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At least 1,711 records · Page 95Linked to original sources
PubMed1994

Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells.

Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Genistein sensitivity of calcium transport pathways in serotonin-activated vascular smooth muscle cells.

Recent studies showed that serotonin-activated increases in intracellular Ca2+ in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhibition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+ and tyrosine phosphorylation are functionally coupled. Therefore, we sought to characterize genistein-sensitive Ca2+ transport pathways in rat aortic A10 cells loaded with fura-2. In the presence of extracellular Ca2+, serotonin evoked a transient increase in [Ca2+]i that was followed by a smaller sustained increase. The transient was inhibited 25-40% by L-type Ca2+ channel antagonists and inhibited 90-95% by genistein. The sustained response was unaffected by L-channel antagonists and only slightly inhibited by genistein. In the absence of extracellular Ca2+, the transient was reduced by 50%, while the sustained component was virtually abolished. These results suggest that influx and release pathways are major contributors to the transient component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably involves an L-type Ca2+ channel that is regulated by tyrosine kinase activity. The pathways that participate in the sustained response are different because they are insensitive to l-channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free media was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+ was unaffected by genistein, reduced by caffeine, and essentially eliminated by thapsigargin. Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+ from the sarcoplasmic reticulum, whereas genistein-mediated suppression probably reflects inhibition of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+ and interference with release. Each A10 cell expressed at least two different ryanodine receptors and two different receptors for inositol 1,4,5-trisphosphate.

Animals↗
PubMed1993

Enhancement of perforin by retinoic acid is mediated by protein kinase C.

We previously demonstrated that interleukin-2 induced murine lymphokine-activated killer cell activity is augmented by retinoic acid. The enhanced cytotoxicity is significantly correlated with the increase in PKC. In the present study, we have shown that retinoic acid increases the expression of perforin, a potent cytolytic mediator, at both protein and mRNA levels. This enhancement can be induced by a direct stimulation of PKC signaling pathway, as manipulated by a short term incubation of lymphokine-activated killer cells with 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin, and suppressed by PKC inhibitors. These results suggest that PKC plays a regulatory role in the enhancement of the expression of cytolytic mediators in retinoic acid-augmented lymphokine-activated killer cells.

Animals↗
PubMed1993

Involvement of dual signal transduction systems in the stimulation of osteoclast-like cell formation by parathyroid hormone and parathyroid hormone-related peptide.

The present study was performed to examine whether parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) would stimulate osteoclast-like cell formation via soluble factor(s) released from osteoblasts and, if so, to characterize the involvement of PTH/PTHrP-responsive dual signal transduction systems [cAMP-dependent protein kinase (PKA) and calcium/protein kinase C(PKC)]. Osteoblasts-conditioned medium (CM) was obtained from rat osteoblastic osteosarcoma cells (UMR-106 cells), which had been cultured in serum free medium for 24 hrs after treatment with various kinds of reagents. The CM of osteoblasts treated with either 10(-7) M human(h) PTH-(1-34) or 10(-7)M hPTHrP-(1-34) equally stimulated osteoclast-like cell formation from hemopoietic blast cells derived from mouse spleen cells, although the CM treated with 10(-8) M 1,25dihydroxyvitamin D3 failed to affect it. The CM treated with both 10(-4) M dibutyryl-cAMP and a direct PKA activator, 10(-4)M Sp-cAMPS significantly increased osteoclast-like cell formation. The CM treated with a PKC activator, 10(-7)M phorbol 12-myristate 13-acetate (PMA) and calcium ionophores (10(-7)M A23187 and 10(-7)M ionomycin) also significantly enhanced osteoclast-like cell formation. The present study first indicated that osteoblast-mediated stimulation of osteoclast-like cell formation by PTH and PTHrP, and the participation of PTH/PTHrP-responsive dual signal transduction systems of osteoblasts in the stimulation of osteoclast-like cell formation by PTH and PTHrP.

Animals↗
PubMed1994

Conversion to Ca(2+)-independent form of Ca2+/calmodulin protein kinase II in rat pancreatic acini.

