Role of Brain Endothelin Receptor Type B (ETB) in the Regulation of Tyrosine Hydroxylase in the Olfactory Bulb of DOCA-Salt Hypertensive Rats.

BACKGROUND: We previously reported that endothelins (ETs) regulate tyrosine hydroxylase (TH) activity and expression in the olfactory bulb (OB) of normotensive and hypertensive animals. Applying an ET receptor type A (ETA) antagonist to the brain suggested that endogenous ETs bind to ET receptor type B (ETB) to elicit effects. OBJECTIVE: The aim of the present work was to evaluate the role of central ETB stimulation on the regulation of blood pressure (BP) and the catecholaminergic system in the OB of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. METHODS: DOCA-salt hypertensive rats were infused for 7 days with cerebrospinal fluid or IRL-1620 (ETB receptor agonist) through a cannula placed in the lateral brain ventricle. Systolic BP (SBP) and heart rate were recorded by plethysmography. The expression of TH and its phosphorylated forms in the OB were determined by immunoblotting, TH activity by a radioenzymatic assay, and TH mRNA by quantitative real-time polymerase chain reaction. RESULTS: Chronic administration of IRL-1620 decreased SBP in hypertensive rats but not in normotensive animals. Furthermore, the blockade of ETB receptors also decreased TH-mRNA in DOCA-salt rats, but it did not modify TH activity or protein expression. CONCLUSION: These findings suggest that brain ETs through the activation of ETB receptors contribute to SBP regulation in DOCA-salt hypertension. However, the catecholaminergic system in the OB does not appear to be conclusively involved although mRNA TH was reduced. Present and previous findings suggest that in this salt-sensitive animal model of hypertension, the OB contributes to chronic BP elevation.

Release of 6-nitrodopamine modulates vascular reactivity of Pantherophis guttatus aortic rings.

6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels and Chelonoidis carbonaria aortic rings. The synthesis/release of 6-ND is inhibited by either pre-incubation of the vessels with the nitric oxide (NO) synthase inhibitor L-NAME or by mechanical removal of the endothelium. 6-ND causes powerful vasorelaxation, acting as a potent and selective dopamine D2-like receptor antagonist. Basal release of 6-ND from Panterophis guttatus endothelium intact and denuded aortic rings was quantified by LC-MS/MS. In order to evaluate the interaction of 6-ND with other catecholamines, aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. Endothelium intact aortic rings presented basal release of 6-ND, which was significantly reduced by previous incubation with L-NAME (100 µM). In endothelin-1 (3 nM) pre-contracted endothelium intact aortic rings, 6-ND (10pM-1 µM) and the dopamine D2-receptor antagonist L-761,626 (10 pM-1 µM) induced concentration-dependent relaxations, which were not affected by incubation with L-NAME but greatly reduced in endothelium-removed aortic rings. 6-ND (0.1-1 µM) produced significant rightward shifts of the concentration-response curves to dopamine in L-NAME pre-treated endothelium-intact (pA2 7.01) rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND (1 µM). The EFS-induced contractions of L-NAME pre-treated endothelium-intact aortic rings were significantly inhibited by incubation with 6-ND (1 µM). The results indicate that 6-ND released from Pantherophis guttatus aortic rings is coupled to NO release and represents a new mechanism by which NO can modulate vascular reactivity independently of cGMP production.

Role of endothelin in the pathophysiology of migraine: A new view on an old player.

There is cumulating evidence that endothelin-1 (ET-1) may play a role in migraine, however controversial findings still impede a conclusion to be drawn. Herein we tested the hypothesis that endothelin ETB receptors are major contributors to migraine-like responses. ET-1, IRL-1620 (selective ETB receptor agonist) or CGRP were injected into the trigeminal ganglion (TG) of female Wistar rats, and the development of periorbital mechanical allodynia was assessed hourly with von Frey hairs. Twenty-four hours later, rats were exposed to an aversive light for 1 h, after which the reactivation of periorbital mechanical allodynia (indicating photic sensitivity) was assessed up to 4 h. Moreover, the effect of systemic Bosentan (ETA/ETB receptors antagonist) or the selective antagonists of ETA (BQ-123) and ETB (BQ-788) receptors injected into the TG were evaluated against CGRP-induced responses. ET-1 and IRL-1620 injection into the TG induced periorbital mechanical allodynia and photic sensitivity. Bosentan attenuated periorbital mechanical allodynia but failed to affect photic sensitivity induced by CGRP. Selective blockade of ETB receptors in the TG fully prevented the development of periorbital mechanical allodynia and photic sensitivity induced by CGRP, but ETA receptor blockade caused only a slight reduction of periorbital mechanical allodynia without affecting photic sensitivity. ETB receptor-operated mechanisms in the TG may contribute to migraine-like responses in female rats.

