PTH and phospholipase A2 in the aging process of intestinal cells

In this study we analyzed, for the first time, alterations in phospholipase A2 (PLA2) activity and response to parathyroid hormone (PTH) in rat enterocytes with aging. We found that PTH, rapidly stimulate arachidonic acid (AA) release in rat duodenal cells (+1- to 2-fold), an effect that is greatly potentiated by aging (+4-fold). We also found that hormone-induced AA release in young animals is Ca2+-dependent via cPLA2, while AA released by PTH in cells from aged rats is due to the activation of cPLA2 and the Ca2+-independent PLA2 (iPLA2). In enterocytes from 3 months old rats, PTH induced, in a time and dose-dependent fashion, the phosphorylation of cPLA2 on serine 505, with a maximum at 10 min (+7-fold). Basal levels of cPLA2 serine-phosphorylation were higher in old enterocytes, affecting the hormone response which was greatly diminished (+2-fold at 10 min). cPLA2 phosphorylation impairment in old animals was not related to changes of cPLA2 protein expression and did not explain the substantial increase on PTH-induced AA release with aging, further suggesting the involvement of a different PLA2 isoform. Intracellular Ca2+ chelation (BAPTA-AM, 5 microM) suppressed the serine phosphorylation of cPLA2 in both, young and aged rats, demonstrating that intracellular Ca2+ is required for full activation of cPLA2 in enterocytes stimulated with PTH. Hormone effect on cPLA2 was suppressed to a great extent by the MAP kinases ERK 1 and ERK2 inhibitor, PD 98059 (20 microM), the cAMP antagonist, Rp-cAMP, and the PKC inhibitor Ro31820 both, in young and aged animals. Enterocytes exposure to PTH also resulted in phospho-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase substrates reside. Hormone-induced enzyme translocation is also modified by aging where, in contrast to young animals, part of phospho-cPLA2 remained cytosolic. Collectively, these data suggest that PTH activates in duodenal cells, a Ca2+-dependent cytosolic PLA2 and attendant AA release and that this activation requires prior stimulation of intracellular ERK1/2, PKA, and PKC. cPLA2 is the major enzyme responsible for AA release in young enterocytes while cPLA2 and the Ca2+-independent iPLA2, potentiate PTH-induced AA release in aged cells. Impairment of PTH activation of PLA2 isoforms upon aging may result in abnormal hormone regulation of membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Effect of aging on the mechanisms of PTH-induced calcium influx in rat intestinal cells

We have investigated the effects of aging on parathyroid hormone (PTH) modulation of intracellular calcium homeostasis and their relationship to signal transduction pathways in isolated rat duodenal cells (enterocytes). PTH (10(-8)-10(-9) M) increased enterocyte (45)Ca(2+) influx and intracellular Ca(2+) concentration ([Ca(2+)](i)) to a greater extent (twofold and 50%, respectively) in aged (24 months) than in young (3 months) animals. The [Ca(2+)](i) response of old cells to the hormone was slower, lacking the early phase of changes in cytosolic Ca(2+). Ca(2+) influx induced by PTH was prevented by the protein kinase A antagonist Rp-cAMPS in both young and aged enterocytes, whereas neomycin and compound U73122, inhibitors of PLC-catalyzed phosphoinositide hydrolysis, abolished hormone-dependent Ca(2+) influx in young but had no effect on aged cells. Higher basal adenylyl cyclase (AC) activity and cAMP content were detected in old enterocytes. PTH increased the absolute levels of cAMP in aged cells and AC activity of microsomes isolated therefrom to a greater extent (>/= twofold) than in young enterocytes/membranes. In young cells, the hormone also induced a rapid and transient release of inositoltrisphosphate (IP(3)) and diacylglycerol (neomycin-sensitive) at 45 sec, and a delayed phase of DAG at 5 min (neomycin-insensitive). The early formation of IP(3) and DAG was blunted in aged animals. These results suggest that both the PLC and adenylyl cyclase cascades are involved in PTH stimulation of Ca(2+) influx in duodenal cells. During aging, however, only the cAMP pathway is operative, mediating a potentiation of the effects of the hormone. Additional studies are required to establish the relative role of PTH-dependent messenger systems in the regulation of intestinal calcium absorption and age-related abnormalities.     