CCK rapidly converted Ca2+/calmodulin kinase II (CaMK II) to a Ca(2+)-independent form with peak action at 30 sec followed by decline to the basal level at 10 min. The threshold concentration of CCK for this action was 30 pM and maximum effect occurred at 1 nM, which induced a 6-10-fold increase. Bombesin and carbachol similarly induced CaMK II autonomous activity, whereas secretin and JMV 180 did not. Ionomycin induced a more stable elevation of CaMK II autonomous activity and the intracellular Ca2+ chelator, BAPTA/AM, blocked the effect of CCK. In conclusion, pancreatic CaMK II is rapidly activated by a large increase in [Ca2+]i generated by either stimulation of phosphatidylinositol pathway or by an influx of extracellular Ca2+.

Amino Acid Sequence↗
PubMed1994

Distinct inhibition by non-peptide and peptide arginine vasopressin antagonists of vasopressin-induced activation of mitogen-activated protein kinase in cultured rat vascular smooth muscle cells.

The preincubation of vascular smooth muscle cells (VSMC) with a non-peptide arginine vasopressin (AVP) antagonist (OPC-21268) (0.5 mM) for 10 min completely inhibited the 1 microM AVP-induced activation of mitogen-activated protein (MAP) kinase but did not affect the angiotensin II-, protein kinase C activator- or ionomycin-stimulated MAP kinase. Similar results were obtained with the simultaneous administration of AVP and OPC-21268. This inhibitory effect of OPC-21268 completely disappeared after washing the cells pretreated with OPC-21268. In contrast, the preincubation of VSMC with a peptide AVP antagonist, d(CH2)5Tyr(Me)AVP (1 microM), for 10 min completely blocked the 1 microM AVP-activated MAP kinase but the simultaneous administration of d(CH2)5Tyr(Me)AVP with AVP could inhibit that only by 40%. However, the washing procedure did not affect the inhibitory effect of d(CH2)5Tyr(Me)AVP. These results indicate that OPC-21268 is a specific AVP antagonist to inhibit the AVP-induced activation of MAP kinase. The antagonistic effect of OPC-21268 is also suggested to be relatively weak but prompt as compared to that of a peptide AVP antagonist d(CH2)5Tyr(Me)AVP.

Angiotensin II↗
PubMed1994

RU486 and calcium fluxes in human spermatozoa.

RU486 decreases the intracellular free calcium concentration in human spermatozoa incubated in capacitation medium. We investigated the calcium fluxes that this progesterone antagonist acts on. We found that 10(-5) M RU486 slowed down the basal calcium influx while progesterone accelerated it. Moreover, in the same way as RU486 inhibits the calcium increase promoted by progesterone, it inhibited the calcium influx stimulated by 10(-6) M thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor. So we speculated that RU486 does not necessarily compete for the same binding sites as progesterone to exert its inhibitory action. In the absence of extracellular calcium, pretreatment by 10(-5) M RU486 decreased the peak of [Ca2+]i released by 5 microM ionomycin. This indicates that RU486 could also act at the membrane level of some intracellular organelles of calcium storage.

Binding, Competitive↗
PubMed1995

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on agonist-stimulated phasic myometrial contractions.

Phasic myometrial contractions utilize mechanisms involving the cycling of calcium into and out of intracellular calcium stores. These studies were performed to determine the effects of 2,5-di(tert-butyl)-1,4-hydroquinone (tBHQ), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on in vitro isometric myometrial contractions. These studies demonstrated that low concentrations of tBHQ (eg. 10 microM) appear to inhibit intracellular calcium cycling, whereas higher concentrations also inhibit extracellular calcium influx. These combined tBHQ effects markedly suppressed myometrial contractions stimulated in response to various agonists including oxytocin, PGF2 alpha, KCl, ionomycin, and Bay K 8644.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

The effects of thimerosal, a sulfhydryl reagent, on phasic myometrial contractions.

Thimerosal inhibits calcium uptake and IP3-induced calcium release from IP3-sensitive endoplasmic reticulum; this study sought to evaluate the effects of thimerosal on agonist-stimulated phasic myometrial contractions. Thimerosal was found to significantly inhibit phasic contractions stimulated by oxytocin, aluminum fluoride, potassium chloride, ionomycin, and Bay K 8644. These observations provide support for the hypothesis that calcium uptake and IP3-induced calcium release are important events during agonist-stimulated phasic myometrial contractions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Reduction of dopamine beta-hydroxylase gene expression by calcium in bovine chromaffin cells.

