Reactive oxygen species regulate signaling pathways induced by M1 muscarinic receptors in PC12M1 cells.

Activation of the m1 muscarinic receptor subtype in rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors was previously shown to induce morphological changes and growth arrest. However, the signaling pathways which lead to these effects were not identified. In an attempt to characterize the intracellular signaling that might be involved in the muscarinic-induced effects, we investigated the role of reactive oxygen species in the regulation of these processes. Stimulation of the muscarinic receptor in these cells increased the intracellular concentrations of reactive oxygen species. Muscarinic activation induced intracellular signaling pathways that involve activation of Ras, extracellular signal-regulated kinase (ERK), and p38. These pathways were partially blocked when reactive oxygen species (ROS) production was prevented by the antioxidant N-acetylcysteine. Other muscarinic-induced signals, such as activation of c-Jun NH(2)-terminal kinase (JNK) or an increase in the binding activity of the transcription factors nuclear factor-kappa B and activator protein-1, were inhibited by the antioxidant dicoumarol. N-Acetylcysteine also blocked the growth arrest and changes in cell shape induced by stimulation of the muscarinic receptor in PC12M1 cells. These findings suggest that ROS act as second messengers in muscarinic-induced cellular signaling. Moreover, generation of ROS appears to be an early and critical intermediary event, which occurs immediately after stimulation of the muscarinic receptor and affects in a variety of mechanisms the muscarinic-mediated cellular signaling.

Involvement of nuclear factor-kappaB in endothelin-A-receptor-induced proliferation and inhibition of apoptosis.

Endothelins have been implicated in the regulation of cell proliferation, differentiation, and apoptosis, but the mechanisms of these complex events are not yet fully understood. Although the nuclear factor-kappaB (NF-kappaB) was shown to play a prominent role in the above processes, its participation in endothelin receptor A (ET(A)R) signaling has not been previously demonstrated. This study provides evidence that NF-kappaB is involved in ET(A)R-induced proliferation and inhibition of apoptosis. Endothelin (ET)-1, ET-3, and sarafotoxin b induce cell proliferation and prevent apoptosis induced by serum deprivation in a Chinese hamster lung (CCL39) cell line that stably expresses ET(A)R (CCL39ET(A)). Activation of ET(A)R resulted in enhanced DNA-binding activity of NF-kappaB and degradation of IkappaB-alpha. Expression of the dominant negative form of IkappaB-alpha (IkappaB deltaN) inhibited the proliferative activities mediated by ET(A)R as well as its anti-apoptotic activities. Treatment of the cells with prostaglandin A1, an inhibitor of IkappaB kinase-beta, reduced ET-1-induced proliferation and its anti-apoptotic effect. These findings indicate that the regulation of cell proliferation and apoptosis by ET(A)R is mediated by the ET(A)R-activated NF-kappaB.

Functional coupling of G proteins to endothelin receptors is ligand and receptor subtype specific.

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins. 2. Coupling between endothelin A (ET(A)) or endothelin B (ET(B)) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein alpha-subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein alpha-subunits. 3. Unstimulated ET(A) and ET(B) receptors (ET(A)R and ET(B)R, respectively) were barely coupled to Go(alpha). The unstimulated ET(A)R coimmunoprecipitated with Gi3alpha, whereas the unstimulated ETBR was much less strongly coupled to Gi3alpha. The coupling of ETBR to Gi1Gi2 alpha-subunits was much stronger than the coupling of ET(A)R to these alpha-subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET(B)R to Gi3alpha, whereas coupling of the ET(A)R to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET(A)R to G(q)alpha/G11alpha than in the coupling of ET(B)R to these alpha-subunits. Ligand-induced coupling between the receptors and the Gi1 and Gi2 alpha-subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to Go(alpha), except that ET-3 increased the coupling of this alpha-subunit to ET(B)R and decreased the coupling to ET(A)R. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific. 4. It remains to be established whether this diversity of receptor-G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.

Functional role for glycosylated subtypes of rat endothelin receptors.