PTH stimulates PLCbeta and PLCgamma isoenzymes in rat enterocytes: influence of ageing

We previously reported that in rat duodenal cells (enterocytes), parathyroid hormone (PTH [1-34]: PTH) stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP(3)) and diacylglycerol (DAG) and that this mechanism is severely altered in old animals. In the present study, we show that PTH [1-34]-dependent IP(3) release in young rats was blocked to a great extent by an antibody against guanine nucleotide binding protein Galphaq/11, indicating that the hormone activates a beta isoform of PLC coupled to the alpha subunit of Gq/11. In addition, PTH rapidly (within 30 s, with maximal effects at 1 min) stimulated tyrosine phosphorylation of PLCgamma in a dose-dependent fashion (10(-10)-10(-7) M). The hormone response was specific as PTH [7-34] was without effects. The tyrosine kinase inhibitors, genistein (100 microM) and herbimycin (2 microM), suppressed PTH-dependent PLCgamma tyrosine phosphorylation. Stimulation of PLCgamma tyrosine phosphorylation by PTH [1-34] greatly decreased with ageing. PP1 (10 microM), a specific inhibitor of the Src family of tyrosine kinases, completely abolished PLCgamma phosphorylation. The hormone-induced Src tyrosine dephosphorylation, a major mechanism of Src activation, an effect that was blunted in old animals. These results indicate that in rat enterocytes PTH generates IP(3) mainly through G-protein-coupled PLCbeta and stimulates PLCgamma phosphorylation via the nonreceptor tyrosine kinase Src. Impairment of PTH activation of both PLC isoforms upon ageing may result in abnormal hormone regulation of cell Ca(2+) and proliferation in the duodenum.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation and protein synthesis in human breast cancer cells

Carbachol (Cch), a muscarinic acetylcholine receptor (mAChR) agonist, increases intracellular-free Ca(2+) mobilization and induces mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in MCF-7 human breast cancer cells. Pretreatment of cells with the selective phospholipase C (PLC) inhibitor U73122, or incubation of cells in a Ca(2+)-free medium did not alter Cch-stimulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by phorbol 12-myristate acetate (PMA), an activator of protein kinase C (PKC), but Cch-evoked MAPK/ERK activation was unaffected by down-regulation of PKC or by pretreatment of cells with GF109203X, a PKC inhibitor. However, Cch-stimulated MAPK/ERK phosphorylation was completely blocked by myristoylated PKC-zeta pseudosubstrate, a specific inhibitor of PKC-zeta, and high doses of staurosporine. Pretreatment of human breast cancer cells with wortmannin or LY294002, selective inhibitors of phosphoinositide 3-kinase (PI3K), diminished Cch-mediated MAPK/ERK phosphorylation. Similar results were observed when MCF-7 cells were pretreated with genistein, a non-selective inhibitor of tyrosine kinases, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF-7 human breast cancer cells mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen activated kinase kinase. In conclusion, analyses of mAChR downstream effectors reveal that PKC-zeta, PI3K, and Src family of tyrosine kinases, but not intracellular-free Ca(2+) mobilization or conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK activation. In addition, MAPK/ERK are involved in the regulation of growth and proliferation of MCF-7 human breast cancer cells.     

1,25-dihydroxyvitamin D(3) stimulated protein kinase C phosphorylation of type VI adenylyl cyclase inhibits parathyroid hormone signal transduction in rat osteoblastic UMR 106-01 cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic cells was shown previously to attenuate Parathyroid hormone (PTH) response by inhibiting adenylyl cyclase (AC) activity. In this study, we have investigated the mechanism by which 1,25(OH)(2)D(3) inhibits AC in rat osteoblastic UMR 106-01 cells. 1,25(OH)(2)D(3) treatment inhibited both PTH and forskolin-stimulated AC activity by 25%-50% within 12 min in a concentration-dependent manner suggesting a direct inhibition of the AC enzyme. Treatment with 25(OH)D(3) had no effect on basal or stimulated AC activity. We determined the profile of AC subtypes expressed in UMR cells and found AC VI to be the dominant subtype accounting for 50% of AC mRNA. Since AC VI can be inhibited by protein kinase C (PKC) phosphorylation, we examined 1,25(OH)(2)D(3) activation of various PKC isoforms. 1,25(OH)(2)D(3) increased the membrane translocation of PKC-betaI, -delta, and -zeta with a concomitant increase in PKC activity. The translocation of PKC-betaI and -delta was blocked by the PLC inhibitor U73122 whereas that of PKC-zeta was abolished by the PI-3 kinase inhibitor wortmannin. The attenuation of cAMP production by 1,25(OH)(2)D(3) was antagonized by the PKC inhibitors Go6850, calphostin C, and wortmannin, but not by a calmodulin kinase II (CaMKII) inhibitor. Treatment with 1,25(OH)(2)D(3) for 20 min increased AC VI phosphorylation by 10.8-fold and this was blocked partially by Go6850 and partially by wortmannin but was unaffected by CaMKII inhibitor. These results demonstrate that 1,25(OH)(2)D(3) activation of PKC isoforms leads to phosphorylation of AC VI and inhibition of PTH-activation of this pathway in osteoblasts.     

MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction

H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.     

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.     

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used G?6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.     

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.     

Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE‐Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos‐2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH‐induced activation of the CRE. Activators of non‐cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE‐luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H‐89 (10 µM) almost completely blocked PTH‐induced activation of the CRE‐reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time‐dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH‐induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN‐93 had no significant effect on the response to PTH. We conclude that non‐cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH. J. Cell. Biochem. 106: 887–895, 2009. © 2009 Wiley‐Liss, Inc.     