Invitro effect of pine bark extract on melanin synthesis, tyrosinase activity, production of endothelin-1, and PPAR in cultured melanocytes exposed to Ultraviolet, Infrared, and Visible light radiation.

BACKGROUND: French maritime pine bark (Pinus pinaster) extract (PBE), the registered trade name of which is Pycnogenol® , has been studied for its depigmenting action due to its antioxidant, anti-inflammatory, and anti-melanogenic activity. However, the mechanisms through which PBE are still not fully clear. OBJECTIVE: Evaluate the impact of PBE on four in vitro parameters closely associated with cutaneous pigmentation, including melanin synthesis, tyrosinase activity, endothelin-1 (ED1), and production of peroxisome proliferator-activated receptor α, δ, and γ (PPAR α, δ, and γ), by studying the modulation of action of ultraviolet radiation A (UVA)/ultraviolet radiation B (UVB), infrared-A (IR-A), visible light (VL), and association of UVA/UVB, IR-A, and VL (ASS). METHODS: Human melanocytes were incubated in a dry extract solution of PBE, exposed to UVA/UVB, IR-A, VL, and ASS for subsequent quantification of melanin, ED1, and PPAR α, δ, and γ. The effects of PBE on inhibition of tyrosinase activity were also performed by monophenolase activity assay. RESULTS: UVA/UVB, IR-A, VL, and ASS radiation caused significant increases in the synthesis of melanin, ED1, and PPAR α, δ, and γ when compared to baseline control. However, PBE significantly reduced the production of melanin, ED1, and PPAR α, δ, and γ, as well as reducing about 66.5% of the tyrosinase activity. CONCLUSIONS: PBE reduces in vitro melanin production by downregulating tyrosinase and reducing pigmentation-related mediators, such as ED1 and PPAR α, δ, and γ, therefore contributing to the inhibition of pathways associated with skin hyperpigmentation.

Facial hyperalgesia due to direct action of endothelin-1 in the trigeminal ganglion of mice.

OBJECTIVE: This study assessed the ability of endothelin-1 (ET-1) to evoke heat hyperalgesia when injected directly into the trigeminal ganglia (TG) of mice and determined the receptors implicated in this effect. The effects of TG ETA and ETB receptor blockade on alleviation of heat hyperalgesia in a model of trigeminal neuropathic pain induced by infraorbital nerve constriction (CION) were also examined. METHODS: Naive mice received an intraganglionar (i.g.) injection of ET-1 (0.3-3 pmol) or the selective ETB R agonist sarafotoxin S6c (3-30 pmol), and response latencies to ipsilateral heat stimulation were assessed before the treatment and at 1-h intervals up to 5 h after the treatment. Heat hyperalgesia induced by i.g. ET-1 or CION was assessed after i.g. injections of ETA R and ETB R antagonists (BQ-123 and BQ-788, respectively, each at 0.5 nmol). KEY FINDINGS: Intraganglionar ET-1 or sarafotoxin S6c injection induced heat hyperalgesia lasting 4 and 2 h, respectively. Heat hyperalgesia induced by ET-1 was attenuated by i.g. BQ-123 or BQ-788. On day 5 after CION, i.g. BQ-788 injection produced a more robust antihyperalgesic effect compared with BQ-123. CONCLUSIONS: ET-1 injection into the TG promotes ETA R/ETB R-mediated facial heat hyperalgesia, and both receptors are clearly implicated in CION-induced hyperalgesia in the murine TG system.

Dysregulated mitogen-activated protein kinase and matrix metalloproteinase in ethanol-induced cavernosal dysfunction.