Glycosylation of endothelin (ET) receptors was found to occur in rat cerebellar and atrial membranes. Specifically, we investigated whether the ETA and ETB receptor subtypes differed in their sensitivity to deglycosylation treatment and whether the two affinity states (nanomolar and picomolar) observed in each receptor subtype reflect differences in glycosylation states. Pretreatment of cerebellar or atrial membranes with endoglycosidase H (endo H) caused a marked decrease in the number of maximal binding sites that bind ligand with nanomolar affinity, whereas ligand affinity remained the same. The picomolar-affinity binding sites were not affected by endo H. The use of specific antagonists indicated that the receptor subtype most likely to be influenced by glycosylation is ETA. We suggest that in both cerebellar and atrial membranes, the carbohydrate chains of the ETA receptor contribute to the binding of ligand to the nanomolar-affinity binding sites, but not to the picomolar-affinity binding sites.

Endothelin in cerebrospinal fluid and plasma of patients in the early stage of ischemic stroke.

BACKGROUND AND PURPOSE: Endothelin 1 (ET-1), a highly potent endogenous vasoactive peptide, exerts a sustained vasoconstrictive effect on cerebral vessels. Elevation of ET-1 in plasma has been reported 1 to 3 days after ischemic stroke. Since we assumed that a much faster and more intense response may be observed in the cerebrospinal fluid (CSF) and since an increase in concentration of ET-1 in the CSF may cause constriction of cerebral vessels and eventually influence the neurological outcome, we measured ET-1 values in the CSF within 18 hours of stroke onset and compared the values with those in the plasma. METHODS: Twenty-six consecutive patients with acute stroke were clinically evaluated according to the modified Matthew Scale and underwent two repeat CT scans. Within 5 to 18 hours of stroke onset, lumbar puncture and blood samples were concomitantly obtained and tested; ET-1 levels in CSF and plasma of these patients were analyzed by radioimmunoassay and compared with the levels of a control group of patients with no neurological disease. RESULTS: The mean CSF concentration of ET-1 in the CSF of stroke patients was 16.06 +/- 4.9 pg/mL, compared with 5.51 +/- 1.47 pg/mL in the control group (P < .001). It was significantly higher in cortical infarcts (mean, 17.7 +/- 4.1 pg/mL) than in subcortical lesions (mean, 10.77 +/- 4.1 pg/mL) (P < .001) and significantly correlated with the volume of the lesion (P = .003). The correlation between ET-1 levels in the CSF and the Matthew Scale score was less significant (P = .05). Plasma ET-1 level was not elevated in any group. CONCLUSIONS: ET-1 is found to be significantly elevated in the CSF of stroke patients during the 18 hours after stroke. No elevation was demonstrated in plasma at this time period. ET-1 may be used as an additional indicator of ischemic vascular events in the early diagnosis of stroke. The dissimilarity between the CSF and plasma ET-1 concentrations may lead also to an hypothesis that there is a vasoconstrictive effect on the cerebral vessels or a neuronal effect caused by ET-1 in the mechanism of the progression of brain ischemia.

cGMP formation in rat atrial slices is ligand and endothelin receptor subtype specific.

Involvement of a cGMP pathway in signal transduction stimulated by endothelins(ETs) and sarafotoxins (SRTXs) was examined in rat atrial slices. These peptides activated different receptor-binding sites (ET-1 and SRTX-b reacted with picomolar binding sites of the ET(A) receptor, and ET-3 and SRTX-c reacted with the nanomolar binding sites of the ET(B) receptor) to produce cGMP. ET-1 and SRTX-b stimulated an increase in cGMP levels via a Ca2+-dependent NO pathway involving a pertussis toxin-insensitive G protein, whereas ET-3 and SRTX-c elevated cGMP levels via a Ca2+-independent CO pathway involving a pertussis toxin-sensitive G protein. These results can best be explained in terms of formation of different ligand-receptor-G-protein complexes. The ligands had no effect on ventricular slices, indicating that these signal transduction mechanisms are unique to the atria.

Endothelin receptor heterogeneity, G-proteins, and signaling via cAMP and cGMP cascades.

1. The endothelins (ETs) are potent vasoactive peptides which are involved in diverse biological processes, such as contraction, neuromodulation, and neurotransmission, as well as in certain pathophysiological conditions including cardiac and renal failure. 2. The diversity of action of ETs may be attributed to (i) the existence of a number of receptor subtypes, and (ii) the G-protein-mediated activation of different signal transduction pathways. 3. The combined action of these two variables modulates the response, since different receptor subtypes can stimulate and/or inhibit the cAMP and cGMP cascades.

