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.

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.

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.

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-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.

Paradoxical signal transduction mechanism of endothelins and sarafotoxins in cultured pituitary cells: stimulation of phosphoinositide turnover and inhibition of prolactin release.

Endothelins (ET-1, ET-2, ET-3 and vasoactive intestinal contractor, VIC) and sarafotoxins (SRTX-b and SRTX-c) appear to bind with high affinity to a homogeneous class of binding sites in cultured rat pituitary cells. All of these ligands seem to interact with the same receptor (ETA-R), except for SRTX-c which apparently binds to a separate receptor. Binding was followed by phosphodiesteric cleavage of phosphoinositides, resulting in the formation of inositol phosphates. No consistent effect on basal or gonadotropin-releasing hormone (GnRH)-induced release of luteinizing hormone (LH) was exerted by ET or SRTX during 2 h of static incubation. On the other hand, both groups of vasoactive peptides inhibited basal and thyrotropin-releasing hormone (TRH)-induced prolactin secretion. Surprisingly, activation of phosphoinositide turnover by TRH in pituitary mammotrophs led to stimulation of prolactin secretion, whereas activation of the same pathway by ET or SRTX resulted in inhibition of prolactin secretion. ET and SRTX stimulated inositol phosphate formation in GH3 cell line and in the gonadotroph-like cell line alpha T-3 (which is capable of producing the alpha subunit of the gonadotrophins), indicating that the peptides interact with both pituitary mammotrophs and gonadotrophs. The very low concentrations (nM range) needed to stimulate phosphoinositide turnover and to inhibit prolactin secretion, as well as the recent finding that ETs are present in the hypothalamo-pituitary axis suggest that ET might participate in the neuroendocrine modulation of pituitary functions. One such possibility is that ETs might be members of the prolactin inhibiting factors (PIFs) family.

A novel subtype of endothelin receptors.

A new subtype of endothelin receptors with binding properties typical of "super-high" affinity sites, i.e. with affinities in the picomolar range, were identified and characterized in several rat brain regions and atrium. The pharmacological profile of these sites is indicative of the endothelin receptor type B (ETB-R). These sites differ from the "conventional" high affinity sites (nanomolar range) in several respects; they do not induce phosphoinositide hydrolysis (whereas the high affinity sites do), and they are affected differently by deglycosylation. Thus, there appear to be at least two subtypes of the ETB-R, namely ETB1-R (super-high affinity sites) and ETB2-R (high affinity sites). We suggest the possibility that the super-high affinity sites are related to the vasodilatation property of endothelins, whereas the high affinity sites participate in their vasoconstrictive action.

Endothelin/sarafotoxin receptor heterogeneity: evidence for different glycosylation in receptors from different tissues.

Neuraminidase was used in an attempt to determine whether the endothelin (ET)/sarafotoxin (SRTX) receptor subtypes are glycoproteins and, if so, to determine the role of the carbohydrate moiety in the binding of ligands to the receptor. Incubation of rat cerebellar membranes with neuraminidase was accompanied by a decrease in the capacity of the receptors to bind ET-1 and SRTX-b. In contrast, treatment of the rat caudate putamen and strium or of guinea pig ileum with the enzyme did not affect the binding properties of these receptors. Following exposure of [125I]-ET-1 affinity-labeled receptor to neuraminidase, gel electrophoresis and autoradiography revealed a decrease in molecular mass in the cerebellar and atrial preparations of about 2.5-2.8 kDa. These data indicate that some of the ET/SRTX receptors are glycoproteins and that the sugar moiety is important for ligand binding. Thus, glycosylation might be responsible for the observed heterogeneity among ET/SRTX receptors.

Contractile effects and binding properties of endothelins/sarafotoxins in the guinea pig ileum.

