Intracerebroventricular administration of anti-endothelin-1 IgG selectively upregulates endothelin-A and κ opioid receptors

Neuroscience 129 (2004) 751–756

INTRACEREBROVENTRICULAR ADMINISTRATION OF ANTIENDOTHELIN-1 IgG SELECTIVELY UPREGULATES ENDOTHELIN-A AND ␬ OPIOID RECEPTORS X. WANG, H. XU AND R. B. ROTHMAN*

The global distribution of ET and its binding sites in the brain suggests that, in addition to being a vasoconstrictor, it may be acting as an important neuropeptide in the CNS. For example, some studies (Raffa et al., 1991, 1996) characterize ET-1-induced nociception, and indicate that this effect can be reversed by central administration of morphine. Consistent with these findings, Piovezan et al. (2004) reported that ETs contribute toward nociception induced by antigen in mice, and that an ET-A receptor antagonist attenuates tactile allodynia in a diabetic rat model of neuropathic pain (Jarvis et al., 2000). More recent studies also suggest that ET-A receptor antagonists can enhance morphine antinociception and restore morphine analgesia in morphine tolerant rats (Bhalla et al., 2002, 2003). Supporting this idea, opioids inhibit ET-mediated DNA synthesis in rat C6 glioma cells (Barg et al., 1994). Viewed collectively, these studies suggest that central ET and opioid systems interact at a functional level. Recent work in our laboratory highlights the possibility that substances in the cerebrospinal fluid (CSF) mediate regulatory functions in the CNS (Goodman et al., 1998, 1999; Rothman et al., 2002, 2003). In these studies, we administered IgG directed against various neuropeptides via the i.c.v. route and documented changes in the expression of various receptors in rat brain. Thus, in the present study, we hypothesized that endogenous ET-1 will regulate components of the endogenous opioid system.

Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, P.O. Box 5180, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA

Abstract—Endothelin (ET) type A receptor antagonists enhance morphine-induced antinociception and restore morphine analgesia in morphine tolerant rats [Peptides 23 (2002) 1837; Peptides 24 (2003) 553]. These studies suggest that the central ET and opioid systems functionally interact. To explore this idea further, we determined the effect of i.c.v. administration of anti-ET-1 IgG (rabbit) on brain opioid receptor and ET receptor expression. Three days after implanting cannula into the lateral ventricle, male Sprague–Dawley rats were administered 10 ␮l (i.c.v.) of either control rabbit IgG (2.5 ␮g/ ␮l) or anti-ET IgG (2.5 ␮g/␮l) on day 1, day 3, and day 5. On day 6, animals were killed and the caudate and hippocampus collected. Anti-ET IgG had no significant effect on expression, measured by Western blots, of ␮, ␦ or ET-B receptors, but increased ␬ opioid (59%) and ET-A (33%) receptor protein expression in the caudate. [35S]-GTP-␥-S binding assays demonstrated that anti-ET IgG decreased [D-Ala2-MePhe4, Gly-ol5]enkephalin efficacy, but not potency in the caudate. Control experiments showed that there was no detectable rabbit IgG in caudate and hippocampal samples. These results suggest that ET in the CSF negatively regulates ␬ opioid and ET-A receptors in certain brain regions. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between ET and the opioid receptor system. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

EXPERIMENTAL PROCEDURES Key words: opioid receptor, endothelin, endothelin receptor, cerebrospinal fluid, neuropeptide.

Animals Male Sprague–Dawley rats weighing 300 –350 g were singly housed (lights on: 07:00 –19:00 h) with food and water freely available. Rats were maintained in facilities accredited by the American Association of the Accreditation of Laboratory Animal Care, and the procedures described herein were carried out in accordance with the Animal Care and Use Committee of the National Institute on Drug Abuse (NIDA) Intramural Research Program. We used the minimum number of animals needed to conduct these experiments and took all appropriate steps to minimize the suffering and/or discomfort of the animals. Animals were anesthetized using sodium pentobarbital (60 mg/ kg, i.p.). Rats were placed into a stereotaxic apparatus, and a craniotomy was performed. A small hole was drilled through the skull using the following coordinates, anterior/posterior, 0.9 mm and medial/lateral, 1.5 mm relative to bregma according to the coordinates of Paxinos and Watson 1982). A 52 mm cannula made from PE-20 tubing was inserted into this opening and slowly lowered into the brain, so that the tip of the cannula resided within the lateral ventricle. Three small holes were also drilled into the skull for placement of three anchor screws (size 0 – 80⫻1/8 inch length). A dental cement