We evaluated the effects of ethanol consumption on the mitogen-activated protein kinases (MAPK) and metalloproteinases (MMP) pathways in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) for 6 weeks. Quantitative real-time polymerase chain reaction experiments showed that ethanol consumption did not alter mRNA levels of p38MAPK, SAPK/JNK, ERK1/2, MMP-2, or MMP-9 in the rat CSM. Western immunoblotting experiments revealed decreased protein expression of p38MAPK and phosphorylation of SAPK/JNK in the CSM from ethanol-treated rats. Additionally, ethanol consumption decreased the expression of MMP-2. Functional assays showed that SP600125, an inhibitor of SAPK/JNK, prevented the increase in endothelin (ET)-1-induced contraction in the CSM from ethanol-treated rats. Treatment with ethanol decreased MMP-2 activity, but did not change net MMP activity in the rat CSM. Ethanol consumption increased the circulating levels of MMP-2, MMP-9, and TIMP-2 as well as the MMP-9/TIMP-1 ratio. The major finding of our study is that ethanol consumption down-regulates both MAPK and MMP pathways in the rat CSM, whereas it increases the circulating levels of MMP-9. Additionally, we found that SAPK/JNK plays a role in ethanol-induced increase on ET-1 contraction in the isolated rat CSM.

Mechanisms involved in facial heat hyperalgesia induced by endothelin-1 in female rats.

OBJECTIVE: Pronociceptive responses to endothelins in the trigeminal system seem to be mediated by ETA and ETB receptors, which have been shown to be expressed in neurons of the trigeminal ganglion of humans and rats. The present study aimed to evaluate the ability of endothelin-1 (ET-1) to induce facial heat hyperalgesia in female rats, the contribution of ETA and ETB receptors to this response, as well as the mechanisms underlying heat hyperalgesia induced by ET-1. DESIGN: ET-1 (100pmol/50µL) was injected into the upper lip and heat hyperalgesia was evaluated for up to 6h. Facial heat hyperalgesia induced by ET-1 was assessed in rats pre-treated locally with BQ-123 or BQ-788 (selective ETA and ETB receptor antagonists, respectively, 30nmol/50µL); BCTC (TRPV1 receptor antagonist; 300µg/50µL); anti-NGF (3µg/50µL); K252a (TrkA inhibitor, 1µg/50µL); or in rats that received intraganglionar resiniferatoxin injection (RTX, 200ng/10µL) to promote C-fibers ablation. RESULTS: ET-1 induced facial heat hyperalgesia that persisted up to 6h and was prevented by BQ-123, BQ-788 or by intraganglionar RTX injection. Likewise, local pre-treatment with BCTC abolished ET-1 induced facial heat hyperalgesia up to 3h. Local pre-treatment with anti-NGF or K252a was effective to prevent ET-1 induced heat hyperalgesia. CONCLUSIONS: In conclusion, ET-1 is able to induce heat hyperagelsia in trigeminal primary afferents of female rats, which is mediated by ETA and ETB receptors. Activation of TRPV1 receptors and NGF-signaling pathways may contribute to heat hyperalgesia induced by ET-1.

Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.

Urocortin 1 expression and secretion by human umbilical vein endothelial cells: In vitro effects of interleukin 8, interferon γ, lipopolysaccharide, endothelin 1, prostaglandin F-2α, estradiol, progesterone and dexamethasone.

Urocortin 1 (Ucn1) is a 40-amino-acid peptide that has vasodilatory activity and displays immunomodulatory and antioxidant properties. Maternal and cord plasma Ucn1 levels are increased in preeclampsia and preterm labor, but the mechanisms of such increase are poorly known. Thus, we investigated Ucn1 localization in human umbilical cord and assessed some potential stimuli to Ucn1 release by human umbilical vein endothelial cells (HUVEC). Human umbilical cords were obtained at uncomplicated term pregnancy (n=11). Ucn1 localization was assessed by immunohistochemistry and quantified. HUVEC were grown in vitro to confluence, then incubated with serial concentrations of interleukin (IL)-8, interferon (INF)-γ, lipopolysaccharide (LPS), endothelin (ET)-1, prostaglandin (PG)F-2α, estradiol, progesterone and dexamethasone and Ucn1 concentrations were measured in the supernatants. Ucn1 was immunolocalized with similar intensity in umbilical cord arteries, vein and Wharton's jelly. Ucn1 mRNA was detected in all HUVEC cultures and Ucn1 peptide was detectable in culture medium from untreated cells at different time points. Incubation with IFN-γ increased Ucn1 secretion in a dose-dependent manner. Treatments with IL-8, LPS, ET-1 and dexamethasone were able to increase three to fourfold Ucn1 release from cultured endothelial cells. In conclusion, umbilical vessels express Ucn1 and may be a contributive source of Ucn1 release into fetal-placental circulation. IL-8, IFN-γ, LPS, ET-1 and dexamethasone promote Ucn1 secretion from cultured HUVEC.

