Involvement of prostaglandins in the response of frog adrenocortical cells to muscarinic receptor activation

The role of prostaglandins (PGs) in the mechanism of action of acetylcholine (ACh) on frog adrenocortical cells has been examined. Administration of a single dose of ACh (5 × 10⁻⁵ M) to perifused frog interrenal fragments, for 20 min, stimulated the production of corticosterone, aldosterone, PGE₂ and 6-keto-PGF_1α. In contrast ACh did not significantly alter TXB2 production. The effect of ACh could be mimicked by muscarine (10⁻⁵ M). Conversely, nicotine (10⁻⁶ to 10⁻⁴ M) was totally inactive. The increase in PG biosynthesis preceded the peak of corticosteroid release. Repeated 20-min pulses of ACh (5 × 10⁻⁵ M) or muscarine (10⁻⁵ M) given at 130-min intervales induced a desensitization phenomenon. In presence of indomethacin (5 × 10⁻⁶ M), the effect of ACh on PG and steroid secretion was totally abolished. In calcium-free medium, the effect of ACh on PG and corticosteroid production was completely blocked. These results indicated that, in the frog, ACh stimulates corticosteroid secretion through a PG-dependent mechanism.     

Regulation of pulmonary venous tone in response to muscarinic receptor activation

We investigated cellular mechanisms that mediate or modulate the vascular response to muscarinic receptor activation (ACh) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E-). Tension and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured simultaneously in fura-2-loaded E- PV strips. In the absence of preconstriction, ACh (0.01-10 microM) caused dose-dependent contraction in E+ and E- rings. ACh contraction was potentiated by removing the endothelium or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methyl ester, P = 0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E- PV rings (P = 0.013 and P = 0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated Ca(2+) channels (nifedipine, P < 0.001), inositol-1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release (2-aminoethoxydiphenyl borate, P = 0.001), PKC (bisindolylmaleimide I, P = 0.001), Rho-kinase (Y-27632, P = 0.002), and tyrosine kinase (TK; tyrphostin 47, P = 0.015) in E- PV rings. ACh (1 microM) caused a leftward shift in the [Ca(2+)](i)-tension relationship (P = 0.015), i.e., ACh increased myofilament Ca(2+) sensitivity. Inhibition of PKC, Rho-kinase, and TK attenuated the ACh-induced increase in myofilament Ca(2+) sensitivity (P < 0.001, P < 0.001, and P = 0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway and involves Ca(2+) influx through voltage-operated Ca(2+) channels and IP(3)-mediated Ca(2+) release. In addition, ACh induces increased myofilament Ca(2+) sensitivity, which requires the PKC, Rho-kinase, and TK pathways.     

Dopamine inhibits corticosteroid secretion in frog adrenocortical cells: evidence for the involvement of prostaglandins in the mechanism of action of dopamine

We have investigated the possible involvement of arachidonic acid metabolites in dopamine-induced inhibition of adrenocortical steroidogenesis. Administration of dopamine (5 x 10(-5) M) for 20 min to perifused frog adrenal slices caused a marked reduction of the release of both prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin (PGI2). Dopamine also induced a significant inhibition of corticosterone and aldosterone secretion. A lag period of 20 min was observed between inhibition of prostanoid and corticosteroid releases. Prolonged dopamine infusion did not prevent the stimulatory effect of PGE1, PGE2 or arachidonic acid on corticosteroid secretion. These observations indicate that activation of dopaminergic receptors in adrenocortical cells is linked to an inhibition of arachidonic acid metabolism. Our data also suggest that the inhibitory effect of dopamine occurs at a step preceding arachidonic acid formation.     

Non-neuronal muscarinic receptor activation prevents apoptosis of endothelial cells induced by homocysteine

