Norepinephrine release from spinal synaptosomes: auto-alpha2 -adrenergic receptor modulation.
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BACKGROUND: Clonidine produces analgesia after spinal injection by activating alpha2-adrenergic receptors. Recently, clonidine has been demonstrated to increase spinal release of norepinephrine (NE) in vivo, in contrast to that anticipated by classic presynaptic autoinhibition. The purpose of the current study was to determine if clonidine could inhibit release of NE in a preparation of spinal cord tissue lacking synaptic circuits. METHODS: Crude synaptosomes were prepared from male Sprague-Dawley rat spinal cord, loaded with [3H]NE, and stimulated by potassium chloride to release [3H]NE. Samples were incubated with clonidine in the absence or presence of various inhibitors. To study the effect of alpha2a-adrenergic receptor subtypes, some animals were pretreated with an oligodeoxynucleotide (ODN) composed of a sense or antisense sequence to a portion of this receptor. RESULTS: Potassium chloride produced a concentration-dependent increase in [3H]NE release, and this release was inhibited by clonidine with a concentration producing 50% maximal inhibition (IC50) of 1.3 microm. The effect of clonidine was inhibited by the alpha2-adrenergic antagonists, yohimbine and idazoxan, but not by alpha1-adrenergic, muscarinic, or opioid antagonists. Intrathecal pretreatment with antisense ODN to alpha2A-adrenergic receptors reduced alpha2A-adrenergic receptor protein expression compared with sense ODN control and also reduced clonidine-induced inhibition of [3H]NE release. CONCLUSIONS: These data demonstrate the existence of classic autoinhibitory alpha2-adrenergic receptors in the spinal cord, probably of the alpha2Asubtype. They further suggest that clonidine-induced stimulation of spinal NE release must occur from indirect actions, presumably due to activation of a spinal circuit. (+info)
Antinociception induced by amitriptyline and imipramine is mediated by alpha2A-adrenoceptors.
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The involvement of alpha2-adrenoceptors in the antinociception induced by the tricyclic antidepressants amitriptyline and imipramine was investigated in mice by using the hot-plate and abdominal constriction tests. The antinociception produced by amitriptyline (15 mg/kg, i.p.) and imipramine (15 mg/kg, i.p.) was prevented by reserpine (2 mg/kg, i.p.) and yohimbine (3-10 mg/kg, i.p.) but not by naloxone (1 mg/kg, i.p.), atropine (5 mg/kg, i.p.), CGP 35348 (100 mg/kg, i.p.) and prazosin (1 mg/kg, i.p.). On the basis of the above data, it can be postulated that amitriptyline and imipramine exerted their antinociceptive effect by activation of alpha2-adrenoceptors. Administration of the alpha2A-adrenoceptor antagonist BRL 44408 (1 mg/kg, i.p.) prevented amitriptyline and imipramine antinociception, whereas the alpha2B/C-adrenoceptor antagonist ARC 239 (10 mg/kg, i.p.) was ineffective. These data indicate that the enhancement of the pain threshold produced by amitriptyline and imipramine is mediated by activation of alpha2A-adrenoceptors. Neither tricyclic antidepressants nor the antagonists used impaired mouse performance evaluated by the rota-rod and hole-board tests. (+info)
Comparison of the effects of clonidine and yohimbine on pupillary diameter at different illumination levels.
