A Role for Bombesin in Sensory Processing in the Spinal Cord1

Bombesin (BN)-containing neuronal processes were demonstrated in laminae I and II of the dorsal horn of the cat, rat, and mouse spinal cord by immunocytochemistry and radioimmunoassay. Dorsal rhizotomy in the cat resulted in a marked decrease in BN immunoreactivity in the dorsal horn indicating that BN is contained in primary sensory afferents. BN-binding sites were also localized in superficial laminae of the dorsal horn. The presence of both BN and BN-binding sites in the dorsal horn suggested that BN may be involved in sensory processing in the spinal cord. Consistent with this hypothesis, it was demonstrated that an injection of BN into the spinal cord caused a biting and scratching response indicative of sensory stimulation. The effect was similar to that observed after injection of substance P into the cord with the exception that the BN effect lasted about 100 times longer than that induced by substance P. Taken together, these data indicate that BN may be a neurotransmitter of primary sensory afferents to the spinal cord. Bombesin (BN) is a tetradecapeptide amide originally isolated and sequenced from the skin of the frog Bombina bombina. More recently, however, BN immunoreactivity has been identified in the mammalian central nervous system (Brown et al., 1978; Moody and Pert, 1979; Walsh et al., 1979; Moody et al., 1980, 1981a, c; Roth et al., 1982), sympathetic nervous system (Schultzberg, 1983), gastrointestinal tract (Polak et al., 1976; Dockray et al., 1979; Walsh et al., 1979; Buffa et al., 1982; Furness et al., 1982), human fetal lung (Wharton et al., 1978; Track and Cutz, 1982; Tsutsumi et al., 1983), adult lung (Cutz et al., 1981; Polak and Bloom, 1982; Track and Cutz, 1982; Tsutsumi et al., 1983), and oat cell carcinoma of the lung (Wood et al., 1981, Erisman et al., 1982; Sorensen et al., 1982; Moody et al., 1981b, 1983; Tsutsumi et al., 1983). The precise structure of mammalian BN is currently unknown, but it has been suggested that the mammalian form of BN may be gastrinreleasing peptide (GRP), a recently identified peptide that has structural and biological similarities to BN (McDonald et al., 1979,1983; Brown et al., 1980; Erspamer et al., 1981; Yanaihara i We thank Drs. F. Cuttitta and J. Minna at NC1 for providing the bombesin antiserum and Dr. Robert T. Jensen for helpful comments on an early draft of this manuscript. We also thank Mr. John F. Bishop for expert technical assistance, Ms. Donna Young for secretarial aid, and Mr. Earl Henderson and Mrs. Muriel Branford for assistance with surgical procedures. This research was supported in part by Grant BN27923451 from the National Science Foundation to V. J. M. and Grant NS17073 from the National Institutes of Health to T. W. M. * To whom correspondence should be addressed. et al., 1981), or neuromedin B, a recently identified BN-like peptide (Minamino et al., 1983). Although the function of BN in anurans is largely unknown, a number of pharmacological actions have been identified in mammals. Peripheral actions of BN include stimulatory effects on gut and uterine smooth muscle contraction (Bertaccini et al., 1974a, b; Caprilli et al., 1975; Barthio et al., 1982; Davison, 1983), stimulation of release of gastrin and cholecystokinin from gut (Bertacinni et al., 1974a, b; Erspamer et al., 1974; Hirschowitz and Molina, 1983), growth hormone from the pituitary gland (Bicknell and Chapman, 1983), amylase and trypsinogen from the pancreas (Jensen et al., 1978; Andriulli et al., 1983), and also induction of tumor growth (Rosengurt and Sinnett-Smith, 1983). Central pharmacological actions of BN which have been reported are modulation of anterior pituitary hormone release (Rivier et al., 1978; Tache et al., 1979; Westendorf and Schonbrunn, 1982); inhibition of gastric acid release