Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation.

OBJECTIVE: Basic calcium phosphate (BCP) crystals, including octacalcium phosphate (OCP), carbonated-apatite (CA) and hydroxyapatite (HA) crystals are associated with destructive forms of osteoarthritis. Mechanisms of BCP-induced cartilage breakdown remain incompletely understood. We assessed the ability of BCP to induce changes in intracellular calcium (iCa(2+)) content and oscillations and the role of iCa(2+) in BCP-induced cartilage degradation. METHODS: Bovine articular chondrocytes (BACs) and bovine cartilage explants (BCEs) were stimulated with BCP or monosodium urate (MSU) crystals. iCa(2+) levels were determined by spectrofluorimetry and oscillations by confocal microscopy. mRNA expression of matrix metalloproteinase 3 (MMP-3), a disintegrin and metalloprotease with thrombospondin-like motifs 4 (ADAMTS-4) and ADAMTS-5 was assessed by quantitative real-time PCR. Glycosaminoglycan (GAG) release was measured in the supernatants of BCE cultures. RESULTS: All three BCP crystals significantly increased iCa(2+) content. OCP also induced iCa(2+) oscillations. Rate of BACs displaying iCa(2+) oscillations increased over time, with a peak after 20 min of stimulation. OCP-induced iCa(2+) oscillations involved both extracellular Ca(2+) (eCa(2+)) influx and iCa(2+) stores. Indeed, OCP-induced iCa(2+) oscillations decreased rapidly in Ca(2+)-free medium. Both voltage- and non-voltage-dependent Ca(2+) channels were involved in eCa(2+) influx. BCP crystal-induced variation in iCa(2+) content was associated with BCP crystal-induced cartilage matrix degradation. However, iCa²(+) was not associated with OCP crystal-induced mRNA expression of MMP-3, ADAMTS-4 or ADAMTS-5. CONCLUSION: BCP crystals can induce variation in iCa(2+) content and oscillations in articular chondrocytes. Furthermore, BCP crystal-induced changes in iCa(2+) content play a pivotal role in BCP catabolic effects on articular cartilage.

DT56a stimulates gender-specific human cultured bone cells in vitro.

DT56a found to have SERM-like properties is used for the treatment of menopausal symptoms and osteoporosis. In vivo experiments demonstrated that DT56a displayed selective estrogenic activity; it stimulated creatine kinase (CK) specific activity in the skeletal tissues but not on the uterus of ovariectomized rats. DT56a, when applied together with estradiol-17beta (E(2)), completely inhibited the E(2)-stimulated CK, as demonstrated by other SERMs. DT56a stimulated bone formation in a rat model as measured by histological and histomorphometrical parameters. In a clinical study, administration of DT56a (Femarelle) resulted in a considerable elevation of bone mineral density and relief of menopausal symptoms. The aim of the present study was to analyze the effects of DT56a in vitro on human-derived bone cultured osteoblasts (Ob), by measuring its effects, at different concentrations, on DNA synthesis, CK and alkaline phosphatase (ALP) specific activities as well as changes in intracellular [Ca(2+)](i) concentrations. DT56a stimulated CK and DNA synthesis in both pre- and post-menopausal female Ob with maximal effect at 100 ng/ml for both age groups. In addition, DT56a stimulated ALP in Ob from both pre- and post-menopausal women with maximal effect at lower dose of 50 ng/ml, with higher response of pre-menopausal cells. Raloxifene (Ral) inhibited all DT56a-stimulated changes in Ob from both age groups. DT56a, when given together with E(2), completely antagonized E(2)-stimulated effects demonstrating its nature as a phyto-SERM. DT56a also, dose dependency, stimulated the intracellular levels of [Ca(2+)](i) with maximal effect at 10 ng/ml. Male-derived Ob did not respond to DT56a in any parameter. In summary, DT56a stimulated sex-specifically female-derived Ob, indicating its unique nature compared to the compounds currently used for postmenopausal osteoporosis by being bone-forming and not only an anti-resorptive agent.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A.

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

Immunolocalization of S100A2 calcium-binding protein in cartilage and bone cells.

S100A2 protein, a Ca2+ binding protein, was investigated by immunocytochemistry in the epiphyseal cartilage and bone cells of growing rats, and in primary cultures of osteoblasts. S100A2 was detected in the chondrocytes and in the extracellular cartilage matrix. In the later however, its presence only in the calcifying areas of the epiphyseal cartilage suggests that it could be involved in the process of calcification of cartilage.

Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate.

It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0.5 to 1.7 mM increased DNA synthesis (P < 0.005) and the number of Ki 67-positive cells (P < 0.005). However, NPS R-467 (0.01-1 microM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0.5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10-10-10-7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3.5 mM) in the medium led to a significant (P < 0.05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression.

Age and gender specific stimulation of creatine kinase specific activity by gonadal steroids in human bone-derived cells in culture.

We previously reported a non-enzymatic method for isolation of human bone cells in culture that display osteoblastic features and respond to 1,25 dihydroxy vitamin D (1,25) and to parathyroid hormone (PTH). The present study was undertaken to analyze the response of cultured human bone cells to 17beta-estradiol (E2) and to dihydrotestosterone (DHT) as a function of gender and age. Cultured human bone cells, obtained from biopsies during orthopedic surgery, were divided into four groups defined by gender and age: pre- and post-menopausal healthy non-osteoporotic women that were not under hormone replacement therapy (HRT) and mature (<55-year-old) and older (>60-year-old) men. We found gender specific responses to gonadal steroids using the specific activity of the brain type (BB) isozyme of creatine kinase (CK) as a response marker. Constitutive levels of CK activity did not change with age or gender and the enzyme extracted from cells from the different sexes and ages did not respond to either progesterone (P) or to 1,25. CK from the different cells responded to gonadal steroids in a gender specific manner, i.e. CK from female derived cells responded to E2 only and the enzyme from male derived cells responded to DHT only. In female derived cells the response to E2 declined significantly with age, while the response to DHT in CK from male derived cells did not vary with age. This may be due to either decreased proportion of mature osteoblasts and/or their differentiation state and/or changes in the levels of estrogen receptor(s), coactivators or corepressors in these cells. These results extend our knowledge of human osteoblast biology (beyond murine cells) and are therefore more relevant for developing models for treatment of human metabolic bone diseases such as post-menopausal osteoporosis.

Estradiol signaling via sequestrable surface receptors.

Estradiol (E(2))-signaling is widely considered to be exclusively mediated through the transcription-regulating intracellular estrogen receptor (ER) alpha and ERbeta. The aim of this study was to investigate transcription-independent E(2)-signaling in mouse IC-21 macrophages. E(2) and E(2)-BSA induce a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) of Fura-2 loaded IC-21 cells as examined by spectrofluorometry. These changes in [Ca(2+)](i) can be inhibited by pertussis toxin, but not by the ER-blockers tamoxifen and raloxifene. The E(2)-signaling initiated at the plasma membrane is mediated through neither ERalpha nor ERbeta, but rather through a novel G protein-coupled membrane E(2)-receptor as revealed by RT-PCR, flow cytometry, and confocal laser scanning microscopy. A special feature of this E(2)-receptor is its sequestration upon agonist stimulation. Sequestration depends on energy and temperature, and it proceeds through a clathrin- and caveolin-independent pathway.

Parathyroid hormone-related peptide stimulates proliferation of highly tumorigenic human SV40-immortalized breast epithelial cells.

Parathyroid hormone-related protein (PTHrP) is the main mediator of humoral hypercalcemia of malignancy (HHM) and it is produced by many tumors, including breast cancers. Breast epithelial cells as well as breast cancer tumors and cell lines have been reported as expressing PTHrP and the PTH/PTHrP receptor, suggesting that PTHrP may act as an autocrine factor influencing proliferation or differentiation of these cell types. We investigated PTHrP gene expression, PTH/PTHrP receptor signaling, and PTHrP-induced mitogenesis in three immortalized human mammary epithelial cell lines that exhibit differential tumorigenicity. The most tumorigenic cells expressed the highest levels of PTHrP messenger RNA (mRNA) and protein. We used reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting to detect the PTH/PTHrP receptor transcripts and proteins in all of the three cell lines. Treatment with human PTHrP(1-34) [hPTHrP(1-34)] and hPTH(1-34) increased intracellular cyclic adenosine monophosphate (cAMP) but not free Ca2+ in the nontumorigenic line. These agonists increased both cAMP and free Ca2+ levels in the moderately tumorigenic line, but only increased free Ca2+ in the highly tumorigenic line. Application of the PTH/PTHrP receptor antagonist [Asn10,Leu11,D Trp12]PTHrP(7-34) or PTHrP antibodies reduced [3H]thymidine incorporation in a dose-dependent fashion in the highly tumorigenic cell line but did not affect the other lines. Thus, treatment with a PTH/PTHrP receptor antagonist reduced cell proliferation, suggesting that PTHrP signaling mediated by the phospholipase C (PLC) pathway stimulates proliferation of a highly tumorigenic immortalized breast epithelial cell line.

