[3H]Resiniferatoxin binding to pig dorsal horn membranes displays positive cooperativity
Abstract
In the present report we have reevaluated specific [3H]resiniferatoxin (RTX) binding, thought to represent the vanilloid (capsaicin) receptor, to whole spinal cord and dorsal horn membranes of the pig using a modified [3H]RTX binding assay. The high nonspecific [3H]RTX binding of the original protocol was reduced by the addition of α1-acid glycoprotein (AGP), a plasma protein that binds RTX, to the assay mixture after the binding reaction had been terminated. Specific [3H]RTX binding to pig whole spinal cord and dorsal horn membranes followed sigmoidal saturation kinetics indicating apparent positive cooperativity. The cooperativity index determined by fitting the data to the Hill equation was 2.31±0.24 in the spinal cord and 2.27±0.13 in the dorsal horn. The apparent dissociation constants in spinal cord and dorsal horn membranes were 87.8±2.7 and 103.9±1.9 pM; the receptor densities were 23±3 and 203±5 fmol/mg protein, respectively. In parallel experiments, rat spinal cord membranes bound [3H]RTX with 2 - 3 fold higher affinity, equal positive cooperativity, and a 49±6 fmol/mg receptor density. As predicted by the modified Hill equation, at low receptor occupancy nonradioactive RTX produced biphasic competition curves. Capsaicin and the competitive antagonist capsazepine also fully displaced specifically bound [3H]RTX from pig dorsal horn membranes with Ki values of 9.7±1.7 μM and 6.8±0.7 μM, respectively; the corresponding Hill coefficients were 1.81±0.17 and 2.32±0.11. [3H]RTX binding was not inhibited by resiniferonol 9, 13, 14-orthophenylacetate, the biologically inactive parent diterpene of RTX. These findings suggest that the vanilloid receptor present in the dorsal horn of the pig, like those present in human and in the rat, is a receptor cluster in which the subunits cooperate.
References (35)
- N. Jancso
- P. Holzer
Neurosci.
(1988) - A. Szallasi et al.
Neurosci.
(1989) - A. Szallasi et al.
Brain Res.
(1991) - A. Szallasi et al.
Brain Res.
(1990) - A. Szallasi et al.
Life Sci.
(1990) - K. Maderspach et al.
Biochim. Biophys. Acta
(1982) - S.M. Sine et al.
J. Biol. Chem.
(1991) - M. Covarrubias et al.
J. Biol. Chem.
(1986) - K. Maderspach et al.
Brain Res.
(1988)
Brain Res.
J. Mol. Biol.
J. Biol. Chem.
J. Med. Biol.
Life Sci.
FEBS Lett.
Neurosci. Lett.
Cited by (17)
Pharmacokinetics of eugenol and its effects on thermal hypersensitivity in rats
2007, European Journal of PharmacologyNeuropathic pain is a type of chronic pain following central or peripheral nervous system lesions that cause allodynia (pain initiated by a non-painful stimulus) and hyperalgesia (increased pain sensation following a painful stimulus). The first objective of the study was to evaluate the pharmacokinetics of eugenol, the principle chemical constituent of clove oil, following a gavage administration (40 mg/kg) in male Sprague–Dawley rats. The second objective was to evaluate the effect of repeated oral administrations of eugenol on hyperalgesia and allodynia using an experimental model of neuropathic pain in rats. Thermal and mechanical sensitivity (Hargreave's test and von Frey filaments) were determined in sciatic nerve cuff-implanted rats. Sensitivities were assessed following repeated oral administrations of 40 mg/kg of eugenol or saline for 5 days (n = 6 per group). Pharmacokinetic parameters were calculated using noncompartmental methods. Serial blood samples were collected over 24 h. Concentrations of eugenol in blood and plasma peaked rapidly following oral administration. Mean T1/2 values of eugenol in plasma and blood were long (14.0 and 18.3 h, respectively), suggesting a potential accumulation of the drug following repeated administrations. Reaction time to thermal stimuli appeared to increase constantly following repeated administrations of eugenol. On the last day of treatment, eugenol treatments resulted in a statistically significant prolongation of the reaction time to thermal stimuli in rats compared to the saline group (Mean ± S.E.M.: 11.4 ± 1.23 vs. 6.1 ± 0.53 s, P < 0.01). These results support the hypothesis that eugenol may alleviate neuropathic pain and that the cumulative effect of the drug may be in part responsible for this effect following repeated daily administrations.
Resiniferatoxin
2007, xPharm: The Comprehensive Pharmacology ReferenceIn 1975, the irritant principle of the Moroccan cactus-like plant, Euphorbia resinifera, was isolated and identified as resiniferatoxin (RTX) …
Stimulatory effect of centrally injected capsaicin, an agonist of vanilloid receptors, on gastric acid secretion in rats
2001, European Journal of PharmacologyCapsaicin, the main pungent ingredient in chilli peppers, acts through specific vanilloid receptors on sensory neurons. The vanilloid receptors have been localized in the brain. We describe here a stimulatory effect of centrally injected capsaicin on gastric acid secretion in urethane-anesthetized rats. Injection of capsaicin (10–30 nmol per rat) into the lateral cerebroventricle markedly stimulated the secretion. Injection of capsazepine (30 nmol) or ruthenium red (30 nmol), antagonists for vanilloid receptors, into the lateral cerebroventricle inhibited the secretion induced by capsaicin, although these antagonists alone significantly stimulated the secretion. Injection of capsaicin into the fourth cerebroventricle also stimulated gastric acid secretion. The effects of centrally injected capsaicin into the lateral and fourth cerebroventricle were mediated via the vagus cholinergic nerve, because the effects were abolished by bilateral vagotomy at the cervical level. The present findings showed that central injection of capsaicin stimulated gastric acid secretion, via vanilloid receptors in the central nervous system (CNS), and through vagus nerve mechanisms in the perfused stomach of urethane-anesthetized rats.