and stimulation of gastric mucus secretion (Tache et al., 1981; Tache, 1982; Tache and Collu, 1982); stimulation of neuronal electrical activity (Phillis and Limacher, 1974; Phillis and Kirkpatrick, 1979; Suzue et al., 1981; Tartara et al., 1982); stimulation of sympathetic outflow (Tache and Brown, 1982); and induction of analgesia (Pert et al., 1980), hyperglycemia (Brown and Vale, 1976; Brown et al., 1979), poikilothermia (Brown et al., 1977; Pittman et al., 1980; Tache et al., 1980), and a stereotypic scratching behavior (Brown et al., 1977). BN may also induce a number of pharmacological effects by both peripheral and central actions. The best examples of this are the central and peripheral actions of BN on induction of satiety (Gibbs et al., 1979; Martin and Gibbs, 1980; Baile and DellaThe Journal of Neuroscience Bombesin and Sensory Processing in the Spinal Cord 2957 Ferra, 1981; Gibbs and Fauser, 1981; Morley and Levine, 1981; Smith et al., 1981; Stein and Woods, 1981; Kulkosky et al., 1982; West et al., 1982; Hsaio and Spencer, 1983) and gastric emptying (Tepperman and Evered, 1980; Tache and Brown, 1982; Hsaio and Spencer, 1983). Recent results have demonstrated that, within the central nervous system, the dorsal horn of the spinal cord has relatively high concentrations of BN-immunoreactive peptides (Moody et al., 1981c; Massari et al., 1983). The fact that some of the BN in the dorsal horn is derived from primary sensory afferents (Fuxe et al., 1983; Massari et al., 1983; Panula et al., 1983) suggested the possibility that BN may play a role in the transmission of sensory information to the central nervous system. Alternatively, other sources of BN to the dorsal horn might modulate neural transmission of primary sensory afferents. In the present study, we investigated the physiology and anatomy of BN and BN receptors in the spinal cord. It was found that BN and BN receptors were localized in superficial laminae of the dorsal horn of the spinal cord and that microinjection of BN into the cord induced a vigorous scratching response indicative of sensory stimulation. Materials and Methods Dorsal rhizotomy. Dorsal rhizotomy was performed 10 days before cats were sacrificed, essentially as described previously (Massari et al., 1983). Three cats were anesthetized with pentobarbital (35 mg/kg) and operated on using sterile procedures. The dorsal roots of Ll through S3 were exposed by performing a hemilaminectomy on the right lumbosacral vertebrae. The dorsal roots were cut extradurally using a small pair of dissecting scissors under a dissecting microscope, taking care to leave the dorsal radicular arteries and ventral root intact. The area was then gently packed in Gelfoam and sutured in layers. All cats received 600,000 units of Bicillin, injected intramuscularly, postoperatively. Radioimmunoassay. Spinal cords from unoperated cats, rats, and mice were removed and frozen on dry ice and stored at -70°C. Cervical spinal cord slices of 300 to 500 pm in thickness were cut in a cryostat at -10°C. Sections were then microdissected as described previously (Moody et al., 1981a, c; Massari et al., 1983). Samples were dissected into 2 N acetic acid on ice. The acidified tissues were boiled for 10 min and homogenized by sonication using a Kontes cell disrupter. An aliquot was removed for protein determination (Lowry et al., 1951). BN immunoreactivity was measured as described previously (Moody et al., 1983). Homogenates were centrifuged at 10,000 x g for 10 min, and the supernatants were removed and dried by vacuum centrifugation (Savant Inc, Hicksville, NY). The samples were resuspended in radioimmunoassay (RIA) buffer which consisted of phosphate-buffered saline (PBS), pH 7.4, containing 0.25% bovine serum albumin. A rabbit anti-BN serum at l:lOO,OOO dilution was incubated with sample or standard for 1 hr at 