Involvement Of Calmodulin Research Articles

Involvement of calmodulin in neuronal cell death

A large body of evidence indicates that disturbances of Ca 2+ homeostasis may be a causative factor in the neurotoxicity following cerebral ischemia. However, the mechanisms by which Ca 2+ overload leads to neuronal cell death have not been fully elucidated. Calmodulin, a major intracellular Ca 2+-binding protein found mainly in the central nervous system, mediates many physiological functions in response to changes in the intracellular Ca 2+ concentration, whereas Ca 2+ overload in neurons after excitotoxic insult may induce excessive activation of calmodulin signaling pathways, leading to neuronal cell death. To determine the role of calmodulin in the induction of neuronal cell death, we generated primary rat cortical neurons that express a mutant calmodulin with a defect in Ca 2+-binding affinity. Neurons expressing the mutant had low responses of calmodulin-dependent signaling to membrane depolarization by high KCl and became resistant to glutamate-triggered excitotoxic neuronal cell death compared with the vector or wild-type calmodulin-transfected cells, indicating that blocking calmodulin function is protective against excitotoxic insult. These results suggest that calmodulin plays a crucial role in the processes of Ca 2+-induced neuronal cell death and the possibility that the blockage of calmodulin attenuates brain injury after cerebral ischemia.

Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 microM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 microg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPgammaS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPgammaS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.

Fas Binding to Calmodulin Regulates Apoptosis in Osteoclasts

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.

Translocation of ErbB receptors into the nucleus

The ErbB receptors are tyrosine kinases that bind a wide variety of ligands. They are implicated in cell proliferation, generation of anti-apoptotic signals, differentiation, and cell migration. The over-expression of different Erb B receptors and/or the expression of aberrant mutant forms are responsible for the development of many human cancers. A simplified view of the mechanism of action of these receptors is that they are triggered when located at the cell surface and/or when present in endosomes. In this review we will discuss a newly emergent, and more complex, picture in which ErbB receptors and their ligands translocate to the cell nucleus where they assume additional functions. The possible involvement of calmodulin in the translocation process will be discussed, as well. The development of new therapeutic strategies targeting the nuclear translocation system and/or the nuclear functions of ErbB receptors could help to control the rapid growth of certain types of tumor cells.

Influence of W-7, a calmodulin antagonist on phospholipid biosynthesis in Candida albicans.

This study was undertaken to investigate the role of calmodulin in phospholipid biosynthesis in Candida albicans using W-7, a calmodulin antagonist. Cells were grown as shake cultures in the absence and presence of W-7 at different concentrations. Changes in cell mass, phospholipid content and incorporation of labelled precursor into phospholipid and activities of respective enzymes have been studied. Decreased incorporation of labelled acetate into total lipids and phospholipids was observed in the presence of 40 microm of W-7 which was not as a consequence of altered growth of Candida in the presence of calmodulin antagonist. Further, a significant decrease in the levels of calmodulin and CaM dependent protein kinase activity was observed in cells grown with different concentrations of W-7. This was accompanied by decreased/increased activity of phosphatidic acid phosphatase and phospholipase A, respectively in W-7 grown cells as compared to controls. These findings suggest definite involvement of calmodulin in the regulation of phospholipid metabolism in Candida albicans.

Calmodulin antagonists suppress gap junction coupling in isolated Hensen cells of the guinea pig cochlea.

The effect of calmodulin (CaM) antagonists W7, trifluoperazine (TFP) and a calmodulin inhibitory peptide on gap junction coupling in isolated Hensen cells of the organ of Corti was analysed by the double whole-cell patch-clamp technique. Addition of the conventional antagonists W7 and TFP in the micromolar range caused a rapid decrease of gap junction conductance after a delay of a few minutes in a dose-dependent manner. Fluorescence spectroscopy of cytoplasmic free calcium concentration ([Ca(2+)](i)) by Fura-2 showed no significant change of [Ca(2+)](i) by W7. Chelation of [Ca(2+)](i) by 10 mM BAPTA or use of nominally Ca(2+)-free external bath did not suppress the W7-induced gap junction uncoupling. The results suggest that W7 and TFP induce gap junction uncoupling at unchanged global [Ca(2+)](i) in Hensen cells. To obtain additional evidence for an involvement of CaM in regulating gap junction conductance a calmodulin inhibitory peptide, the MLCK peptide (250 nM), was added to the standard pipette solution. Again gap junction uncoupling was observed, but on a significantly slower time scale. This is the first study of an effect of calmodulin antagonists on gap junction coupling in isolated Hensen cells. The question whether the effect of calmodulin inhibitors is specific and involves CaM-dependent gating of gap junction coupling in Hensen cells is discussed.