Objective Endothelial apoptosis plays an important role in the initiation of atherosclerosis. It would be useful to clarify whether activation of non-neuronal muscarinic receptor （NNMR） could prevent endothelial apoptosis and atherosclerosis. We investigated the effects of NNMR activation on regulating rat aortic endothelial cells （RAECs） apoptosis induced by homocysteine, an independent risk factor of atherosclerosis, and further studied its molecular mechanism. Methods RAECs were incubated using homocysteine at the concentration of 2.7 mmol/L for 36 h. RAECs were also pre-treated with carbachol or arecoline to examine their effects. RT-PCR was used to assess changes in the gene expression related to cell apoptosis. Results Incubation of RAECs with homocysteine at the concentration of 2.7 mmol/L resulted in morphologic changes, such as cellular shrinkage, membrane blebbing, chromatin condensation and margination. These could be attenuated by pretreatment with carbachol and arecoline at the concentration of 10 μmol/L for 12 h. Homocysteine induced apoptosis in RAECs and the molecular mechanisms were associated with the regulation of fas, fas-L and caspase-8 in the death receptor pathway, bcl-2, bcl-xL and bax in the mitochondrial pathway, caspase-22 in the endoplasmic reticulum pathway and caspase-3, caspase-6 and p53 as downstream effectors. Carbachol and arecoline attenuated the effects of homocysteine on genes in the death receptor pathway, in the mitochondrial pathway and in the downstream pathway. Atropine could reverse all of the effects of arecoline. Conclusion Activation of NNMR by carbacol and arecoline inhibits homocysteine-induced endothelial cell apoptosis mainly through regulation of death receptor pathway, mitochondrial pathway and downstream effectors.     

Global gene expression response to telomerase in bovine adrenocortical cells

The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state.     

Involvement of prostaglandins in paraquat intoxication

Paraquat is known to cause severe lung damage through pulmonary edema as its initial feature of toxicity. The purpose of this study was to investigate the toxicity of paraquat in rabbits intraperitoneally injected with 2 or 4 mg/kg/day of the herbicide for a period of 7 days. In the lung, prostaglandin levels of the intoxicated rabbits showed a significant increase in PGF2 alpha. This increase was dose dependent. However, a nonsignificant change in the 6-keto-PGF1 alpha was also observed. Plasma and serum levels of thromboxane-B2 were also significantly elevated but the levels of 6-keto-PGF1 alpha were affected nonsignificantly. The pathology of elevated levels of PGF2 alpha and TXB2 in the lung and blood, in response to paraquat toxicity, is discussed.     

Role of thromboxane receptor activation in the bronchospastic response to endothelin

The selective TxA2/PGH2 (TP) receptor antagonist, SQ 30,741, was used to test the hypothesis that TP-receptor activation contributes to the reactivity of airways and isolated trachea to endothelin-1 (ET-1). Dose-dependent contractions of guinea pig tracheal strips to ET-1 in vitro were unaffected by either SQ 30,741 (1 microM) or indomethacin (2.8 microM). In contrast, maximal bronchospastic responses (increases in airways resistance and decreases in dynamic lung compliance) of anesthetized guinea pigs to ET-1 (0.5 and 1.5 nmole/kg i.v.) in vivo were blocked greater than 90% by SQ 30,741 (1 mg/kg i.v.). Concurrent increases in arterial blood pressure and decreases in leukocyte counts induced by ET-1 were unaffected by SQ 30,741. In rats, ET-1 (1.5 nmole/kg i.v.) did not affect lung mechanics, but did cause biphasic blood pressure and leukopenia responses which were unaltered by SQ 30,741. These data demonstrate that there is considerable species variability in the bronchospastic response to ET-1, and that in guinea pigs, this response is caused predominantly by the activation of TP-receptors.     

Role of thromboxane receptor activation in the bronchospastic response to endothelin

The selective TxA₂/PGH₂ (TP) receptor antagonist, SQ 30, 741, was used to test the hypothesis that TP-receptor activation contributes to the reactivity of airways and isolated trachea to endothelin-1 (ET-1). Dose-dependent contractions of guinea pig tracheal strips to ET-1 in vitro were unaffected by either SQ 30, 741 (1 μM) or indomethacin (2.8 μM). In contrast, maximal bronchospastic responses (increases in airways resistance and decreases in dynamic lung compliance) of anesthetized guinea pigs to ET-1 (.05 and 1.5 nmole/kg i.v.) in vitro were blocked >90% by SQ 30, 741 (1 mg/kg i.v.). Concurrent increases in arterial blood pressure and decreases in leukocyte counts induced by ET-1 were unaffected by SQ 30, 741. In rats, ET-1 (1.5 nmole/kg i.v.) did not affect lung mechanics, but did cause biphasic blood pressure and leukopenia responses which were unaltered by SQ 30, 741. These data demonstrate that there is considerable species variability in the bronchospastic response to ET-1, and that in guinea pigs, this repsonse is caused predominantly by the activation of TP-receptors.     

Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cgamma2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.     

TNF-alpha potentiates the ion secretion induced by muscarinic receptor activation in HT29cl.19A cells

Chronic gastrointestinaldiseases such as ulcerative colitis and Crohn's disease arecharacterized by severe diarrhea. Mucosal biopsies of these patientsshow enhanced levels of cytokines, secreted by infiltrated inflammatorycells. In this study, we investigated the effect of the cytokine tumornecrosis factor- (TNF-) on ion secretion in human intestinalepithelial cells. The conventional microelectrode technique in the cellline HT29cl.19A was used, which allows for simultaneous measurements oftransepithelial potential difference and intracellular potentialdifference across the apical membrane. Preincubation (2-78 h) with10 ng/ml TNF- did not change basal secretory activity. However, thesecretory response to the muscarinic receptor agonist carbachol wasstrongly increased after exposure to TNF-. Application of theprotein kinase C (PKC) inhibitor GF 109203X (bisindolylmaleimide I)inhibited the response to carbachol as well as the TNF--potentiatedresponse, indicating that PKC mediates the effect of carbachol in thiscell line. Propranolol, a substance that inhibits the phospholipase D(PLD) pathway, strongly reduced the response to muscarinic stimulation and its potentiation by TNF-. The results indicate that activation of PLD is involved in ion secretion induced by muscarinic receptor activation and that TNF- can potentiate this pathway.

     

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.     

Involvement of the electrophile responsive element and p53 in the activation of hepatic stellate cells as a response to electrophile menadione

The cytotoxic effects of menadione and hydrogen peroxide were examined in two hepatic stellate cell lines derived from normal or cirrhotic rat liver. The cirrhotic fat-storing cells (CFSC) were found more resistant than the normal fat-storing cells (NFSC) to menadione cytotoxicity. No significant differences were observed in hydrogen peroxide toxicity in these two cell lines. Although protein levels and enzymatic activities of catalase, Cu,Zn-SOD, Mn-SOD, and NADPH cytochrome c reductase were similar in these cell lines, 20-fold increases of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzymatic activity and protein levels were detected in CFSC compared to those of NFSC. Gel mobility shift assays and functional analysis using transient transfection experiments indicated the involvement of the electrophile responsive element (EPRE) in the up-regulation of the NQO1 expression. Antibody supershift analysis revealed that, although Nrf2 is a member of the EPRE-binding complex in both NFSC and CFSC, Nrf1 was identified as a part of the protein/DNA complex only in CFSC. Expression of p53 tumor suppressor gene was found in higher levels in CFSC than in NFSC. We conclude that activation of the EPRE-signaling pathway, which up-regulates several phase II genes and affects p53 stabilization, may offer resistance to hepatic stellate cells against oxidative damage during hepatic injury. This resistance may be a part of the activation process of the hepatic stellate cells and could contribute to their increased proliferation and production of extracellular matrix.     

Involvement of prostaglandins in the local action of endotoxin

Endotoxin from Gram-negative bacteria increases the permeability coefficient of albumin in isolated rat mesenteries used as a separating membrane between the two halves of a diffusion cell. Endotoxin also promotes cyclic AMP accumulation in similar mesenteric sheets. These effects are dose-related, and are inhibited by indomethacin. As shown by direct assay, Prostaglandin E-immunoreactive material is synthesized in the presence of endotoxin and is responsible for the increase in albumin permeability and for the increase in cyclic AMP.     

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.     

Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells.

Syk has been shown to be activated by osmotic stress, however, the mechanisms involved are largely unknown. In this study, we demonstrated that cell shrinkage, rather than osmolarity, was responsible for osmotic stress-induced Syk activation. Osmotic stress-induced Syk activation depended partly upon aggregation of surface receptors. Moreover, intracellular reactive oxygen species were involved in mediating osmotic stress-induced Syk activation, with osmotic stress-induced Syk activation being inhibited by the pretreatment of cells with N-acetyl-cysteine and reduced glutathione. When cells were treated with the combination of sodium chloride and hydrogen peroxide, there was a synergistic activation of Syk. In conclusion, osmotic stress-induced Syk activation required suramin-inhibitable surface receptor aggregation and accumulation of intracellular reactive oxygen species.