4°C. Then 5,000 cpm (about 4 fmol) of [iz51-Tyr4] BN were added, and the solution was incubated for 16 hr at 4°C; the total volume was 0.4 ml. After incubation, 200 ~1 of normal rabbit serum were added at 1:200 dilution followed by 100 ~1 of goat antirabbit serum at 1:20 dilution. After 30 min, 200 ~1 of 12% polyethylene glycol were added to enhance precipitation, and the samples were centrifuged at 1,000 x g for 20 min. The supernatant was removed and the pellet was counted for radioactivity in an LKB gamma counter (LKB Instruments, Inc., Gaithersburg, MD). Immunocytochemistry. Rats and mice were anesthetized and perfused through the ascending aorta with ice-cold PBS containing 0.5% sodium nitrite followed by 200 to 300 ml of ice cold 4% formaldehyde in PBS, pH 7.4. Normal and operated cats were anesthetized and perfused through the abdominal aorta according to the method of Loren et al. (1980) using 2% glyoxylic acid in the preperfusion buffer and 2% paraformaldehyde in the perfusion buffer. The spinal cords were rapidly removed, cut into 3.0-mm sections, and postfixed for 30 min in the same fixative. The tissue slices were rinsed for 48hr in PBS containg 20% sucrose (w/v), frozen on dry ice, cut into 20.pm coronal sections in a cryostat, mounted on chrome-alum-coated slides, and processed for the indirect immunohistochemical procedure of Coons (1958). The antibody used was a monoclonal antibody generated in mice. Sections were incubated overnight in the BN antibody diluted 1:lOOO in PBS containing 0.3% Triton X-100 (v/v). The sections were washed three times for 5 min each in PBS with 0.3% Triton X-100 and then incubated for 30 min in fluorescein isothiocyanate-conjugated goat anti-mouse IgG (N. L. Cappel Laboratories, Cochranville, PA), diluted 1:300 in PBS with 0.3% Triton X-100. The sections were washed three times for 5 min each, rinsed in PBS, and mounted in Gelvatol (Monsanto Chemical Co., Indian Orchard, MA). Sections were examined under a Leitz Orthoplan fluorescence microscope equipped with a Ploem illuminator. Preabsorption of BN antisera overnight with synthetic BN eliminated all staining, whereas preabsorption with substance P (SP) had no effect (peptides from Peninsula Laboratories, San Carlos, CA). Autoradiography. Frozen, unfixed spinal cords from rats, cats, and mice were cut into 20-Km sections in a cryostat at -20°C and mounted on chrome-alum-coated slides. BN autoradiography was performed as described nrevouslv (Wolf et al.. 1983). Brieflv. slides were incubated in a buffercomposed of 10 mM HEPES, pH 7.4; 130 mM NaCl, 4.7 mM KCl, 5 mM MgClx, 1 mM EGTA, 0.1% bovine serum albumin, 0.07 mM bacitracin, and 3 nM [iZ51-Tyr4]BN in the presence or absence of 1 pM unlabeled BN. Incubations were performed for 60 min at 22”C, followed by two consecutive 4-min washes in buffer at 4°C. Slices then were either counted by scraping into test tubes and assaying radioactivity in a Micromedic gamma counter or prepared for autoradiography using tritium-sensitive film (Ultrofilm, LKB) as described previously (Quirion et al., 1983). The exposure time was 48 hr. After this exposure, films were processed in Kodak D19 at 22°C for 4 min and then fixed for 5 min. Intrathecal injections and behavioral analysis. Intrathecal injections (5 ~1) into the lumbar region of unanesthetized male CD1 mice (20 to 25 gm) and analysis of the biting and scratching response were performed as described previously (Hylden and Wilcox, 1981). BN, GRP, or SP (100 pmol; Peninsula Laboratories) or vehicle (0.01 N acetic acid in saline, pH 3.5) at a concentration of 100 pmol/5 ~1 was injected and the number of biting or scratching responses per period was counted. A response was defined as either the nose touching the abdomen or the hindlimb touching the body. The behavioral response was analyzed both quantitatively and qualitatively.