Activation of muscarinic receptors inhibits apoptosis in PC12M1 cells.

Previous studies have shown that PC12 cells depend on growth factors for their survival. When deprived of growth factors, the cells undergo a dying process termed "apoptosis" (programed cell death). We show here that muscarinic agonists inhibited the apoptotic death of growth factor-deprived PC12M1 cells (PC12 cells stably expressing cloned m1 muscarinic acetylcholine receptors). This protective effect of the muscarinic agonists was observed in both proliferating and neuronal PC12M1 cells, was blocked by the muscarinic antagonist atropine, and was not observed in PC12 cells lacking m1 receptors. Muscarinic receptors therefore mediate inhibition of apoptosis in these cells. In addition to its effect on survival, the muscarinic agonist oxotremorine induced inhibition of DNA synthesis as well as growth arrest of exponentially growing PC12M1 cells at the S and G2/M phases of the cell cycle. Muscarinic receptors in these cells may therefore mediate inhibition of cell cycle progression.

Endothelin receptor subtypes and their role in transmembrane signaling mechanisms.

The endothelins (ETs) are potent vasoactive peptides that appear to be involved in diverse biological actions, for example, contraction, neuromodulation, and neurotransmission, as well as in various pathophysiological conditions, such as renal and heart failure. The diversity of actions of ETs may be explained in terms of (1) the existence of several receptor subtypes and (2) the activation of different signal transduction pathways. This review summarizes the state of the art in this intensively studied field, with particular focus on structural aspects, receptor heterogeneity, coupling of receptors to G-proteins, and signal transduction mechanisms mediated by the activation of ET-receptors.

Cyclic GMP formation in rat cerebellar slices is stimulated by endothelins via nitric oxide formation and by sarafotoxins via formation of carbon monoxide.

Involvement of a cyclic GMP pathway in signal transduction stimulated by endothelins (ETs) and sarafotoxins (SRTXs) was explored using rat cerebellar slices. These peptides activated the same receptor binding sites (ET-1 and SRTX-b at the picomolar sites; ET-3 and SRTX-c at the nanomolar sites) to produce cyclic GMP, but their signaling pathways differed. The endothelins (ET-1 and ET-3) were found to signal via nitric oxide formation and to involve pertussis toxin-sensitive G-protein(s). The SRTXs (b and c), while also stimulating cyclic GMP production, did so via a pathway which is not L-arginine-dependent, i.e., carbon monoxide formation, and did not involve pertussis-toxin-sensitive G-protein(s). This is the first demonstration that the signaling pathways of endothelins and sarafotoxins may differ, even though they share the same binding sites.

Ligand-specific stimulation/inhibition of cAMP formation by a novel endothelin receptor subtype.

The possible involvement of a cAMP pathway in endothelin (ET) signal transduction was explored using rat atrial slices. We show that ET-1 induces both stimulation and inhibition of cAMP formation, depending on its concentration. Unexpectedly, the effects of ET-3 and of sarafotoxins b and c (SRTX-b and SRTX-c) on this pathway differ from that of ET-1. Moreover, we show that the ET-1-induced formation of cAMP results from catecholamine release in a process mediated by a Ca2+ channel coupled to a pertussis toxin sensitive G-protein. It is concluded that this pathway is mediated by a new ETA receptor subtype (probably presynaptic), for which ET-1 is an agonist and ET-3, SRTX-b, and SRTX-c are antagonists.

Endothelins and sarafotoxins: receptor heterogeneity.

1. Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of 21-amino-acid peptides comprising at least eight isoforms. 2. ET exerts multiple pharmacological effects through its receptors. 3. This review summarizes the observations and findings pointing to the existence of receptor subtypes and leading to their identification. 4. Two receptor subtypes have been cloned and stably expressed. 5. The existence of at least two more is predicted by dissimilar ligand potencies in different tissues, kinetics of receptor-ligand interactions, and cross-linking of receptors and radiolabeled ligands.

The non-psychotropic cannabinoid (+)-(3S,4S)-7-hydroxy-delta 6- tetrahydrocannabinol 1,1-dimethylheptyl (HU-211) attenuates N-methyl-D-aspartate receptor-mediated neurotoxicity in primary cultures of rat forebrain.