The ability of 17 ß-estradiol to attenuate intrahepatic vasoconstriction to endothelin-1 in female rats is lost in cirrhosis.

BACKGROUND AND RATIONALE: The control of Endothelin-1 (ET-1)-mediated intrahepatic vasoconstriction in cirrhosis is beneficial for the alleviation of relevant complications. Cirrhosis is accompanied by hypogonadism and altered sex hormone status. Besides, sex hormones have vasoactive effects, but it is unknown if they influence vascular function in cirrhosis. This study aimed to investigate the roles of sex hormones in hepatic vascular reactions to ET-1 in cirrhosis. Liver cirrhosis was induced in Spraque-Dawley male and female rats with common bile duct ligation (BDL). Sham-operated (Sham) rats were controls. On the 43rd day after operations, intrahepatic vascular concentration-response curves to ET-1 were obtained with the following preincubatioins: 1) vehicle; 2) 17ß-estradiol; 3) progesterone; 4) testosterone. Livers from sham and BDL rats were dissected for real-time polymerase chain reaction analysis of estrogen, progesterone and testosterone receptors. RESULTS: Compared with sham males perfused with vehicle, sham females presented higher perfusion pressure changes to ET-1 which was reversed only by 17 ß-estradiol. In cirrhosis, compared with males, 17 ß-estradiol no longer attenuated vascular responsiveness to ET-1 in females. In females, BDL rats had lower hepatic estrogen receptor α(ERßα) mRNA expression than that in sham rats. CONCLUSIONS: The sham females showed a stronger intrahepatic vascular constrictive effect to ET-1 than sham males, which could be reversed by 17ß-estradiol. However, the influence of 17 ß-estradiol was lost in cirrhotic females, which may be attributed, at least partly, to intrahepatic ER α down-regulation in females with cirrhosis.

Endothelin-1 and -3 induce choleresis in the rat through ETB receptors coupled to nitric oxide and vagovagal reflexes.

We have reported previously that centrally applied ET (endothelin)-1 and ET-3 induce either choleresis or cholestasis depending on the dose. In the present study, we sought to establish the role of these endothelins in the short-term peripheral regulation of bile secretion in the rat. Intravenously infused endothelins induced significant choleresis in a dose-dependent fashion, ET-1 being more potent than ET-3. Endothelins (with the exception of a higher dose of ET-1) did not affect BP (blood pressure), portal venous pressure or portal blood flow. ET-1 and ET-3 augmented the biliary excretion of bile salts, glutathione and electrolytes, suggesting enhanced bile acid-dependent and -independent bile flows. ET-induced choleresis was mediated by ET(B) receptors coupled to NO and inhibited by truncal vagotomy, atropine administration and capsaicin perivagal application, supporting the participation of vagovagal reflexes. RT (reverse transcription)-PCR and Western blot analysis revealed ETA and ET(B) receptor expression in the vagus nerve. Endothelins, through ET(B) receptors, augmented the hepatocyte plasma membrane expression of Ntcp (Na⁺/taurocholate co-transporting polypeptide; Slc10a1), Bsep (bile-salt export pump; Abcb11), Mrp2 (multidrug resistance protein-2; Abcc2) and Aqp8 (aquaporin 8). Endothelins also increased the mRNAs of these transporters. ET-1 and ET-3 induced choleresis mediated by ET(B) receptors coupled to NO release and vagovagal reflexes without involving haemodynamic changes. Endothelin-induced choleresis seems to be caused by increased plasma membrane translocation and transcriptional expression of key bile transporters. These findings indicate that endothelins are able to elicit haemodynamic-independent biological effects in the liver and suggest that these peptides may play a beneficial role in pathophysiological situations where bile secretion is impaired.

Mineralocorticoid receptor activation is crucial in the signalling pathway leading to the Anrep effect.