Seven of the eight known isopeptides of the endothelin/sarafotoxin (ET/SRTX) family were tested on the isolated guinea pig ileum and found to cause a concentration-dependent increase in basal tone. The rate or the amplitude of the spontaneous rhythmic contractions of the ileal smooth muscle were essentially not affected by any of the peptides. The maximum contraction elicited by vasoactive intestinal contractor (VIC) was slightly stronger than that induced by endothelin-1 (ET-1) or sarafotoxin-b (SRTX-b), and significantly stronger than the maximal contractions elicited by sarafotoxin-a (SRTX-a), sarafotoxin-c (SRTX-c), or endothelin-3 (ET-3). Sarafotoxin-d (SRTX-d) caused, essentially, no contraction but a rather marked relaxation. The potencies of the various peptides to induce the increase in tension, in terms of EC50 values (cumulative effective concentrations that induce half-maximum response), ranged between 6 and 95 nM depending on the peptide. VIC, ET-1, SRTX-b and SRTX-a had similar potencies and were significantly more potent than SRTX-c and ET-3. A high concentration of SRTX-b elicited no additional response when applied to the organ bath after one of the other peptides had shown a maximal effect. Binding experiments with ileal membranes revealed similar binding properties for the various peptides. Competition with iodinated SRTX-b showed no meaningful differences between the various peptides. It is concluded that all the ET/SRTX peptides compete for the same receptor subtype in the ileum. In terms of efficacy, VIC can be considered as a full agonist of this receptor, SRTX-d is probably an antagonist, while all the other peptides behave as partial agonists.

Kinetic and cross-linking studies indicate different receptors for endothelins and sarafotoxins in the ileum and cerebellum.

Kinetics of ligand/receptor interactions using ET-1, ET-3 and SRTX-b were studied and cross-linking experiments carried out in guinea pig ileum and rat cerebellar preparations. Dissociation studies indicate that the two regions are characterized by different receptor subtypes and different modes of ligand binding. Autoradiographic patterns obtained following cross-linking of ET-1 and ET-3 to the different tissues support these conclusions.

Endothelin/sarafotoxin receptor induced phosphoinositide turnover: effects of pertussis and cholera toxins and of phorbol ester.

The induction of phosphoinositide hydrolysis (PI) by endothelin/sarafotoxin (ET/SRTX) receptors in rat heart myocytes was investigated by the use of bacterial toxins as well as a phorbol ester. Both pertussis- and choleratoxin enhanced the stimulation of PI hydrolysis. Phorbol ester treatment of the myocytes for short periods distinguished between two types of PI-hydrolysis, the one induced by endothelins and the other by sarafotoxins. The possible mediation of G-protein (s) in the induction by ET/SRTX receptors of PI-hydrolysis is discussed.

Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu6 bond, followed by cleavage at the amino side of Ile19. The nicked peptide exhibited biochemical activities comparable to those of the intact peptide--i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was approximately 1 hr, while it was approximately 4 hr for SRTX-b and even higher for SRTX-c. These in vitro findings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs.

Different pathways of endothelin/sarafotoxin-stimulated phosphoinositide hydrolysis in myocytes.

Aging of rat heart myocytes in culture is accompanied by approximately 50% reduction in endothelin (ET)/sarafotoxin (SRTX) receptor-binding capacity as well as in the induction of phosphoinositide (PI) hydrolysis. Treatment of aged cultures under conditions yielding myocytes with a lipid composition similar to that in young cultures restored all the ET/SRTX receptors; at the same time it re-established only the endothelin-induced but not the sarafotoxin-induced PI-hydrolysis response. Thus more than one mechanism may stimulate PI metabolism.

Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes.

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.

Carboxyl residue(s) at the ligand-binding site of rat muscarinic receptors.

Chemical modification of muscarinic receptors of rat cerebral cortex, brain stem and atria by a carboxyl-group-specific reagent, namely trimethyloxonium ion (TMO+) reduces the number of tritium-labeled antagonist- and agonist-binding sites in a dose-dependent way. No such effect is observed when modification is carried out in the presence of atropine, oxotremorine or carbamylcholine. These findings suggest that TMO+ specifically methylates the carboxyl residue(s) positioned at the binding site in members of the M1 and M2 receptor family.

Bisquaternary pyridinium oximes as presynaptic agonists and postsynaptic antagonists of muscarinic receptors.

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes.

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [3H]-N-methyl-4-piperidyl benzilate [( 3H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [3H]-4NMPB, with the highest potency (KI = 1.7 - 6 microM) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (Bmax) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.