3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel calmodulin antagonist, reduces brain edema through the inhibition of enhanced blood-brain barrier permeability after transient focal ischemia.

An alteration of the blood-brain barrier (BBB) permeability contributes to the development of brain edema after stroke. In this study, we evaluated the effects of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel calmodulin antagonist, on brain edema formation and BBB integrity in rats subjected to transient focal ischemia. DY-9760e (1 mg/kg/h) was intravenously infused for 6 h, starting immediately after reperfusion of a 1-h middle cerebral artery occlusion. Treatment with DY-9760e significantly suppressed the increase in water content and the extravasation of Evans blue dye after transient focal ischemia. Analysis of a magnetic resonance imaging method revealed that DY-9760e significantly prevented the development of brain edema in the cortical region of the ipsilateral hemisphere. Trifluoperazine, a calmodulin antagonist that is structurally different from DY-9760e, also attenuated brain edema elicited by transient focal ischemia. Furthermore, DY-9760e and trifluoperazine reduced tumor necrosis factor-alpha-induced hyperpermeability of inulin through a cultured brain microvascular endothelial cell monolayer, suggesting an involvement of calmodulin in the regulation of brain microvascular barrier function. The present results demonstrate that DY-9760e ameliorates brain edema formation and suggest that this effect may be mediated in part by the inhibition of enhanced BBB permeability after ischemic insults. Thus, DY-9760e is expected to be a therapeutic drug for treatment of acute stroke patients.

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [ 3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 μM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.

Calmodulin Activates Intersubunit Electron Transfer in the Neuronal Nitric-oxide Synthase Dimer

Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH. Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups. We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer. A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as full-length subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation. The E592A mutation prevented Arg binding to the oxygenase domain in which it was present. It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit. However, it had no effect when present in the full-length subunit (i.e. the subunit containing the reductase domain). The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin. This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS. In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.

Involvement of calmodulin in glucagon-like peptide 1(7-36) amide-induced inhibition of the ATP-sensitive K+ channel in mouse pancreatic beta-cells.

The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (K(ATP)) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic beta-cells. Membrane potential, single channel and whole-cell currents through the K(ATP) channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic beta-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the K(ATP) channels in mouse pancreatic beta-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane K(ATP) conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 microM) completely reversed the inhibitory actions of GLP-1 on the membrane K(ATP) conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the K(ATP) channel activity by 20 nM GLP-1 and its restoration by 20 microM W-7 or 10 microM calmidazolium at the single channel level. Bath application of 20 microM W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the K(ATP) channel activity accompanied by the initiation of electrical activity in mouse pancreatic beta-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system.

Multiple calmodulin genes exhibit systematically differential responses to chronic ethanol treatment and withdrawal in several regions of the rat brain

Ethanol induces profound alterations in the neuronal signaling systems, including the calcium (Ca 2+) signaling. Prolonged exposure to ethanol evokes adaptive changes in the affected systems as they strive to restore the normal neuronal function. We investigated the involvement of calmodulin (CaM) genes, coding for the major mediator protein of intracellular Ca 2+ signals, in these adaptive processes at the mRNA level. The changes induced in the regional abundances of the CaM I, II, and III mRNA classes by chronic ethanol treatment and withdrawal were examined by means of quantitative in situ hybridization, employing gene-specific [ 35S]cRNA probes on rat brain cryostat sections. Regional analysis of the resulting changes in mRNA levels highlighted brain areas that belong in neuronal systems known to be especially sensitive to the action of ethanol. The results revealed systematically differential regulation for the three mRNA classes: the CaM I and CaM III mRNA levels displayed increases, and CaM II levels decreases in the affected brain regions, in both chronic ethanol- and withdrawal-treated animals. As regards the numbers of brain regions undergoing significant alterations in mRNA content, the CaM I mRNA levels exhibited changes in most brain areas, the CaM II levels did so in a lower number of brain regions, and the CaM III levels changed in only a few brain areas. These results suggest a differential regulation for the CaM genes in the rat brain and may help towards elucidation of the functional significance of the multiple CaM genes in the mammalian genome.

Involvement of calmodulin and protein kinase C in cholecystokinin release by bombesin from STC-1 cells.