We determined the effect of Ca++ influx on dopamine beta-hydroxylase (DBH) gene expression in primary cultured bovine chromaffin cells. The Ca++ ionophore A23187 (100nM) specifically reduced DBH mRNA to 15 +/- 10% of the untreated control while tyrosine hydroxylase was induced (142 +/- 15%) and phenylethanolamine N-methyltransferase was unchanged (107 +/- 11%). This effect on DBH was also observed with ionomycin, reversed by EGTA and unaffected by cycloheximide. Depolarization by potassium also resulted in DBH reduction which was reversed by the voltage dependent Ca++ channel blockers nifedipine and verapamil. DBH mRNA level reduced by A23187 could be re-induced by forskolin and DBH induced by forskolin could be reduced by the ionophore, suggesting that the cAMP and Ca++ pathways might act independently in regulating DBH gene expression.

Animals↗
PubMed1995

Selection and partial characterization of calcium ionophore (A23187) resistant cells.

We have selected calcium ionophore (A23187)-resistant cells (AR-7) in a human promyelocytic leukemia cell line, HL-60. AR-7 showed approximately 8.5-fold resistance to A23187 compared with the wild type cells after continuous exposure for 3 days. AR-7 had cross resistance to ionomycin (4.6-fold) and thapsigargin (340-fold) which can also increase intracellular Ca++. Similar magnitude of resistance to apoptosis, as defined by characteristic morphology and internucleosomal DNA fragmentation, induced by these agents was observed after 4 hr of incubation. However, both the elevation of intracellular Ca++ following stimulation with various concentrations of A23187 and the sensitivity to anti-cancer agents including etoposide, 1-beta-D arabinofuranosylcytosine, and hydroxyurea were comparable between the two cell types. This cell line is considered to be useful for exploring the mechanism(s) of cell death, especially apoptosis, induced by calcium ionophore.

Antineoplastic Agents↗
PubMed1995

Requirement of p56lck in T-cell receptor/CD3-mediated apoptosis and Fas-ligand induction in Jurkat cells.

Interactions of Fas (CD95) and its ligand (Fas-L) has been shown to play a pivotal role in T cells receptor (TCR)/CD3 activation-induced cell death via apoptosis. Although several lines of evidence suggest involvement of protein tyrosine kinase (PTK) activity in this process, the role of src family PTK p561ck (lck) is not known. We report here that, contrary to wild type Jurkat, the lck-deficient mutant JCaM is resistant to anti-CD3-induced apoptosis and fails to express Fas-L mRNA upon anti-CD3 treatment. However, both Jurkat and JCaM were found to constitutively express Fas and were equally sensitive to anti-Fas-mediated apoptosis. If stimulated with PMA plus ionomycin, JCaM expressed Fas-L mRNA and underwent apoptosis. These findings indicate that p56lck is required for TCR/CD3-mediated Fas-L induction but not for the transduction of Fas receptor-mediated death signal.

Antigens, Surface↗
PubMed1996

Adult alveolar type II cells lack cAMP and Ca(2+)-activated Cl-channels.

We investigated the presence of Ca(2+)-activated Cl-channels in adult rat alveolar type II (ATII) using patch-clamp techniques. Only one active channel each, with a single channel conductance of 50 pS and an opening probability (Po) of 0.76 was found among 130 successful cell-attached and 5 inside-out patches. Addition of CPT-cAMP into the bath (500 microM) induced one active patch from 33 silent cell-attached patches. Incubation of 9 ATII cells, with ionomycin (1 microM), failed to elicit chloride single currents in 9 cell-attached patches. Cl- currents were also absent from 35 whole cell patches, even after the addition of 10 microM terbutaline in the bath or 1 mM ATP and 5 mM MgCl2 in the pipette. These results indicate that only a very small fraction of adult rat ATII cells express CFTR and suggest that Cl- ions are passively transported across the cell junctions.

Adenosine Triphosphate↗
PubMed1997

Ca2+-ATPase inhibitors and Ca2+-ionophore induce osteoclast-like cell formation in the cocultures of mouse bone marrow cells and calvarial cells.

