br Results br Discussion Previous studies have shown that al

Results
Discussion Previous studies have shown that all the components of ET system are expressed by various neuronal and non-neuronal structures throughout the CNS (McCumber et al., 1990; Giaid et al., 1991). In effect, endothelin-converting enzyme activity, mRNA, ET-1 and ET-3 immunoreactivity has been described within specific central nuclei, suggesting multiple roles of brain ET (Sluck et al., 1999). Endothelin sc9 are widely dispersed throughout the CNS, reflecting ET-1 and ET-3 peptide localization. Brain distribution of endothelin receptors using ligand binding or receptor autoradiography studies has shown high density of binding in several brain areas within circumventricular structures among them the SFO and median eminence (Kohzuki et al., 1991; Sokolovsky, 1992). In general, the brain appears to contain predominantly, if not exclusively, endothelin ETB receptors (Hori et al., 1992). However, using a specific antibody against ETA receptor (Kurokawa et al., 2000) it was found it to be widely distributed on cell bodies and neuron terminals in several parts of the rat brain, including a number of medullary regions, the arcuate nucleus of the hypothalamus, ventral tegmental area and in the locus coeruleus, an area rich in noradrenergic neurons, which are known to be activated by physiological challenges and stimuli that are considered stressors. Consistent with the presence of specific receptors, in high density in the SFO and ME, our current observation of an elevation of InsP1 accumulation in whole ME and SFO demonstrates that ETs exert their effects by activation of an ET receptor linked to a second messenger system involving polyphosphoinositide hydrolysis. These results are compatible with previous reports on ET-stimulation of PI turnover in rat neuroblastoma–glioma hybrid cells (Chau et al., 1993), glial cells (Marsault et al., 1990), in cerebellar granule and glioma cells (Lin et al., 1991) and whole adrenal medulla and pineal gland (Garrido and Israel, 1997, Garrido and Israel, 1999). In addition, their confirm our previous report for ET-3 (Garrido and Israel, 1994). Little is known about which cell type are endothelin receptors subtypes associated with. Although ETB receptors have been found associated with neuronal fibers immunoreactive for luteinizing hormone-release hormone (LHRH) in rat hypothalamus (Yamamoto et al., 1997), it has been shown that ETB, which is the major receptor among ET receptors in the brain, is predominantly expressed on astrocytes (Gebke et al., 2000; Hori et al., 1992). As for the subtype of receptor involved in ETs action in ME and SFO, our results point to the ETB receptor subtype as responsible for ETs-mediated increase in InsP1 accumulation in this two rat brain localized areas, since in both structures, addition of the endothelin isopeptide (ET-1 or ET-3) increased InsP1 accumulation in a dose dependent-manner with similar EC50 values. Furthermore, our data using selective agonist and antagonists, allowed us to characterize the receptor subtype involved in the ETs induced increase in PI turnover. The fact that ETs-mediated increase in InsP1 accumulation was not affected by two selective ETA receptor antagonist, and it was mimicked by IRL 1620 an ETB receptor agonist, and these effects were blocked by BQ 788, a selective ETB receptor antagonist, leads to the suggestion that in the SFO and ME, ET signaling through PI is mediated by endothelin ETB receptor. Our results are in agreement with previous reports, of the presence of high affinity receptors for ET-3 in endothelial cells from spontaneously hypertensive rats coupled to PI turnover (Araki et al., 1989) and the stimulation of PI turnover by ET-1 and ET-3 with similar potency in cultured cerebellar granule cells and astrocytes (Lin et al., 1991; Marsault et al., 1990). Inside the brain tissue, ETs play numerous important biological roles through the stimulation of specific receptor subtype in different cell types. Most physiological known effects of ETs are attributed to the stimulation of the ETA receptor (Calogero et al., 1994; Salazar et al., 1998; Wall and Ferguson, 1992). There is evidence which suggest that ETB receptors in the CNS appear to play an important role, even under basal conditions, in counterbalancing ETA mediated functional actions and also in the control of extracellular ET-1 levels. Thus, some ET actions could oppose ETA mediated effect through the stimulation of ETB receptors. Indeed, it was reported that vasodepression following injection of ET-1 into lateral ventricles (Tadepalli and Hashim, 1995) and depressor response induced by ET-1 microinjection into superior colliculus of the rat are predominantly mediated by ETB receptor (D'Amico et al., 1998). In an experimental rat model devoid of functional ETB receptors (Ehrenreich et al., 1999), the sl/sl rat, it was reported a measurable functional consequences in astrocytes, reflected as a reduced astrocytic ET-1 eliminatory capacity and an increased rate of bigET-1 conversion into mature, biologically active ET-1. These latter effects may result in an elevated extracellular ET-1 concentration, which acting from the adventitial side of cerebral vessels, may cause potent vasoconstriction.