Neurochemical Research, Vol. 24, No. 10, 1999, pp. 1203-1207
Multiphasic Morphine Modulation of Substance P Release from Capsaicin-Sensitive Primary Afferent Fibers Georgina Cano,2,4 Jose Luis Arcaya,2 Gerber Gomez,1 William Maixner,3 and Heberto Suarez-Roca1,5 (Accepted April 21, 1999)
Morphine produces a multiphasic modulation of K+-evoked substance P release from trigeminal slices and dorsal root ganglion neurons in culture. We now found that the C-fiber stimulant, capsaicin (1 UM), evoked release of substance P that was inhibited, enhanced and inhibited by 0.1 nM, 1 UM, and 10 UM morphine, respectively. This morphine's multiphasic effect was blocked by naloxone (100 nM). Neonatal treatment with capsaicin produced thermal hypoalgesia and abolished the multiphasic effect of morphine on substance P release evoked by 50 mM K+. These findings suggest that the multiphasic modulation of substance P release by morphine is dependent on C-type afferents and may be of relevance to nociception. KEY WORDS: Substance P; capsaicin; primary afferents; release; naloxone; morphine; trigeminal nucleus.
INTRODUCTION
by different types of stimuli (3-6). We have previously reported that a wide range of morphine concentrations (5 log units) produce a concentration-dependent multiphasic modulation (inhibitory and facilitatory) of K+evoked substance P release from trigeminal nucleus slices (7). Using subtype-selective opioid receptor antagonists and agonists, we showed that the multiphasic effects of morphine are likely due to the sequential activation of inhibitory and excitatory U, inhibitory 8 and excitatory K opioid receptors (7-9). A limitation in these studies relates to the use of K+ to depolarize the slices since K+ releases substance P from at least three sources: primary afferents, intrinsic neurons, and descending fibers (10,11). Thus, we were not able to determine which source of substance P was modulated by the various opioid receptor agonists. We now evaluated whether morphine alters the release of substance P evoked by capsaicin, a neurotoxin that selectively stimulates primary afferent C-fibers (12). In addition, we examined the ability of morphine to produce a multiphasic effect on K+-evoked substance P release from trigeminal nucleus caudalis slices obtained from rats neonatally treated with capsaicin. This treatment with
The release of substance P has been postulated to be involved in the transmission of nociceptive information by primary afferent C-fibers, from the periphery to the dorsal spinal cord and trigeminal nucleus caudalis (1,2). The release of substance P from these primary afferents is under the inhibitory control of presynaptic opioid receptors. Indeed, opioid analgesics, such as morphine, have been found to inhibit the in vitro and in vivo release of substance P from rat dorsal spinal cord and trigeminal nucleus caudalis evoked Sec. of Pharmacology, Instituto de Investigaciones Clinicas, Univ. del Zulia. 2 INBIOMED, Maracaibo, Apartado 1151, Venezuela: 3 Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455. 4 Current address: *Dept. of Neuroscience, 446 Crawford Hall, Univ. of Pittsburgh, Pittsburgh, PA 15260. 5 Heberto Suarez-Roca, M.D., Ph.D., University of Zulia, Instituto de Investigaciones Clinicas, Section of Pharmacology, Apartado Postal 1151, Maracaibo 4001-A, Venezuela. Phone: (58)(61)-931859, Fax: (58)(61)-597248, E-mail:
[email protected] or heberto_suarez@ hotmail.com 1
1203 0364-3190/99/1000-1203$16.00/0 © 1999 Plenum Publishing Corporation
1204 capsaicin is known to produce a relatively selective destruction of primary afferent C-fibers (11), allowing to evaluate the effect of morphine on substance P release from sources resistant to neonatal capsaicin, i.e., intrinsic neurons and descending fibers.