The increase in myocardial reactive oxygen species after epidermal growth factor receptor transactivation is a crucial step in the autocrine/paracrine angiotensin II/endothelin receptor activation leading to the slow force response to stretch (SFR). Since experimental evidence suggests a link between angiotensin II or its AT1 receptor and the mineralocorticoid receptor (MR), and MR transactivates the epidermal growth factor receptor, we thought to determine whether MR activation participates in the SFR development in rat myocardium. We show here that MR activation is necessary to promote reactive oxygen species formation by a physiological concentration of angiotensin II (1 nmol l(-1)), since an increase in superoxide anion formation of ~50% of basal was suppressed by blocking MR with spironolactone or eplerenone. This effect was also suppressed by blocking AT1, endothelin (type A) or epidermal growth factor receptors, by inhibiting NADPH oxydase or by targeting mitochondria, and was unaffected by glucocorticoid receptor inhibition. All interventions except AT1 receptor blockade blunted the increase in superoxide anion promoted by an equipotent dose of endothelin-1 (1 nmol l(-1)) confirming that endothelin receptors activation is downstream of AT1. Similarly, an increase in superoxide anion promoted by an equipotent dose of aldosterone (10 nmol l(-1)) was blocked by spironolactone or eplerenone, by preventing epidermal growth factor receptor transactivation, but not by inhibiting glucocorticoid receptors or protein synthesis, suggesting non-genomic MR effects. Combination of aldosterone plus endothelin-1 did not increase superoxide anion formation more than each agonist separately. We found that aldosterone increased phosphorylation of the redox-sensitive kinases ERK1/2-p90RSK and the NHE-1, effects that were eliminated by eplerenone or by preventing epidermal growth factor receptor transactivation. Finally, we provide evidence that the SFR is suppressed by MR blockade, by preventing epidermal growth factor receptor transactivation or by scavenging reactive oxygen species, but it is unaffected by glucocorticoid receptor blockade or protein synthesis inhibition. Our results suggest that MR activation is a necessary step in the stretch-triggered reactive oxygen species-mediated activation of redox-sensitive kinases upstream NHE-1.

Schistosomiasis differentially affects vasoconstrictor responses: up-regulation of 5-HT receptor-mediated aorta contraction.

Schistosomiasis, classified by the World Health Organization as a neglected tropical disease, is an intravascular parasitic disease associated to a chronic inflammatory state. Evidence implicating inflammation in vascular dysfunction continues to mount, which, broadly defined, reflects a failure in the control of intracellular Ca2+ and consequently, vascular contraction. Therefore, we measured aorta contraction induced by 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), two important regulators of vascular contraction. Isometric aortic contractions were determined in control and Schistosoma mansoni-infected mice. In the infected animals, 5-HT induced a 50% higher contraction in relation to controls and we also observed an increased contraction in response to Ca2+ mobilisation from sarcoplasmic reticulum. Nevertheless, Rho kinase inhibition reduced the contraction in response to 5-HT equally in both groups, discarding an increase of the contractile machinery sensitivity to Ca2+. Furthermore, no alteration was observed for contractions induced by ET-1 in both groups. Our data suggest that an immune-vascular interaction occurs in schistosomiasis, altering vascular contraction outside the mesenteric portal system. More importantly, it affects distinct intracellular signalling involved in aorta contraction, in this case increasing 5-HT receptor signalling.

Endothelins participate in the central and peripheral regulation of submandibular gland secretion in the rat.

We previously reported that endothelins (ETs) are involved in the rat central and peripheral regulation of bile secretion. In this study we sought to establish whether ET-1 and ET-3 modulated submandibular gland secretion when locally or centrally applied. Animals were prepared with gland duct cannulation to collect saliva samples and jugular cannulation to administer sialogogues. ETs were given either into the submandibular gland or brain lateral ventricle. Intraglandularly administered ETs failed to elicit salivation per se. However, ET-1, but not ET-3, potentiated both cholinergic- and adrenergic-evoked salivation through ET(A) receptors. ET-1 decreased cAMP content but increased phosphoinositide hydrolysis, whereas ET-3 attenuated both intracellular pathways. The expression of ET(A) and ET(B) receptor mRNAs as well as that of ETs was revealed in the submandibular gland by RT-PCR. Immunohistochemical studies showed that ET(A) receptor staining was localized around the interlobular ducts and acini, compatible with the myoepithelial cells' location, whereas ET(B) receptor staining was restricted to small blood vessels. When applied to the brain, both ETs induced no salivation but enhanced cholinergic- and adrenergic-evoked salivary secretion through parasympathetic pathways. ET-1 response was mediated by brain ET(A) receptors, whereas that of ET-3 was presumably through nonconventional ET receptors. Present findings show that ETs are involved in the brain regulation of cholinergic- and adrenergic-stimulated submandibular gland secretion through the activation of distinct brain ET receptors and parasympathetic pathways. However, when ETs were administered into the gland, only ET-1 enhanced cholinergic and adrenergic salivation likely through myopithelial cell contraction by activating ET(A) receptors coupled to phospholipase C. The presence of ETs and ET receptors suggests the existence of an endothelinergic system in the submandibular gland.