Parathyroid hormone increases thiol proteinase activity by activation of protein kinase C in cultured kidney tubule cells (OK)

Chronic exposure (24 h) to parathyroid hormone (PTH) increases the intracellular proteolytic activity in cultured opossum kidney cells 2-fold at physiological PTH concentrations (10(-12) mol/l). This increase can be blocked by E-64, an inhibitor of thiol proteinases. The phorbol ester TPA mimicks the effect of PTH, whereas the calcium ionophore A23187 reduces the intracellular proteinase activity. Forskolin and dibutyrylic cAMP do not elevate proteinase activity. The protein kinase C inhibitor staurosporine is equally effective in blocking the TPA- and PTH-induced proteinase activity increase. These data indicate that PTH increases the intracellular thiol proteinase activity by an activation of protein kinase C and not by the cAMP dependent way.     

Cross-talk of parathyroid hormone-responsive dual signal transduction systems in osteoblastic osteosarcoma cells: Its role in PTH-induced homologous desensitization of intracellular calcium response

The present study was designed to characterize the cross-talk of parathyroid hormone (PTH)-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [PKC]) and its participation in PTH-induced homologous desensitization of intracellular calcium ([Ca²⁺]i) in osteoblastic UMR-106 cells. Although our recent study revealed that prolonged (more than 2 h) pretreatment with PKC-activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) significantly decreased the PTH-stimulated cAMP production, pretreatment with PMA (10^?7 and 10^?6 M) but not 10^?6 M 4alphaphorbol 12,13-didecanoate (PDD), incapable of activating PKC for 30 min significantly augmented 10^?7 M hPTH-(1-34)-stimulated cAMP production. H-7 (50 uM), a PKC inhibitor, significantly antagonized this PMA-induced effect. Pretreatment with 10^?6 M PMA for 30 min did not affect PTH receptor binding but significantly augmented a cAMP responsiveness to 10^?5 M forskolin and 1 ug/ml cholera toxin. Pertussis toxin (0.5 ug/ml) did not affect the PMA-induced augmentation of the PTH-stimulated cAMP production. PTH caused a complete homologous desensitization of [Ca²⁺]i response within 30 min. Pretreatment with 10^?4 M dibutyryl cAMP for 30 min and 6 h significantly reduced and completely blocked the PTH-induced increase in [Ca²⁺]i, respectively. Pretreatment with 10^?4 M Sp-cAMPS, a direct PKA activator, for 30 min completely blocked the PTH-induced increase in [Ca²⁺]i. Rp-cAMPS (10^?4 M), an antagonist of PKA, slightly but significantly antagonized the PTH-induced homologous desensitization of [Ca²⁺]i response. The present study indicates that the time of exposure to PKC activation is a critical determinant in modulating the cAMP system, while PKA activation counterregulatorily acts on the [Ca²⁺]i system, and that PKA activation is linked to the PTH-induced homologous desensitization of [Ca²⁺]i response. © 1994 Wiley-Liss, Inc.     

Dopamine stimulates 45Ca2+ uptake through cAMP, PLC/PKC, and MAPKs in renal proximal tubule cells

We have examined the effect of dopamine on Ca(2+) uptake and its related signaling pathways in primary renal proximal tubule cells (PTCs). Dopamine increased Ca(2+) uptake in a concentration (>10(-10) M) and time- (>8 h) dependent manner. Dopamine-induced increase in Ca(2+) uptake was prevented by SCH 23390 (a DA(1) antagonist) rather than spiperone (a DA(2) antagonist). SKF 38393 (a DA(1) agonist) increased Ca(2+) uptake unlike the case with quinpirole (a DA(2) agonist). Dopamine-induced increase in Ca(2+) uptake was blocked by nifedipine and methoxyverapamil (L-type Ca(2+) channel blockers). Moreover, dopamine-induced increase in Ca(2+) uptake was blocked by pertussis toxin (a G(i) protein inhibitor), protein kinase A (PKA) inhibitor amide 14/22 (a PKA inhibitor), and SQ 22536 (an adenylate cyclase inhibitor). Subsequently, dopamine increased cAMP level. The PLC inhibitors (U 73122 and neomycin), the PKC inhibitors (staurosporine and bisindolylmaleimide I) suppressed the dopamine-induced increase of Ca(2+) uptake. SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a MAPKK inhibitor) also inhibited the dopamine-induced increase of Ca(2+) uptake. Dopamine-induced p38 and p42/44 MAPK phosphorylation was blocked by SQ 22536, neomycin, and staurosporine. The stimulatory effect of dopamine on Ca(2+) uptake was significantly inhibited by the NF-kappaB inhibitors SN50, TLCK, and Bay 11-7082. In addition, dopamine significantly increased the level of NF-kappaB p65, which was prevented by either SQ 22536, neomycin, staurosporine, PD 98059, or SB 203580. Thus, dopamine stimulates Ca(2+) uptake in PTCs, initially through by G(s) coupled dopamine receptors, PLC/PKC, followed by MAPK, and ultimately by NF-kappaB activation.     

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.