EXPERIMENTAL PROCEDURE Animals. Male Sprague-Dawley rats (Centro Biotecnologico IVIC, Caracas, Venezuela), weighing 150-300 grams, were handled according to the guidelines of the National Institute of Health (USA) for the care and use of laboratory animals. All efforts were made to reduce animal suffering and the number of animals used. Neonatal Treatment with Capsaicin. A group of 80 rats was treated neonatally with capsaicin as described by Nagy et al. (11). Briefly, 2-3 day old animals were injected subcutaneously, while under ether anesthesia, with capsaicin (25 or 50 mg/kg; 30-50 Ul). A second group of 40 animals was treated with the vehicle (10% Tween 80 and 10% ethanol v/v in 0.9% NaCl). The animals were used for the experiments 3-4 months after the pretreatment with capsaicin or its vehicle. A third group of about 80 naive rats were used for the experiments in which the modulatory effect of morphine on capsaicin-evoked substance P release was evaluated. Assessment of Thermal Nociception. Thermal nociception was assessed one day before the superfusion experiments by measuring the response latency (i.e., licking or jumping) to a hot plate maintained at 52.5 ± 0.1 °C. A 45-s response cutoff time was employed. Superfusion Experiments. Rats were killed by decapitation and the trigeminal nuclei were immediately dissected under cold (4°C) oxygenated superfusion medium as previously described (SuarezRoca et al. 1992). Twelve to 16 trigeminal slices (300 Um thick) from 3-4 rats were transferred to 0.1-ml superfusion chambers (Brandel, MD), which were submerged in a water bath at 37°C, and superfused at a flow rate of 0.35 ml/min with a medium containing: 118 mM NaCl, 4.8 mM KC1, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4, 10 mM glucose, 0.57 mM ascorbic acid, 20 UM bacitracin and 0.1% bovine serum albumin, gassed continuously with 95% 02/5% CO2 to maintain a pH of 7.4. Collection of the superfusate started after 30 min of superfusion with either drug-free medium or naloxone-containing buffer. Superfusate fractions (0.7 ml/ 2 min) were collected at 4°C and the samples were kept at -20°C until the radioimmunoassay was initiated. After 2 and 28 min of the beginning of collection, trigeminal slices from naive rats were exposed to two 2-min periods of stimulation: the first stimulation period (S1) was done with 50 mM K+ whereas the second stimulation period (S2) was carried out with 1 UM capsaicin. For rats neonatally treated with capsaicin or its vehicle, the stimulation protocol was similar to that of naive rats, except that in a set of experiments used to evaluate morphine effect, S2 was done with 50 mM K+ (instead of 1 UM capsaicin). In all the experiments, the modulatory effect of morphine on substance P release from trigeminal slices was examined by superfusing the opioid agonist, over a concentration range of 0.1 nM -30 UM, from 15 min before S2 to the end of the superfusion. SI served as an internal control for S2. The effect of each morphine concentration was evaluated in four to eight independent experiments, i.e., using different set of slices from distinct rats. In each superfusion experiment, two chambers served as controls in which SI and S2 were carried out in the absence of morphine. The effect of morphine (0.1 nM, 1 UM and 10 UM) was also examined in
Cano, Arcaya, Gomez, Maixner, and Suarez-Roca the presence of the opioid receptor antagonist, naloxone (100 nM) during S2. In two chambers, that served as controls for the antagonist experiments, naloxone was superfused from 30 min before collection until the end of the superfusion. Substance P Radioimmunoassay. At the completion of each experiment, substance P was extracted from slices and the amount of the peptide in each superfusate fraction and tissue extract was determined by radioimmunoassay as previously described (7). The assay sensitivity was typically 0.25 pg/100 Ul, and intra-assay and interassay variation were typically around 3% and 11%, respectively. Drugs, at the concentrations used in this study, did not interfere with the assay. Calculations and Statistical Analysis. The amount of substance P released was expressed as a percentage of substance P content in the tissue at the initiation of each stimulation (7). The effects of morphine was evaluated by expressing the results as ratios of the stimulationevoked release in the presence of morphine (S2) over that in the absence of morphine (S1). Results were expressed as a percentage of the S2/S1 ratios obtained from control (i.e., no morphine exposure) slices with control responses assigned a value of 100 %. Statistical evaluation of the S2/S1 ratios or the percentages respect to control were performed by either an analysis of variance (ANOVA) followed by post hoc analysis with Duncan's multiple range test or two-tailed student's t-test. The results of these analyses were corroborated with the non-parametric Kruskal-Wallis test. Significance was assumed at p < 0.05.