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus.

BACKGROUND AND PURPOSE: Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE(2) content in plasma, CSF and hypothalamus induced by either LPS or ET-1. EXPERIMENTAL APPROACH: Rats were given (i.p.) dipyrone (120 mg·kg(-1)) or indomethacin (2 mg·kg(-1)) 30 min before injection of LPS (5 µg·kg(-1), i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE(2) levels were determined in the plasma, CSF and hypothalamus by elisa. KEY RESULTS: LPS or ET-1 induced fever and increased CSF and hypothalamic PGE(2) levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE(2) but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE(2) levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE(2) levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE(2) levels. Dipyrone also reduced the increase in the venous plasma PGE(2) concentration induced by LPS. CONCLUSIONS AND IMPLICATIONS: These findings confirm that PGE(2) does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE(2) synthesis.

Mechanisms involved in the long-term modulation of tyrosine hydroxylase by endothelins in the olfactory bulb of normotensive rats.

The olfactory bulbs play a relevant role in the interaction between the animal and its environment. The existence of endothelin-1 and -3 in the rat olfactory bulbs suggests their role in the control of diverse functions regulated at this level. Tyrosine hydroxylase, a crucial enzyme in catecholamine biosynthesis, is tightly regulated by short- and long-term mechanisms. We have previously reported that in the olfactory bulbs endothelins participate in the short-term tyrosine hydroxylase regulation involving complex mechanisms. In the present work we studied the effect of long-term stimulation by endothelins on tyrosine hydroxylase in the rat olfactory bulbs. Our findings show that endothelin-1 and -3 modulated catecholaminergic transmission by increasing enzymatic activity. However, these peptides acted through different receptors and intracellular pathways. Endothelin-1 enhanced tyrosine hydroxylase activity through a super high affinity ET(A) receptor and cAMP/PKA and CaMK-II pathways, whereas, endothelin-3 through a super high affinity atypical receptor coupled to cAMP/PKA, PLC/PKC and CaMK-II pathways. Endothelins also increased tyrosine hydroxylase mRNA and the enzyme total level as well as the phosphorylation of Ser 19, 31 and 40 sites. Furthermore, both peptides stimulated dopamine turnover and reduced its endogenous content. These findings support that endothelins are involved in the long-term regulation of tyrosine hydroxylase, leading to an increase in the catecholaminergic activity which might be implicated in the development and/or maintenance of diverse pathologies involving the olfactory bulbs.

Endothelin-1 and -3 modulate the neuronal norepinephrine transporter through multiple signalling pathways in the rat posterior hypothalamus.

We have previously reported that endothelin-1 and -3 modulate different steps of noradrenergic transmission in the hypothalamus. We showed that endothelins modify neuronal norepinephrine transport activity through the regulation of the kinetic constant and internalization. In the present work we sought to define the endothelin receptors and intracellular mechanisms involved in the down-regulation of neuronal norepinephrine uptake induced by endothelin-1 and -3 in the rat posterior hypothalamic region. Results showed that endothelin-1 reduced norepinephrine uptake through ET(B) receptors, whereas endothelin-3 through a non-conventional or atypical endothelin receptor. In both cases, the effect on norepinephrine uptake was coupled to protein kinase A and C as well as nitric oxide pathways. However, neither protein kinase G nor intracellular or extracellular calcium and calcium/calmodulin-dependent protein kinase II were involved. In addition, the same intracellular mechanisms participated in the reduction of nisoxetine binding (norepinephrine transporter internalization index) induced by both endothelins. Present findings reveal the underlying mechanisms involved in the regulation of the neuronal norepinephrine transporter by endothelins and further support the role of these peptides in the modulation of noradrenergic transmission at the presynaptic nerve endings in the posterior hypothalamus.

Roles of endothelin ETA and ETB receptors in nociception and chemical, thermal and mechanical hyperalgesia induced by endothelin-1 in the rat hindpaw.