The non-psychotropic cannabinoid (+)-(3S,4S)-7-hydroxy-delta 6- tetrahydrocannabinol 1,1-dimethylheptyl (HU-211), a stereoselective inhibitor of the N-methyl-D-aspartate (NMDA) receptor, protects primary cultures of rat forebrain against NMDA receptor-mediated neurotoxicity. Cell mortality produced by exposure for 10 min to NMDA or glutamate was reduced to approximately 18 or 27%, respectively, by application of 50 microM HU-211 for 10-15 min during or after exposure of cultures to excitatory amino acid. This effect of HU-211 was dependent on its concentration (EC50 = 8.7 +/- 4 microM). HU-211 also reduced the toxicity induced by brief exposure (10 min) to kinase or quisqualate, though less effectively. HU-211 may therefore prove useful as a non-psychoactive drug that protects against neurotoxicity mediated by the NMDA receptor.

BQ-123 identifies heterogeneity and allosteric interactions at the rat heart endothelin receptor.

Studies of the binding of endothelin-1 (ET-1) to its receptors in the rat heart, as well as kinetic measurements in the presence and absence of the specific antagonist BQ-123, appear to exclude a scheme of simple competitive inhibition and an interaction of ET-1 with a homogeneous population of receptors. Studies with BQ-123 established the presence of three subtypes of endothelin receptors and an allosteric interaction, suggesting the possible existence of a specific site for BQ-123 that interacts and/or interferes with the properties of endothelin-binding sites.

Blockade of 45Ca2+ influx through the N-methyl-D-aspartate receptor ion channel by the non-psychoactive cannabinoid HU-211.

The effects of the synthetic non-psychoactive cannabinoid (+)-(3S,4S)-7-hydroxy-delta 6-tetrahydrocannabinol 1,1-dimethylheptyl (HU-211) on the activity of the N-methyl-D-aspartate (NMDA) receptor/ion channel were examined. HU-211 non-competitively blocks the increase in binding of [3H]N-[1-(2-thienyl)-cyclohexyl]piperidine ([3H]TCP) induced by the polyamines spermine and spermidine or by glutamate and glycine. HU-211 does not, however, affect the direct binding of [3H]glycine and [3H]glutamate to their binding sites on the NMDA receptor, which suggests that the effects of HU-211 are not mediated via the binding sites of glutamate-, glycine- and phencyclidine-like drugs or of polyamines. HU-211 can also block 45Ca2+ uptake through the NMDA-receptor/ion channel in primary cell cultures of rat forebrain. All of the above inhibitory effects of HU-211 on the NMDA-receptor/ion channel activity are stereospecific, since the (-)(3R,4R) enantiomer (HU-210) is ineffective.

Impaired binding properties of endothelin-1 receptors in human endometrial carcinoma tissue.

BACKGROUND: Endothelins, potent stimulators of smooth muscle tissue activity, were recently shown to also function as mitogens for numerous cell types. The authors investigated the properties of endothelin-1 (ET-1) receptors in human endometrial tissue compared with human endometrial carcinoma tissue. METHODS: Tissue samples from 13 patients with endometrial carcinoma and from 12 women undergoing hysterectomy due to uterus myomatous were obtained immediately after surgical removal. Binding properties of the endothelin receptors were studied using 125I-labeled ET-1. RESULTS: A significant difference was demonstrated between binding properties of ET-1 receptors of these two groups. The mean maximal density (Bmax) value of the normal endometrial samples was 2029 +/- 341 fmol/mg protein, whereas that of the neoplastic samples was 356 +/- 121 fmol/mg protein. No differences were found, however, between the mean dissociation constant (Kd) values of these groups. CONCLUSIONS: These results might be compatible with the increased blood flow that characterizes malignant endometrial tissue. However, they do not indicate an important mitogenic role for ET-1 in the development of endometrial cancer.

Endothelin receptors in rat cerebellum: activation of phosphoinositide hydrolysis is transduced by multiple G-proteins.