The endothelins comprise a family of three 21-amino acid peptides, each encoded by separate genes. As described in several review articles (Anggard et al., 1990; van den Buuse and Webber, 2000), endothelins (termed endothelin-1 [ET-1], ET-2 and ET-3) are widely distributed in the brain and peripheral organs, and interact with two G-protein-linked receptors in mammals (Held et al., 1998), termed the ET-A and ET-B receptors (Sakurai et al., 1992). The brain expresses mostly ET-B receptors (van den Buuse and Webber, 2000), with much lower amounts of ET-A on both neuronal and non-neuronal cell types (Held et al., 1998). *Corresponding author. Tel: ⫹1-410-550-1487; fax: ⫹1-410-5502997. E-mail address: [email protected] (R. B. Rothman). Abbreviations: CSF, cerebrospinal fluid; DAMGO, [D-Ala2-MePhe4, Gly-ol5]enkephalin; ET, endothelin; NIDA, National Institute on Drug Abuse.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.09.004

751

752

X. Wang et al. / Neuroscience 129 (2004) 751–756

Fig. 1. Western blots of opioid receptor expression in control and anti-ET-1 IgG-treated rat brain. Each value is the mean⫾S.D. (n⫽3). * P⬍0.05 when compared with control.

stage was then constructed around each cannula and allowed to harden, thereby securing the cannula in place. Starting 3 days after implanting cannula, rats received i.c.v. injections (10 ␮l) of either control rabbit IgG (2.5 ␮g/␮l) or rabbit anti-ET-1 IgG (2.5 ␮g/␮l) on day 1, day 3, and day 5. On the day 6, animals were killed. The brains were collected and placed on an ice-cold glass plate for dissection: the cerebellum is removed and the brain is divided into two halves by cutting the corpus callosum and brain stem with a glass manipulator. The cortex is then peeled back to reveal the hippocampus, which is then gently removed using glass manipulators. The caudate nucleus is then gently teased out. The brain parts are then quickly frozen on dry ice and stored at ⫺80 °C. The 2.5 ␮g/␮l dose was chosen on the basis of previous experience with this experimental paradigm (Goodman et al., 1998, 1999; Rothman et al., 2002, 2003).

Western blot analysis Tissues were homogenized by sonication in cold RIPA buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mg/ml aprotinin, 1 mM sodium orthovanadate in PBS buffer, pH 7.4). Following protein measurements using the Pierce BCA Protein Assay Reagent Kit (Rockford, IL, USA), the homogenates were diluted to a concentration of 2 mg/ml with 2⫻ SDS-PAGE loading buffer (Invitrogen, Carlsbad, CA, USA). Samples were then boiled for 8 min. Sample protein

(30 ␮g/lane) was separated on 8 –16% polyacrylamide minigels (Invitrogen). Proteins separated by electrophoresis were transferred to Immobilon-PVDF membranes (Millipore Corporation, Bedford, MA, USA) using a semi-dry apparatus (Bio-Rad, Hercules, CA, USA). Membranes were incubated for 60 min at 25 °C in PBS solution containing 5% nonfat dry milk to reduce nonspecific binding. Membranes were then probed by overnight incubation with antibodies to: ␮, ␦ and ␬ opioid receptors (1: 1000 dilution; Calbiochem, San Diego, CA, USA), and ET-A and ET-B receptors (1:200 dilution; Calbiochem). In other control experiments, membranes were probed with mouse monoclonal anti-rabbit IgG (1:200 dilution; Sigma Chemical, St. Louis, MO, USA) to detect the presence of rabbit IgG. The membranes were then rinsed three times with PBS, and then incubated with 1:4000 dilution of horseradish peroxidase-labeled secondary antibody in PBS solution, containing 0.25% nonfat dry milk for 60 min at room temperature. After washing three more times, antibody complex were visualized by chemiluminescence using a kit from Pierce Biotechnology.

Radioimmunoassay All dissected tissues were immediately frozen on dry ice and stored at ⫺80 °C until peptide extraction. Samples were heated at 95 °C for 10 min in 2 M acetic acid, cooled on ice, homogenized

Fig. 2. Effects of chronic i.c.v. administration of anti-ET-1 IgG on agonist-stimulated [35S]GTP-␥-S binding. Each value is the mean⫾S.D. (n⫽3).