Evidence on the relative roles of endothelin ET(A) and ET(B) receptors in mediating the nociceptive and hyperalgesic actions of endothelin-1 is still fragmented and conflicting, due to variations between species and/or models. This study assesses the participation of ET(A) and ET(B) receptors on the nociceptive behavior and hyperalgesia to chemical (formalin), mechanical and thermal stimuli evoked by endothelin-1 injected into the rat hind-paw. Intraplantar (i.pl.) injection of endothelin-1 (1-30 pmol, 50 microl) induced dose-dependent nociceptive behaviors over the first hour. Endothelin-1 (3-30 pmol) also potentiated both phases of nociception induced by a subsequent ipsilateral i.pl. injection of formalin (0.5%, 50 microl). Endothelin-1, at 10 pmol, increased responses of the first phase (0-10 min) by 97% and of the second phase (15-60 min) by 120%, and similar degrees of potentiation were observed following 30 pmol of the peptide. Endothelin-1 (1-30 pmol) caused slowly developing long-lasting thermal and mechanical hyperalgesia with maximum effects at 10 and 30 pmol, respectively, reaching significance at 2-3h and remaining elevated for up to at least 8h after injection. Treatment with the selective ET(A) and ET(B) peptidic antagonists BQ-123 and BQ-788 (i.pl., both at 10 nmol, 3.5h after ET-1 injection) or with the non-peptidic antagonists atrasentan and A-192621 systemically (i.v., 10 and 20mg/kg, respectively) each caused significant reductions in endothelin-1-induced nociception, as well as chemical, thermal and mechanical hyperalgesia. Thus, the nociceptive and hyperalgesic effects induced by i.pl. endothelin-1 seem to be mediated by both ET(A) and ET(B) receptors.

Effect of inhibitors of mitogen-activated protein kinase kinase on alpha(1B)-adrenoceptor phosphorylation.

1 Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on alpha(1B)-adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2'-amino-3'-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased alpha(1B)-adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid. 2 Staurosporine (IC(50) approximately 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC(50) approximately 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced alpha(1B)-adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases. 3 Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with alpha(1B)-adrenoceptors. Association of phospho-ERK 1/2 to alpha(1B)-adrenoceptors increased not only in response to agonist but also in response to agents that increase alpha(1B)-adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect. 4 Our data show that blockade of MEK activity results in increased alpha(1B)-adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor-ERK1/2 interaction.

Aging and total stenosis triggers differential responses of carotid and basilar arteries to endothelin-1 and phenylephrine.

Our aim was to investigate the effects of ageing on the vascular contractility of carotid and basilar arteries from guinea-pigs, in a model of total stenosis. Moreover, we attempted to identify whether total stenosis of the left common carotid (stenosed) in adult guinea-pigs, would affect the contractions of contralateral carotid (intact) and basilar arteries to different vasoconstrictors. With this purpose, the left carotid was occluded with a silk thread at a position close to its origin. Vascular reactivity experiments using standard muscle bath were performed 7, 15, 30, and 90 days after carotid occlusion. Reactivity of carotid and basilar arteries to endothelin-1, phenylephrine and KCl was reduced with ageing in naive guinea-pigs. The endothelin-1 and KCl-induced contractions were unaltered in arteries from SHAM-operated animals. Moreover, phenylephrine-induced contractions were reduced in both carotids from 7 days SHAM-operated guinea-pigs, when compared to naive group. Stenosis induced progressive reduction in the contraction induced by endothelin-1, phenylephrine and KCl in the stenosed carotid, when compared to their respective age-matched naive and SHAM groups. Interestingly, an increased contractile-response to vasoconstrictor agents in all the contralateral carotids was observed. Stenosis (30 and 90 days) also induced an increase in the contractions induced by endothelin-1 in the basilar artery. Conversely, phenylephrine and KCl-induced contractions were reduced in basilar arteries 7, 15, 30 and 90 days after stenosis. These results showed that stenosis in adult guinea-pigs induce alterations of vascular reactivity in arteries distant from the site of injury. Thus, in spite of the common use of contralateral carotid as control, it must be aware of the potential alteration induced by stenosis in the vascular motility of such vessels. Additionally, it was verified a relationship between the period of stenosis and the alterations in the vascular reactivity to these vasoconstrictors.