Capsazepine, a vanilloid receptor antagonist, inhibits nicotinic acetylcholine receptors in rat trigeminal ganglia
1997, Neuroscience LettersVanilloid receptors are activated by capsaicin, the pungent ingredient in hot pepper. They are also specifically and competitively inhibited by capsazepine (CPZ). To determine whether CPZ is specific to vanilloid receptors, its effects were tested on the currents evoked by nicotine in rat trigeminal ganglia. We found that 10 μM CPZ, a concentration frequently used to inhibit capsaicin's physiological responses attributed to capsaicin, reversibly inhibits (40%) the magnitude of the currents activated by 100 μM nicotine. We conclude that 10 μM capsazepine can alter the effects of channels other than those activated by capsaicin, and thus caution must be used in attributing all the CPZ-sensitive physiological effects to those only produced by blocking of vanilloid receptors.
Specific [3H]resiniferatoxin (RTX) binding detects the vanilloid (capsaicin) receptors and provides a biochemical means for exploring their pharmacology. In the present study we demonstrate specific vanilloid (RTX) binding sites in various brain areas not known to be innervated by primary afferent neurons. Specific high-affinity binding of [3H]RTX could be detected in membrane preparations of the posterior (“hypothalamic”) and anterior (“septal”) parts of the preoptic area, locus ceruleus, medial hypothalamus, brainstem reticular formation and ventral thalamic nuclei from naive rats. The determined levels of binding at 4 nM [3H]RTX were 23.0 ± 4.5, 7.1 ± 1.6, 29.9 ± 2.3, 23.5 ± 2.4, 9.9 ± 2.2 and 8.1 ± 1.9 f, respectively; unfortunately, the high levels of non-specific binding (higher than 80%) in the present experiments made it impossible for us to characterize fully the binding properties of the receptors. However, no detectable specific [3H]RTX binding was present in membranes of brain nuclei from rats pretreated with 300 capsaicin, a treatment which causes loss of response to capsaicin. Significant specific [3H]RTX binding was also absent in membrane preparations of the midbrain central gray matter, somatosensory cortex and cerebellum either from naive or capsaicin treated rats. In human brain specific [3H]RTX binding measured at 4 nM [3H]RTX showed a pattern of distribution similar to that in the rat brain. The corresponding levels of specific [3H]RTX binding in the preoptic area, locus ceruleus, medial hypothalamus, reticular formation and ventral thalamus were 44.9 ± 2.4, 50.6 ± 3.0, 36.1 ± 2.9, 9.4 ± 2.8 and 8.4 ± 2.4 f, respectively. Our findings corroborate previous biological evidence that vanilloid receptors are present in brain as well as in sensory afferent neurons.
Binding of neuroleptic drugs ( trifluoperazine and rimcazole) to vanilloid receptors in porcine dorsal horn
1996, European Journal of PharmacologyNeuroleptic drugs were reported to modulate [3H]resiniferatoxin binding to vanilloid receptors in the spinal cord, with marked differences between rat and man. In the present study, we have used a [3H]resiniferatoxin binding assay using porcine dorsal horn membranes to explore further species differences in the interaction of neuroleptic drugs at spinal vanilloid receptors. Specific binding of 13 pM [3H]resiniferatoxin to porcine dorsal horn membranes (corresponding to a 7% fractional receptor occupancy) was affected by trifluoperazine in a bi-phasic fashion, with an initial 90% enhancement of binding preceding inhibition: a fit to the modified Hill equation yielded a cooperativity index of 1.8 and a Ki of 5 μM. Under similar conditions, rimcazole, by contrast, had a monophasic effect: it enhanced but, up to 100 μM, did not inhibit [3H]resiniferatoxin binding. These results are in accord with previous findings in human spinal cord but contrast with those in the rat. In experiments in which the concentration of [3H]resiniferatoxin was varied, 20 μM trifluoperazine reduced the Bmax by 33% (from 181 ± 9 fmol/mg protein to 121 ± 5 fmol/mg protein) without a measurable change in affinity or cooperativity. In parallel experiments, by contrast, neither capsaicin nor capsazepine (both at a concentration of 10 μM) affected the Bmax or cooperativity but, as expected, reduced the affinity from 61 ± 8 pM to 120 ± 11 pM or to 101 ± 7 pM, respectively. Whereas vanilloid receptor agonists (resiniferatoxin and capsaicin) affected [3H]resiniferatoxin binding at low (approximately 7%) fractional receptor occupancies by the radioligand in a bi-phasic fashion, the competitive vanilloid receptor antagonist capsazepine failed to induce the initial binding enhancement. Thus, capsazepine appears to bind to vanilloid receptors in a non-cooperative fashion, or at least with much reduced positive cooperativity in this system. The mechanism by which neuroleptic drugs modulate resiniferatoxin binding is yet to be clarified and is clearly complicated as well as species-dependent; nonetheless, the reduced Bmax at higher concentrations suggests that it may at least in part be non-competitive.