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3.

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.

Membrane signalling and progesterone in female and male osteoblasts. I. Involvement Of intracellular Ca(2+), inositol trisphosphate, and diacylglycerol, but not cAMP.

Bone is a target tissue of progestins, but the mechanisms by which they act are still unclear. We examined the early (5-60 s) effects of progesterone and progesterone covalently bound to BSA (P-CMO BSA), which does not enter the cell, on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the formation of inositol 1,4,5 trisphosphate (InsP3) and diacylglycerol (DAG) in confluent female and male rat osteoblasts. P-CMO BSA like free progesterone increased [Ca(2+)]i via Ca(2+) influx through L-type Ca(2+) channels and Ca(2+) mobilization from the endoplasmic reticulum. Both progestins increased InsP(3) and DAG formation within 10 s, and the increase was blocked by phospholipase C inhibitors (neomycin and U-73122). Progesterone and P-CMO BSA mobilized calcium from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis toxin-insensitive G-protein in both osteoblast types, and this process was controlled by protein kinase C. Neither progestin had any effect on cAMP formation in male and female osteoblasts. The membrane effects were not blocked by a progesterone nuclear antagonist. They were independent of the concentration of nuclear receptors and not linked to gender. Thus, progesterone appears to act in female and male rat osteoblasts via unconventional cell-surface receptors which belong to the class of membrane receptors coupled to phospholipase C via a pertussis toxin-insensitive G-protein. The bifurcating pathways leading to the formation of InsP(3) and DAG may provide a certain flexibility in controlling cell responses, both by their nature and by their rates of formation and degradation.

Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: gender and age differences.

We present a model for isolating human cell culture derived from biopsies obtained during orthopedic surgery. Four donor groups were defined by gender and age: pre- and postmenopausal women (<50 and >55 years, respectively), and younger (30-55 years) and older (>60 years) men. Bone-derived cells were identified as osteoblasts by major osteoblastic characteristics; that is, high alkaline phosphatase (ALP) activity, dose-dependent increase of ALP by 1,25(OH)2D3, high levels of parathyroid hormone (PTH)-induced cyclic AMP, and 1,25-(OH)2D3-induced osteocalcin. In all cells, levels of osteocalcin were significantly elevated (p < 0.05 and 0.01). In cells derived from men, no significant age differences were found in ALP and osteocalcin values of basal activity and in fold stimulation 1,25(OH)2D3. Cells from postmenopausal women showed a nonsignificant lower basal ALP activity than premenopausal cells. In postmenopausal cells, ALP responded less to 1,25(OH)2D3 (33% increase, p < 0.05) than the premenopausal cells (100% increase, p < 0.05). In cells from either age group, ALP did not respond to the gonadal steroids 17beta-estradiol (E2) and dihydrotestosterone (DHT) or progesterone. Basal levels of osteocalcin were higher in cells of premenopausal origin as compared with postmenopausal cells (p = 0.05), but response to 1,25(OH)2D3 was the same. PTH significantly stimulated cAMP (p = 0.001) in all age and gender groups analyzed. In all groups, no differences were found in either basal activity or in PTH response. Unlike men, cells derived from the bone of women were more susceptible to age changes. We postulate that the postmenopausal cell population had a decreased number of osteoblasts, or cells in a lower differentiation stage. These results extend our knowledge of bone biology found in animal models and reveal that human osteoblasts from men do not show the same age-dependent differences observed in women.

Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages.

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca(2+)], which is due to release of Ca(2+) from intracellular Ca(2+) stores. This Ca(2+) mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Galpha(q/11) and gbetagamma proteins and membrane signaling of calcitriol and estradiol.