The mouse intestinal neuroendocrine tumor cell line STC-1 secretes cholecystokinin (CCK) and other hormones. We investigated the role of Ca2+, calmodulin (CaM), and protein kinase C (PKC) in the regulation of CCK release from STC-1 cells. Phorbol 12-myristate 13-acetate (TPA) significantly stimulated CCK release. Staurosporine significantly inhibited CCK release from STC-1 cells stimulated by TPA in a dose-dependent manner. The absence of extracellular calcium completely inhibited CCK release from TPA-stimulated STC-1 cells. Neurotensin did not stimulate CCK release from these cells. W-7, a CaM antagonist, reduced CCK release from STC-1 cells stimulated by bombesin in a dose-dependent manner. These findings suggest that CaM and PKC play an important role in the regulation of CCK release from STC-1 cells stimulated by bombesin.

Calcium-induced ERK activation in human T lymphocytes occurs via p56 Lck and CaM-kinase

We previously demonstrated that stimulation of human T-lymphocytes with calcium ionophores induced the phosphorylation and enzymatic activation of ERK2. We now report on the mechanism by which calcium-ionophore-induced activation of ERK1 and 2 occurs in these cells. The activation of ERK1 and 2 by increases in intracellular calcium was inhibited by calmidazolium suggesting the involvement of calmodulin in this response. To further elucidate the mechanism by which calcium-induced ERK activation occurs, we used the CaM-kinase inhibitor KN-93 and an inactive analog of KN-93 (KN-92). KN-93, but not KN-92, blocked ionomycin-induced activation of ERK1 and 2 in human T lymphocytes. We previously demonstrated that stimulation of T lymphocytes with ionomycin or A23187 resulted in a CaM-kinase-dependent shift in the mobility of p56 Lck. To determine if p56 Lck was involved in calcium-induced ERK activation, we stimulated the p56 Lck negative Jurkat cell derivatives, J.CaM1.6 and J.CaM1/Rep3, with ionomycin. In these p56 Lck negative cell lines, activation of ERK1 and 2 in response to ionomycin was only minimally detected. When J.CaM1 cells were reconstituted with p56 Lck, ionomycin induced ERK1 and 2 activation. Treatment of Jurkat cells with PP2, an inhibitor of p56 Lck, inhibited calcium-induced, but not PMA-induced, ERK1 and 2 activation. Treatment of Jurkat cells with the MEK inhibitor PD98059 blocked ionomycin-induced ERK activation, but not the shift in the mobility of p56 Lck. Our data suggests that increases in intracellular calcium induce the activation of ERK1 and 2 in human T lymphocytes via sequential activation of CaM-kinase and phosphorylation of p56 Lck.

Signal Mediators for Phytoalexin Production in Defense Response of Oats Elicited by Victorin as a Specific Elicitor

Signals mediating phytoalexin (PA) production were analyzed in primary leaves of oats cv. Iowa X469 treated with an elicitor victorin. Production of the PA avenanthramide A was inhibited by DPI, an inhibitor of NADPH oxidase/nitric oxide synthase (NOS) and the NOS inhibitors l-NMMA and 1,3-PBIT. However, catalase and superoxide dismutase (SOD) hardly suppressed it. From the data, NO functions as a major reactive oxygen species in signal transduction leading to PA production in the defense response of oats. EGTA, verapamil and ruthenium red inhibited PA production, suggesting that Ca2+ influx into the cytoplasm and intracellular Ca2+ movement are involved in the defense response. Trifluoperazine, a calmodulin function inhibitor, and K-252a, a serine/threonine kinase inhibitor, also suppressed the accumulation, whereas okadaic acid, a serine/threonine phosphatase inhibitor, did not suppress it, suggesting the involvement of calmodulin and protein kinase, but not of phosphatase in PA production.

Involvement of Calmodulin in 1α,25-Dihydroxyvitamin D3 Stimulation of Store-operated Ca2+ Influx in Skeletal Muscle Cells

The steroid hormone 1alpha,25-dihydroxyvitamin D(3) (1, 25-(OH)(2)D(3)) rapidly modulates Ca(2+) homeostasis in avian skeletal muscle cells by driving a complex signal transduction mechanism, which promotes Ca(2+) release from inner stores and cation influx from the outside through both L-type and store-operated Ca(2+) (SOC) channels. In the present work, we evaluated the involvement of calmodulin (CAM) in 1,25-(OH)(2)D(3) regulation of SOC influx in chick skeletal muscle cells. Treatment with 10(-9) m 1,25-(OH)(2)D(3) in Ca(2+)-free medium resulted in a rapid but transient Ca(2+) rise correlated with the sterol-induced inositol 1,4,5-trisphosphate (IP(3)) production. The SOC influx stimulated by the hormone was insensitive to both CAM antagonists (fluphenazine, trifluoperazine, chlorpromazine, compound 48/80) and the CAM-dependent protein kinase II (CAMKII) inhibitor KN-62 when added after the sterol-dependent Ca(2+) transient, but it was completely abolished when added prior to the IP(3)-induced mobilization of Ca(2+) from endogenous stores. Moreover, in cells microinjected with antisense oligonucleotides directed against the CAM mRNA the sterol-stimulated SOC influx was reduced up to 60% respect to uninjected cells. The present results suggest that the 1, 25-(OH)(2)D(3)-induced (IP(3)-mediated) cytosolic Ca(2+) transient is required for CAM, activation which in turn activates SOC influx in a mechanism that seems to include CAMKII.