X. Wang et al. / Neuroscience 129 (2004) 751–756 Table 1. Opioid agonist-stimulated [35S]-GTP-␥-S bindinga Agonist

DAMGO Control IgG Anti-ET IgG SNC80 Control IgG Anti-ET IgG

EC50 (nM⫾SD)

Table 2. Effects of chronic i.c.v. administration of anti-ET IgG on dynorphin levels in rat caudate and hippocampusa

Emax (% of maximal stimulation) Control IgG Anti-ET IgG

266⫾44 310⫾64

149⫾6 121⫾6*

109⫾22 124⫾24

114⫾5 123⫾5

a Rats received i.c.v. injections of either control rabbit IgG or anti-ET IgG (2.5 ␮g/␮l in 10 ␮l) on days 1, 3 and 5. [35S]-GTP-␥-S binding assays with rat caudate membranes were conducted as described in Experimental Procedures. Each value is the mean⫾S.D. from three experiments, with triplicate determinations. * P⬍0.05 when compared to control.

by sonication (30 s) and centrifuged at 12,000⫻g for 15 min. The supernatants were lyophilized, diluted in 0.5 ml of RIA buffer and kept at ⫺20 °C until analysis. Dynorphin A(1–17) was determined using a commercially available radio-immunoassay kit (Peninsula Laboratories, Belmont, CA, USA). Briefly, samples were reconstituted in phosphate RIA buffer and a standard curve was prepared. All samples and standards were incubated with primary antibody (rabbit antipeptide serum) for 16 –24 h at 4 °C. The following day 125Idynophin A(1–17) was also added and incubated for an additional 16 –24 h at 4 °C. At the end of the incubation period (day 3), SPA beads coated with anti-rabbit secondary antibody (Amersham, Piscataway, NJ, USA) were added at the manufacturer’s recommended concentration and incubated with gentle agitation overnight and counted the next day in a Trilux liquid scintillation counter (PerkinElmer, Torrance, CA, USA).

753

Caudate, pg/mg

Hippocampus, pg/mg

N

1.12⫾0.17 1.44⫾0.13*

1.21⫾0.18 1.20⫾0.15

6 6

a Rats received three i.c.v. injections of either control rabbit IgG or anti-ET IgG (2.5 ␮g/␮l in 10 ␮l) on days 1, 3 and 5. RIA assays were conducted as described in Experimental Procedures. * P⬍0.05 when compared to control.

[35S]GTP-␥-S functional assays [35S]GTP-␥-S binding was determined as described previously (Xu et al., 2001). Rat caudate membranes (10 ␮g) were suspended in 500 ␮l of buffer containing 50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 100 ␮M GDP, 0.1% BSA, 0.05 nM [35S]GTP-␥-S, and varying concentration of test drugs. The reaction was initiated by the addition of membranes and terminated after 3 h by the addition of 3 ml of cold (4 °C) 10 mM Tris–HCl, pH 7.4, followed by rapid vacuum filtration through Whatman GF/B filters. The filters were then washed twice with 4 ml of cold 10 mM Tris–HCl, pH 7.4. Bound radioactivity was counted using a Trilux liquid scintillation counter at 60% efficiency. Nonspecific binding was determined in the presence of 40 ␮M GTP-␥-S. Assays were performed in triplicate and each experiment was performed three times. The EC50 (the concentration of agonist that produces 50% maximal stimulation) and Emax (percent of maximal stimulation) were determined using the program MLAB-PC (Civilized Software, Bethesda, MD, USA). The results are mean⫾S.D.

Chemicals [D-Ala2-MePhe4, Gly-ol5]enkephalin (DAMGO) was provided by Multiple Peptide Systems: San Diego, CA, USA, via the Re-

Fig. 3. Western blots of ET receptor expression in control and anti-ET-1 IgG-treated rat brain. Each value is the mean⫾S.D. (n⫽3). * P⬍0.05 when compared with control.

754

X. Wang et al. / Neuroscience 129 (2004) 751–756

Fig. 4. Western blots of various concentrations of rabbit IgG were run along with rat brain samples prepared from caudate, hippocampus and cortex of rats that had received i.c.v. injections of control rabbit IgG as described in Experimental Procedures. The brain samples had no detectable rabbit IgG. Similar results were obtained in a second experiment.

search Technology Branch, NIDA. SNC80 was provided by Dr. Kenner Rice, Laboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda, MD, USA. Anti-ET-1 peptide IgG was purchased from Peninsula Laboratories. This antibody is raised in rabbits and is affinity purified. As noted in the product specification sheet, Anti-ET-1 peptide IgG is highly selective for rat ET-1. Rabbit IgG and anti-rabbit IgG were purchased from Sigma. Horseradish peroxidase-labeled secondary antibody was purchased from Amersham Corporation. Antibodies directed against ␮, ␦ and ␬ opioid receptors and ET-A and ET-B receptors were obtained from Calbiochem.