17beta-estradiol and 1,25-dihydroxyvitamin D(3)()(calcitriol) rapidly increase (< 5 sec) the concentration of intracellular calcium by mobilizing Ca(2+) from the endoplasmic reticulum and forming inositol 1,4,5-trisphosphate (InsP(3)) and diacylglycerol. Calcitriol increases InsP(3) formation via activation of phospholipase C (PLC)-beta1 linked to a pertussis toxin (PTX)-insensitive G-protein, and estradiol via activation of PLC-beta2 linked to a PTX-sensitive G-protein. Since PLC are effectors of different subunits of various G-proteins, we looked for and identified several G-subunits (Galpha(q/11), Galphas, Galphai, Gbeta and Ggamma) in female rat osteoblasts using Western immunoblotting. The action of calcitriol on InsP(3) formation and Ca(2+) mobilization in Fura-2-loaded confluent osteoblasts involved Galpha(q/11). The membrane effects of estradiol involved Gbetagamma; subunits, and principally Gbeta subunits, but not alpha-subunits. These results may provide additional evidence for membrane receptors of steroid hormones. Since PLC-beta1 is the target effector of Galpha(q/11), whereas PLC-beta2 is only activated by betagamma subunits, this specificity may help to generate membrane receptor-specific responses in vivo.

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.

Phospholipase C-beta and ovarian sex steroids in pig granulosa cells.

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharmacological tools and polyclonal phospholipase C-beta (PLC-beta) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U-73122. Ca2+ mobilization involved PLC-beta1 for progesterone, PLC-beta2 for estradiol and PLC-beta4 for androstenedione. A pertussis toxin-insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L-type Ca2+ channels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC-beta, G-proteins, and calcium channels, and these mechanisms were hormone-specific.

Calcitonin gene-related peptide (CGRP) increases intracellular free Ca2+ concentrations but not cyclic AMP formation in CGRP receptor-positive osteosarcoma cells (OHS-4).

Although calcitonin gene-related peptide (CGRP) may act as a local factor in bone, its mechanisms of action on osteoblasts are not well understood. We previously showed the presence of CGRP transcripts and peptide in human OHS-4 osteoblastic cells. The authors investigated the expression of CGRP receptor (CGRP-R) and its intracellular signalling properties in OHS-4 cells. Semi-quantitative RT-PCR analysis showed that OHS-4 cells express much more CGRP-R than calcitonin (CT)-R transcripts. After amplification of CGRP-R by RT-PCR and cloning of amplified fragments, the predicted CGRP-R sequence in OHS-4 cells was found to share 100% identity with human lung CGRP-R. Biochemical analysis showed that hCGRP did not increase intracellular cAMP levels in synchronized OHS-4 cells whatever was the cell cycle position. However, adenylate cyclase activity was functional, as human parathyroid hormone increased cAMP levels. In contrast, hCGRP induced a rapid, transient and dose-dependent increase in free cytosolic calcium levels. The data show that CGRP increases intracellular free Ca2+concentration but is not coupled to adenylate cyclase in CGRP receptor-positive OHS-4 osteosarcoma cells, suggesting that CGRP induces downstream events driven by phospholipase C in these cells.

Functional testosterone receptors in plasma membranes of T cells.

T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4(+) and CD8(+) subsets of T cells are directly revealed with the impeded ligand testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone-BSA conjugate induces a rapid rise (<5 s) in [Ca2+]i of Fura-2-loaded T cells. This rise reflects influx of extracellular Ca2+ through non-voltage-gated and Ni2+-blockable Ca2+ channels of the plasma membrane. The testosterone-BSA-induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti-AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription-polymerase chain reactions and Western blotting. AR can be visualized with the anti-AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H-R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.

Activation of phospholipase C-beta1 via Galphaq/11 during calcium mobilization by calcitonin gene-related peptide.

Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether beta gamma subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+]i) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-beta isoforms (beta1, beta3, beta4, but not beta2) and G-proteins (Galphaq/11 and Galphas). Only phospholipase C-beta1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-gamma have no effect. Activation of PLC-beta1 by CGRP involves the Galphaq/11 subunit, which is insensitive to pertussis toxin, but not Gbeta gamma subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.

Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro.

We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0. 5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.

Estradiol binding to cell surface raises cytosolic free calcium in T cells.

The Fura-2 method is used to examine a possible action of 17beta-estradiol (E2) on [Ca2+]i of splenic T cells isolated from female C57BL/10 mice. E2 concentrations between 10 fM and 10 nM induce a rapid and dose-dependent increase in [Ca2+]i due to Ca2+ influx and release of Ca2+ from intracellular stores. Ca2+ influx is mediated by Ca2+ channels which are completely blockable by Ni2+ and partly by nifedipine. The antiestrogen tamoxifen does not inhibit the E2-induced rise in [Ca2+]i. Ca2+ influx and Ca2+ release from intracellular stores is also inducible by plasma membrane impermeable E2 conjugated to BSA. E2-BSA-FITC binds to the surface of T cells of both the CD4+ and CD8+ subset. Our data suggest a novel E2-signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E2.