Osteoclasts which derive from hemopoietic cells are multinucleated cells responsible for bone resorption. We found that cyclopiazonic acid (CPA), thapsigargin (TG), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) induced osteoclast-like cell (OCL) formation in cocultures of mouse calvaria-derived stromal cells and hemopoietic cells such as bone marrow cells and spleen cells. OCLs induced by these compounds showed typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase activity and pit forming activity. These compounds are known as endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitors that increase intracellular Ca2+ levels by inhibiting Ca2+-ATPase activity located in the membrane of ER/SR. Ca2+-ionophores such as ionomycin which increase intracellular Ca2+ levels also stimulated OCL formation in the cocultures. Differentiation of hemopoietic cells into OCLs induced by these compounds required the presence of calvarial cells. These results indicate that an increase of intracellular Ca2+ levels may be a part of signaling pathways to induce osteoclast differentiation in the presence of calvarial cells.

Animals↗
PubMed1997

Electrophysiological responses to oxytocin and ATP in monolayers of a human sweat gland cell line.

It was shown that oxytocin (OT) elicits electrophysiological responses in cultured monolayers of NCL-SG3, a human immortalized sweat gland cell line. The response to OT was greater for basal applications. It was also found that monolayers respond to ATP with a transient transepithelial-potential change, with a more pronounced response to apical than to basal applications. The IC50 for the response to OT was 180 nM at room temperature. The response to OT was not due to effects of OT on vasopressin (AVP) receptors as evidenced by three tests: (a) The response was completely blocked by the selective OT-receptor antagonist [Mpa1,D-Tyr(Et)2,Thr4,Orn8]-OT (CAP) applied at equal concentrations (100-1000 nM) to that of OT. (b) The response to OT was similar to that of ionomycin (2 microM) or ATP (150 microM). In contrast, the response to AVP (500 nM) or cAMP (2 mM) were smaller and of a different time course. (c) OT increased but AVP had no effect on the intracellular free calcium. It is suggested that OT may have a role in the regulation of salt balance in sweating.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages.

We found citrulline-containing proteins in mouse peritoneal macrophages undergoing calcium ionophore-induced apoptosis. Such proteins were products of deimination of arginine residues catalyzed by endogenous peptidylarginine deiminase (EC 3.5.3.15) activated by calcium influx. Western blotting analyses of the extract from macrophages incubated with 1 microM ionomycin showed selective deimination of vimentin without detectable degradation. Double immunofluorescence staining of deiminated proteins and vimentin suggested localization of deiminated vimentin around the periphery of round-shaped nucleus, which was thought to be an early morphological sign of apoptosis. The biological implication of vimentin deimination in macrophage apoptosis is discussed.

Animals↗
PubMed1998

A role of calcineurin in coreceptor regulation during differentiation of Cd4+Cd8+ T cells.

The immunosuppressant FK506 inhibits thymocyte positive selection. Calcineurin, a FK506-sensitive Ca2+/calmodulin-dependent protein phosphatase, is presumed to be involved in this event without direct evidence. We have previously shown that moderate stimulation of CD4(+)CD8(+) thymocytes with a combination of the calcium ionophore ionomycin and phorbol myristate acetate mimics positive selection events including downregulation of CD8 expression. Moderate stimulation of a CD4(+)CD8(+) T cell line with the same combination of drugs also induced specific downregulation of CD8 expression. FK506 inhibited the CD8 downregulation in both cell types. The T cell line was transfected with an expression vector encoding an active form of calcineurin. The transfectans remained CD4(+)CD8(+), but became CD4(+)CD8(-) or CD4(+)CD8(low) upon stimulation with phorbol myristate acetate alone. The extent of the CD8 downregulation was correlated with the expression level of the mutant calcineurin. These results suggest that the calcium signal for the CD8 downregulation is mainly delivered through calcineurin activation.

Animals↗
PubMed2000

Modulation of the Ca(2+)-activated Cl(-) channel by 14-3-3epsilon.

We have previously reported an association of 14-3-3epsilon isoform with calmodulin. Using the voltage-clamp technique, the present study investigated the potential role of 14-3-3 in modulating the Ca(2+)-activated Cl(-) channel (CaCC) endogenously expressed in Xenopus oocytes. Injection of 14-3-3epsilon antisense oligodeoxynucleotides resulted in potentiation of the ionomycin-induced Cl(-) current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppressed the antisense-potentiated current. The data suggest that 14-3-3epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin-dependent pathway. The potential role of 14-3-3epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed.

14-3-3 Proteins↗