RESULTS As reported in Fig. 1, administration of anti-ET IgG to rats increased expression of ␬ opioid receptors by 59% in the caudate, but not the hippocampus. Anti-ET IgG did not alter expression of ␮ or ␦ receptors in either brain region. To determine the effect of anti-ET IgG on the functional effects of opioid receptor activation, we determined the ability of agonists to stimulate [35S]GTP-␥-S binding. As reported in Table 1 and Fig. 2, anti-ET IgG decreased the efficacy of DAMGO-stimulated [35S]GTP-␥-S binding without significantly altering the EC50. Anti-ET IgG has no effect on SNC80-stimulated [35S]GTP-␥-S binding. ␬ Receptor-stimulated [35S]GTP-␥-S binding was not tested

because the effect is too small to be detected in rat brain. Anti-ET IgG increased caudate dynorphin levels by 28% without altering dynorphin levels in the hippocampus (Table 2). Anti-ET IgG increased expression of ET-A receptors in the caudate by 33%, but not in the hippocampus, and had no effect on ET-B receptor expression (Fig. 3). To determine if the administered IgG distributes to brain tissue, we attempted to measure rabbit IgG in the brain samples. Although rabbit IgG was detectable at a level of 5 ng per sample, there was no detectable rabbit IgG in the brain homogenates (Fig. 4).

DISCUSSION The brain is abundantly endowed with a wide variety of neuropeptides that function as neurotransmitters and neuromodulators. Because of the communication between the extracellular fluid bathing neurons and the CSF, the CSF will contain almost any substance produced by the brain, including neuropeptides. The CSF, therefore, provides a route for excretion of substances as well as a means to deliver substances to brain regions distant from their site of production (Amin-Hanjani and Chapman, 2001).

X. Wang et al. / Neuroscience 129 (2004) 751–756

For several years, we have tested the hypothesis that neuropeptides in the CSF mediate regulatory functions in the CNS. We showed, for example, that chronic administration of IgG directed against neuropeptide-FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) up-regulated ␮ opioid receptors (Goodman et al., 1998), and that chronic administration of IgG directed against various opioid peptides regulated the density of ␦ opioid receptors (Goodman et al., 1999). More recently, we reported that administration of IgG directed against corticotropin releasing factor up-regulated ␮ opioid receptors and ␤-adrenergic receptors (Rothman et al., 2002), and that administration of anti-CART peptide IgG (the peptide product of cocaine amphetamine-related transcript) regulated the expression of ␮ opioid and 5-HT2A receptors (Rothman et al., 2003). To further test the hypothesis that neuropeptides in the CSF mediate regulatory functions in the CNS, we hypothesized, based on studies reviewed in the introduction, that extracellular ET will regulate components of the endogenous opioid system. To test this hypothesis, we administered (i.c.v.) IgG directed against ET-I. With this experimental method, one can infer the actions of endogenous ET as being the opposite observed with administration of anti-ET IgG. The results demonstrated that anti-ET IgG regulates, in rat caudate, expression of ␬ opioid receptors, dynorphin levels, and ET-A receptors as well as regulating ␮ receptor coupling to G proteins. These observations suggest that endogenous ET, in the extracellular fluid, down-regulates the ␬ opioid system, both by decreased expression of the ␬ opioid receptor and by decreased dynorphin levels, down-regulates ET-A receptors, and increases the efficacy of ␮ agonists. It is relevant to note that the ␬ and ␮ opioid systems often have opposing actions, and the ␬ system acts as an anti-␮ system (Rothman, 1992). A model to explain these findings is that ET produces nociception while at the same time altering neuronal systems in a direction to oppose this action: down-regulating the ␬ opioid system (receptors and dynorphin levels), increased ␮-receptor efficacy and decreased ET-A receptors. An important question is if rabbit IgG distributes into brain tissue following administration into the lateral ventricle and exerts its effects locally, or if it exerts its effects solely by actions in the CSF. Some studies indicate that IgG, when administered into the lateral ventricle, has limited distribution into brain tissue (Moos, 2003). The data reported in Fig. 4 support this finding, since we were unable to detect rabbit IgG in brain homogenates. Thus, the simplest interpretation of our results is that the administered IgG decreases the steady state concentration of the target neuropeptide in the CSF, and by extension, the extracellular fluid bathing neurons. This interpretation implies that the differential effect anti-ET IgG on the caudate and hippocampus does not result from differential penetration of the IgG into the tissue, but more likely from the lower levels of ET-B receptors in rat hippocampus as compared with caudate (Kohzuki et al., 1991).

