Additionally to their peripheral effects evidence indicate a
Additionally to their peripheral effects, evidence indicate a role for ETs in the central nervous system (Mosqueda-Garcı́a et al., 1993). Indeed, using Northern blot analysis and in situ hybridization it has been shown the presence of immunoreactive ET of non-vascular origin and of neuronal location in several Ferulenol areas (McCumber et al., 1990). It has been demonstrated the existence of, at least, two distinct subpopulations of ET receptor in brain localized areas such as the subfornical organ (SFO) and the median eminence (ME) (McCumber et al., 1990; Kohzuki et al., 1991). Functional differences between these two receptor subtypes in the intact brain localized areas and signaling coupled to each receptor subtype is not completely determined. Increasing evidence suggests that phosphoinositide (PI) turnover induced by activation of ET receptors is involved in biological activities elicited by ET-related peptides. It has been shown that ET stimulates PI turnover in peripheral tissue (Araki et al., 1989), in neurons (Lin et al., 1991), in cultured glial cells, cerebellar cells and astrocytes (Marsault et al., 1990) in rat adrenal cortex (Woodcock et al., 1990) and in the pineal gland (Garrido and Israel, 1999).
Stimulation of ET-1 high affinity binding site has been associated to the activation of phospholipase C in rat brain (Yokokawa et al., 1991) and in cultured endothelial cells from human brain microvasculature (Purkiss et al., 1994; Stanimirovic et al., 1994) and to phophoinositide hydrolysis and intracellular calcium mobilization (Goldman et al., 1991). In addition, endothelin have been shown to be involved in phosphorylation of specific proteins from human intact cerebral capillaries (Catalán et al., 1996).
In the present study we assessed endothelin-1 and 3-induced inositol monophosphate (InsP1) accumulation and ET receptor subtype associated with this action, in whole ME and SFO, two brain areas located outside the blood–brain barrier and known to participate in the regulation of water and electrolyte balance and neuroendocrine control (Samson et al., 1991).
Materials and methods Male Sprague–Dawley rats (220–280 g) were housed under alternate periods of dark and light and were given water and chow ad libitum. The animals were killed by decapitation between 09.00 and 10.00 h. Microdissection of fresh SFO was performed according to Summy-Long and Severs (1979) and ME according to Garrido and Israel (1994). Briefly, after decapitation, the brains were immediately removed and placed in an ice-cooled dish in a cold room. The ventral hypothalamic tissue was exposed and the ME was dissected under stereomicroscopic control; immediately the remained hypothalamic tissue was aspirated until the third ventricle was exposed. The choroid plexus from lateral and third ventricles was carefully removed. The SFO was removed by transecting the hippocampal commissure just above the SFO protrusion and making lateral cuts bordering the columns of the fornix. The SFO tissue sample thus contained a small amount of hippocampal commissure and fornix. After removal, tissues were kept in ice-cold Krebs-Ringer buffer (KRB; containing in mM: NaCl 125, KCl 3.5, KH2PO4 1.25, MgSO4 1.2, CaCl2 0.75, NaHCO3 25 and glucose 10) and gassed with 95% O2:5% CO2. The PI hydrolysis was assessed as accumulation of InsP1 in the presence of 10 mM LiCl in whole tissue according to the method described (Garrido and Israel, 1994). Briefly, each individual brain structures (SFO or ME) were labeled for 2 h at 37 °C in 20 ml KRB with 0.5 mCi of myo-[2-3H]-inositol (specific activity 18.8 Ci/mmol) with continuous gassing with 95% O2:5% CO2. After labeling, the tissue were washed in freshly gassed KRB and each sample was transferred into individual 1.5 ml Eppendorf tubes containing 360 μl of LiCl KRB buffer and were preincubated for 10 min at 37 °C with or without the correspondent antagonist (For ETA: BQ 123 and BQ 610, 10−5 M; for ETB: BQ 788, 10−5 M [Davenport, 2002]). According the correspondent protocol, agonists (ET-1, ET-3 or IRL 1620, 10−7 M (Davenport, 2002) (40 μl) or buffer (for control samples) were added, and the tissues were stimulated for 60 min at 37 °C. The incubation was terminated by addition of 100 μl of ice-cold 30% trichloroacetic acid (final concentration 6%) and the samples were transferred to ice. Tissue was sonicated on ice and centrifuged at 10.000g for 15 min at 4 °C. The whole supernatant was used for analysis of inositol-1-phosphate by Dowex anion exchange chromatography (AG 1-X8, 100-200 mesh formate form, BioRad Laboratory, Hercules, CA). Fractions (5 ml) containing InsP1 were collected and counted for radioactivity. To estimate the incorporation of radioactivity into phospholipids, membrane-associated radioactivity was extracted with chloroform–methanol and counted for radioactivity. For individual tissues, the amount of InsP1 was calculated as percentage of the radioactivity originally present in the membrane (InsP1/InsP1+LIPIDS).