RESULTS Neonatal treatment with 50 mg/kg subcutaneous capsaicin, known to deplete substance P releasable pool located in primary afferents (11), almost completely abolished capsaicin-evoked substance P release (S2/S1 = 0.17 ± 0.08) compared to vehicle-treated animals (S2/S1 = 2.09 ± 0.27) (p < 0.01; Fig. 1 A). In contrast, neonatal treatment with capsaicin, that does not affect substance P releasable pool located in intrinsic neurons and descending fibers (11), did not significantly change K+-evoked substance P release (S2) corrected by either the first stimulation (S1) or substance P tissue content (neonatal capsaicin: S2/S1 = 1.41 ± 0.73 and S1 = 2.26 ± 0.25% tissue content; neonatal vehicle: S2/S1 = 1.09 ± 0.25 and S1 = 2.30 ± 0.16% tissue content) (Fig. 1B). Yet, the absolute substance P release peak (sum of fractions 3-5) evoked by the first stimulation with K+ (S1) was markedly reduced by the neonatal treatment with capsaicin (5.44 ± 0.41 pg) compared to controls (21.09 ± 2.72 pg) (p < 0.01, n = 8). Neonatal treatment with 25 and 50 mg/kg subcutaneous capsaicin also prolonged hot plate latency responses by about 82% (17.9 ± 1.3 s) and 148% (24.4 ± 1.6 s), respectively, compared to vehicle-treated rats (9.8 ± 0.7 s) (F2,93 = 25.53; p < 0.01). In addition, the neonatal treatment with 25 and 50 mg/kg capsaicin reduced substance P content in the trigeminal nucleus by
Morphine Effect on Substance P Release
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Fig. 2. Multiphasic effect of morphine on capsaicin-evoked substance P release from trigeminal nucleus slices of naive rats and its antagonism with naloxone. MOR: morphin e superfused alone; MOR + NAL: morphine superfused in the presence of 100 nM naloxone. The straight line represents 100% release of controls (no drug exposure). On the ordinate, substance P release is given as a percentage of control S2/S1 ratio (1.85 ± 0.51). The abscissa shows molar concentrations of morphine in the superfusate. Each point is the mean ± SEM of at least 4 independent experiments or set of slices. *denotes a significant difference compared to morphine alone (p < 0.05; twotailed t-test) and **denotes a significant difference compared to a 100% control (p < 0.01; one-way ANOVA followed by Duncan's test performed on the percent data). Fig. 1. Effect of neonatal treatment with 50 mg/kg capsaicin on stimulation-evoked substance P release from trigeminal nucleus slices. Substance P release was induced by 50 mM K+ (K+) during S1 and by 1 UM capsaicin (CAP) (Panel A) or 50 mM K+ (Panel B) during S2. On the ordinate, substance P release is given as a percentage of substance P content at the initiation of the stimulation. The abscissa shows fractions collected (0.7 ml/2 min). There was a 18-min void period between fraction 5 and 6. Each point represents the mean ± s.e.m. of at least 8 independent experiments. * denotes a significant difference compared to vehicle (p < 0.01; two-tailed t-test).
about 46% (1.74 ± 1.8 pg/mg wet tissue) and 67% (1.05 ± 1.6 pg/mg wet tissue), respectively, compared to vehicle-treated rats (3.17 ± 5.7 pg/mg wet tissue) (F 2 , 1 5 =9,63;p