755

In summary, the results strongly suggest that endogenous ET, circulating in the extracellular fluid of the brain, regulates both the ET and opioid systems, emphasizing both the functional interactions between these two neuropeptide systems, as well as the more general role that CSF neuropeptides play as regulatory substances.

REFERENCES Amin-Hanjani S, Chapman PH (2001) Cerebrospinal fluid and its abnormalities. Encyclopedia of Life Sciences, London: Nature Publishing Group. Anggard EE, Botting RM, Vane JR (1990) Endothelins. Blood Vessels 27:269 –281. Barg J, Belcheva MM, Zimlichman R, Levy R, Saya D, McHale RJ, Johnson FE, Coscia CJ, Vogel Z (1994) Opioids inhibit endothelinmediated DNA synthesis, phosphoinositide turnover, and Ca2⫹ mobilization in rat C6 glioma cells. J Neurosci 14:5858 –5864. Bhalla S, Matwyshyn G, Gulati A (2002) Potentiation of morphine analgesia by BQ123, an endothelin antagonist. Peptides 23:1837–1845. Bhalla S, Matwyshyn G, Gulati A (2003) Endothelin receptor antagonists restore morphine analgesia in morphine tolerant rats. Peptides 24:553–561. Goodman CB, Heyliger S, Emilien B, Partilla JS, Yang HY, Lee CH, Cadet JL, Rothman RB (1998) Regulation of mu binding sites after chronic administration of antibodies directed against specific antiopiate peptides. Peptides 19:1703–1709. Goodman CB, Heyliger S, Emilien B, Partilla JS, Yang HY, Lee CH, Cadet JL, Rothman RB (1999) Chronic exposure to antibodies directed against anti-opiate peptides alter delta-opioid receptor levels. Peptides 20:1419 –1424. Held B, Pocock JM, Pearson HA (1998) Endothelin-1 inhibits voltagesensitive Ca2⫹ channels in cultured rat cerebellar granule neurones via the ET-A receptor. Pflugers Arch 436:766 –775. Jarvis MF, Wessale JL, Zhu CZ, Lynch JJ, Dayton BD, Calzadilla SV, Padley RJ, Opgenorth TJ, Kowaluk EA (2000) ABT-627, an endothelin ET(A) receptor-selective antagonist, attenuates tactile allodynia in a diabetic rat model of neuropathic pain. Eur J Pharmacol 388:29 –35. Kohzuki M, Chai SY, Paxinos G, Karavas A, Casley DJ, Johnston CI, Mendelsohn FA (1991) Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography. Neuroscience 42:245–260. Moos T (2003) Delivery of transferrin and immunoglobulins to the ventricular system of the rat. Front Biosci 8:a102–109. Paxinos G (1982) The rat brain in stereotaxic coordinates. New York: Academic Press. Piovezan AP, D’Orleans-Juste P, Frighetto M, Souza GE, Henriques MG, Rae GA (2004) Endothelins contribute towards nociception induced by antigen in ovalbumin-sensitised mice. Br J Pharmacol 141:755–763. Raffa RB, Schupsky JJ, Lee DK, Jacoby HI (1996) Characterization of endothelin-induced nociception in mice: evidence for a mechanistically distinct analgesic model. J Pharmacol Exp Ther 278:1–7. Raffa RB, Schupsky JJ, Martinez RP, Jacoby HI (1991) Endothelin-1induced nociception. Life Sci 49:PL61– 65 Rothman RB (1992) A review of the role of anti-opioid peptides in morphine tolerance and dependence. Synapse 12:129 –138. Rothman RB, Vu N, Wang X, Xu H (2003) Endogenous CART peptide regulates mu opioid and serotonin 5-HT(2A) receptors. Peptides 24:413– 417. Rothman RB, Vu N, Xu H, Baumann MH, Lu YF (2002) Endogenous corticotropin releasing factor regulates adrenergic and opioid receptors. Peptides 12:2177–2180. Sakurai T, Yanagisawa M, Masaki T (1992) Molecular characterization of endothelin receptors. Trends Pharmacol Sci 13:103–108. van den Buuse M, Webber KM (2000) Endothelin and dopamine release. Prog Neurobiol 60:385– 405.

756

X. Wang et al. / Neuroscience 129 (2004) 751–756

Xu H, Hashimoto A, Rice KC, Jacobson AE, Thomas JB, Carroll FI, Lai J, Rothman RB (2001) Opioid peptide receptor studies: 14. Stere-

ochemistry determines agonist efficacy and intrinsic efficacy in the [35S]GTP-gamma-S functional binding assay. Synapse 39:64 – 69.

(Accepted 1 September 2004) (Available online 18 October 2004)