Induction of phosphoinositide hydrolysis in rat cerebellar slices by endothelins (ET-1 and ET-3) and sarafotoxins (SRTX-b and SRTX-c) was demonstrated by measurement of labelled inositol phosphate generation. Pertussis toxin (PT) enhanced the induction of phosphoinositide hydrolysis by all four peptides. The process seems to be mediated by at least two heterotrimeric G-proteins, the one sensitive and the other insensitive to PT. Measurement of the GTPase activity induced in this preparation indicated that phosphoinositide hydrolysis is stimulated via a functional coupling between the endothelin receptor of the ETB-R subtype and a PT-insensitive G-protein family, i.e. Gq/11. The involvement of PT-sensitive G-proteins, i.e. Gi-like and/or Go-like proteins, in the stimulation of phosphoinositide hydrolysis by ETs and SRTXs is discussed.

Evidence for involvement of the voltage-dependent Na+ channel gating in depolarization-induced activation of G-proteins.

Evidence for activation of pertussis-toxin-sensitive G-proteins by membrane depolarization in rat brainstem synaptoneurosomes was recently reported (Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605; (1991) Neurosci. Lett. 126, 87-90) and is further supported in this study by the observation that the depolarization-induced effect is inhibited when G-proteins are stabilized in the non-activated state with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which was introduced into synaptoneurosomes during the process of permeabilization and resealing. In the present study, agents that either keep the voltage-dependent Na+ channel in persistently activated state (while Na+ currents are blocked) or prevent it from activation were used in an attempt to determine whether the voltage-dependent Na+ channels are involved in the depolarization-induced activation of pertussis-toxin-sensitive G-proteins. The main probe employed was the cardiotonic and antiarrhythmic agent DPI, which is a racemic mixture of two enantiomers, one of which (the R enantiomer) reportedly prevents depolarization-induced activation of the Na+ channel while the other (the S enantiomer) inhibits Na+ channel inactivation. The results suggest that while inactivation of the voltage-dependent Na+ channel does not interfere with the putative depolarization-induced activation of G-proteins, membrane depolarization affects G-proteins and the coupled muscarinic receptors only if the voltage-dependent Na+ channels are capable of being activated. Thus, inhibition of the depolarization-induced activation of Na+ channels was accompanied by inhibition of the depolarization-induced activation of pertussis-toxin-sensitive G-proteins and by modifications of both the coupling of G-proteins to muscarinic receptors and the ADP-ribosylation of Go-proteins. These effects could be counteracted by persistent activation of the voltage-dependent Na+ channels (while Na+ current was blocked). Our observations may suggest that the voltage-dependent Na+ channel gating is involved in the depolarization-induced activation of pertussis toxin-sensitive G-proteins and may provide evidence for a possible mechanism of membrane depolarization signal transduction in excitable cells.

Endothelin-1 receptors in the human myometrium: evidence for different binding properties in post-menopausal as compared to premenopausal and pregnant women.

OBJECTIVE: We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels. SUBJECTS AND METHODS: Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n = 12), myomatous premenopausal women (n = 14) and pregnant women (n = 14), using 125I-labelled endothelin-1. RESULTS: The mean (+/- SD) maximal receptor density (Bmax) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 +/- 196, 1116 +/- 201 and 490 +/- 145 pmol/g protein, respectively). Receptor affinity (Kd) did not differ significantly between these groups. Among the pregnant women, mean Bmax and Kd values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase. CONCLUSIONS: In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.

Endothelin-1 receptors on the human placenta and fetal membranes: evidence for different binding properties in pre-eclamptic pregnancies.

Densities and affinities of tissue protein receptor sites for endothelin-1 in placental and fetal membranes of six preeclamptic and 16 normotensive women in the 36-41st week of gestation were determined by the use of a binding assay with [125I]endothelin-1. The mean maximal density of receptor sites (Bmax) was significantly higher in the placentas of the pre-eclamptic than of the normotensive women (905 +/- 107 vs. 539 +/- 140 fmol/mg protein, p < 0.0001). No differences were found between the two groups with respect to the mean affinity (Kd) of placental receptors, or the mean Bmax and the mean Kd of fetal chorionic samples. In the normotensive group, there were no differences in mean placental Bmax values or in mean Kd values between women who went into spontaneous labor (whether delivered vaginally or abdominally) and those who were electively operated on prior to labor onset. No binding sites were detected in the fetal amniotic membranes of any of the women. Our results suggest that an increase in the maximal density of receptor sites to endothelin-1 in the placenta may play a role in the pathophysiology of pre-eclampsia by contributing to the placental insufficiency that characterizes this disorder.