Evidence for the involvement of calmodulin in mouse sperm capacitation.

Although Ca(2+) is of fundamental importance in mammalian sperm capacitation, its downstream targets have not been definitively demonstrated. The purpose of this study was to use the calmodulin (CaM) antagonists W7 and calmidazolium (CZ) to investigate the possible role of CaM, a Ca(2+)-specific binding protein, in capacitation. Sperm membrane changes associated with capacitation were assessed by the B pattern after chlortetracycline staining and by the ability to undergo the acrosome reaction (AR) in response to lysophosphatidylcholine (LPC). The percentage of B pattern sperm was significantly inhibited by W7 or CZ in a concentration-dependent manner. At 100 microM W7 or 10 microM CZ, these inhibitors also significantly reduced the sperm's ability to undergo the LPC-induced AR. Inhibition of the B pattern and the LPC-induced AR was overcome by exogenous cAMP analogues. Treatment of the sperm with 100 microM W7 also resulted in a significant decrease in their ability to fertilize eggs in vitro. At 100 microM, W5, a less potent dechlorinated W7 analogue, had no effect on the B pattern, LPC-induced AR, or fertilization competence. Sperm viability and protein tyrosine phosphorylation were not substantially affected by 100 microM W7 (relative to 100 microM W5) or 10 microM CZ; however, the percentages of motile and hyperactivated sperm were significantly reduced. The antagonist-inhibited sperm motility was restored by dilution in control medium, but not by cAMP analogues. These results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.

Involvement of calmodulin and protein kinase C in the regulation of K + transport by carbachol across the rat distal colon

The cholinergic agonist carbachol stimulates the apical H +–K +–ATPase and apical as well as basolateral K + channels in the rat distal colon. The effect of carbachol was tested in the presence of different inhibitors of the Ca 2+ signaling pathway in order to characterize the intracellular mechanisms involved. Both carbachol-stimulated Rb +-efflux as well as carbachol-stimulated mucosal Rb +-uptake were dependent on the presence of serosal Ca 2+. The Ca 2+–calmodulin antagonist calmidazolium (10 −7 mol l −1) inhibited the stimulation of mucosal and serosal Rb + efflux by carbachol. A similar effect had KN-62 (10 −5 mol l −1), an inhibitor of the Ca 2+–calmodulin-dependent kinase II, suggesting the regulation of basolateral and apical K + channels by this kinase. Staurosporine (10 −6 mol l −1), which potently inhibits protein kinase C, did not alter the effect of carbachol on Rb + efflux, although the stimulation of apical Rb + efflux by carbachol seemed to be less prolonged, indicating that protein kinase C is not involved in the regulation of K + permeability. In contrast, mucosal Rb + uptake, which is determined by the ouabain- and vanadate-sensitive K + transport via the apical H +–K +–ATPase, was decreased to nearly one third of control values in the presence of calmidazolium. Both calmidazolium and staurosporine, but not KN-62, prevented the stimulatory action of carbachol on the H +–K +–ATPase, suggesting a synergistic control of this ion pump by both Ca 2+–calmodulin and protein kinase C.

Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP‐independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10−8–10−5 M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10−5 M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10−5 M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period. J. Cell. Biochem. 73:36–48, 1999. © 1999 Wiley-Liss, Inc.

Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.

Possible role of calmodulin in excystation of Giardia lamblia.

The protozoan Giardia lamblia initiates infection when trophozoites emerge from a cyst in the hosts by the excystation process. Although this process is crucial to the initiation of infection by G. lamblia, little is known about its regulation. To study the possible involvement of calmodulin (CaM) in excystation we tested the effect of several CaM antagonists (TFP, W-7, and W-5) on this cellular function. Except for W-5 the rest of these compounds inhibited excystation. The protein kinase C inhibitor H-7 had no effect on excystation, suggesting that CaM antagonists acted by selectively inhibiting CaM. Furthermore, CaM was redistributed after the induction of excystation and there was an increase in its fluorescence and activity. These results suggest that a CaM-dependent process is involved in G. lamblia excystation.