in organ culture. One of each pair was subjected to a single 20-minute period of intermittent loading at 0.4 Hz, producing peak longitudinal compressive strains of 650 microstrain (mu epsilon). In the 18-hour culture period following loading, alkaline phosphatase levels in the osteoblasts of the loaded tibiotarsi were maintained whereas in controls they declined. In situ hybridization using a collagen type I cRNA riboprobe showed a substantial increase in expression of mRNA for collagen type I in the periosteal tissue of bones that were cultured for 18 hours after loading compared with that in similarly cultured controls and bones cultured for 4 hours. These results demonstrate that appropriate loading of embryonic chick bones in organ culture elicits adaptive regulation of matrix synthesis as evidenced by increased expression of the gene for type I collagen and alkaline phosphatase activity. This model may be useful as it must contain all the obligatory steps between strain change in the matrix and modified osteogenic activity.

Adaptation, Physiological↗
PubMed1991

Regulation of expression of the genome of bacteriophage M13. Gene V protein regulated translation of the mRNAs encoded by genes I, III, V and X.

With the aid of a binary plasmid in vivo testsystem it was demonstrated that the single-stranded DNA binding protein encoded by gene V of bacteriophage M13 not only regulates the synthesis of its cognate DNA replication proteins at the level of translation, but also of the assembly proteins and the coat proteins encoded by genes I and II, respectively. Furthermore, gene V protein functions as a translational autoregulator of its own synthesis. Comparison of the mRNA levels of genes I and X in the presence and absence of wild-type gene V protein indicated that gene V protein augments the physical stability of these mRNAs. The expression of the Escherichia coli beta-galactosidase gene and of a gene X mutant containing a deletion in the nontranslated mRNA leader sequence was not influenced by gene V protein, lending support to the conclusion that gene V protein exerts its regulatory effect via a specific nucleotide sequence in the leader sequences of the respective M13 mRNAs. We conclude that gene V protein functions as a master regulatory protein of the expression and replication of the M13 genome.

Autoradiography↗
PubMed1991

Biological properties of synthetic human parathyroid hormone: effect of deamidation at position 76 on agonist and antagonist activity.

Recent studies have suggested a role for the carboxyl-terminus of PTH in the binding of the molecule to renal and skeletal receptors, but the functional significance of this binding remains uncertain. We have investigated the possible role of this region by examining the effect of substituting the asparagine residue at position 76 of the native human molecule [Asn76]hPTH-(1-84) with an aspartate residue, [Asp76] hPTH-(1-84) on activity in both renal and skeletal cytochemical (CBA) and adenylate cyclase (AC) bioassays. In the renal CBA, [Asp76]hPTH-(1-84) was considerably less potent than [Asn76]hPTH-(1-84) and produced dose-dependent inhibition of the bioactivity of intact bovine (b) PTH-(1-84), bPTH-(1-34), and [Asn76]hPTH-(1-84). [Asp76]hPTH-(39-84) inhibited the response to intact PTH to a lesser extent, whereas [Asp76]hPTH-(53-84) had no antagonistic activity. In the metatarsal CBA, [Asp76]hPTH-(1-84) inhibited the response to intact PTH, but was less potent than in the renal CBA. In both renal (OK) and skeletal (UMR) cell AC assays [Asp76]hPTH-(1-84) and [Asn76]hPTH-(1-84) were equipotent agonists. Therefore, the CBAs are much more sensitive to modification of the carboxyl end of the molecule than AC assays. The antagonist properties of [Asp76]hPTH-(1-84) appeared to be mediated by phosphodiesterase activation as theophylline abolished the antagonism of this analog. These studies indicate that generation of PTH analogs, modified at the carboxyl-terminal region as well as at the amino-terminus, may be useful for developing potent PTH antagonists.

Adenylyl Cyclases↗