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AIMS: To evaluate the pupillary effects of single doses of the alpha2-adrenoceptor agonist clonidine and the alpha2-adrenoceptor antagonist yohimbine under several illumination conditions. METHODS: Sixteen healthy male volunteers received clonidine 0.2 mg, yohimbine 22 mg, clonidine 0.2 mg + yohimbine 22 mg in a double-blind placebo-controlled, cross-over study. 2 h post drug ingestion pupil diameter was recorded in darkness, and at luminance levels of 6 Cd m-2, 91 Cd m-2 and 360 Cd m-2. The effects of the active treatments on pupil diameter were also expressed as the differences from the placebo condition ('placebo-corrected' data; mean [95% CI]). RESULTS: Clonidine had little effect on pupil diameter in darkness; however, it caused a significant, light-dependent, miosis when the eye was illuminated. On the other hand yohimbine increased pupil size; this increase was significant at 91 and 360 Cd m-2. There were no significant differences between the effects of the combined treatment (clonidine 0.2 mg + yohimbine 22 mg) and the effect of placebo. CONCLUSIONS: The pupillary effects of clonidine and yohimbine are likely to reflect the interaction of these drugs with inhibitory alpha2-adrenoceptors located on central noradrenergic neurones, which in turn would lead to a decrease and an increase, respectively, in sympathetic outflow to the iris. The light dependence of the pupillary effects of these drugs, however, suggests that the parasympathetic light reflex pathway is also involved, which is known to be under inhibitory control from the central noradrenergic neurones. Modulation of parasympathetic outflow seems to play an important role since both drugs had relatively little effect on pupil diameter in darkness when sympathetic activity predominates. (+info)
Effect of brimonidine on rabbit trabecular meshwork hyaluronidase activity.
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PURPOSE: To study the presence of hyaluronidase activity in the rabbit trabecular meshwork and its regulation by brimonidine. METHODS: A spectrophotometric assay that consists of the assessment of N-acetylhexosamine groups released from hyaluronic acid was used to examine hyaluronidase activity. Cyclic adenosine monophosphate (cAMP) levels were assessed by radioimmunoassay. RESULTS: Hyaluronidase activity was detected in the rabbit trabecular meshwork. Its optimal activity was in the acid range of pH 3.8. Brimonidine significantly increased trabecular hyaluronidase-specific activity and decreased cAMP accumulation. Yohimbine significantly inhibited the effect of brimonidine on both hyaluronidase activity and cAMP accumulation. CONCLUSIONS: The finding of endogenous hyaluronidase activity in rabbit trabecular meshwork supports the hypothesis that this tissue can metabolize its own glycosaminoglycan (GAG) products. The present results suggest, however, that the hypotensive effect of brimonidine could be mediated, at least in part, by its ability to increase GAG catabolism, probably through a cAMP-independent mechanism. (+info)
Agonist-directed trafficking of porcine alpha(2A)-adrenergic receptor signaling in Chinese hamster ovary cells: l-isoproterenol selectively activates G(s).
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In this study, we investigated the hypothesis of agonist-directed trafficking of receptor signaling for the alpha(2A)-adrenergic receptor (alpha(2A)-AR). alpha(2A)-ARs couple to both G(s) and G(i) to stimulate or inhibit adenylyl cyclase activity. Chinese hamster ovary-K1 cell lines expressing the porcine alpha(2A)-AR at high (alpha(2A)-H) and low (alpha(2A)-L) levels were used to estimate the relative efficacies (R.e.s) of a series of agonists for the G(s) and G(i) pathways. G(s)-mediated responses were measured after pertussis toxin treatment to inactivate G(i) in alpha(2A)-H, whereas G(i) responses were measured in alpha(2A)-L, where G(s) responses were absent. The full agonist UK-14,304 showed a large receptor reserve for G(i) responses in alpha(2A)-H but little receptor reserve for G(s) responses in alpha(2A)-H or for G(i) responses in alpha(2A)-L. With the exception of l-isoproterenol (ISO), all agonists showed similar R.e.s at the alpha(2A)-AR for G(s) and G(i) responses, with rank orders of R.e.s as follows: l-epinephrine = l-norepinephrine = UK-14,304 > p-aminoclonidine > or = BHT-920 > or = BHT-933 > clonidine = p-iodoclonidine > or = xylazine > or = guanabenz. Interestingly, ISO had the highest efficacy at the alpha(2A)-AR for activating G(s) versus G(i) (9-fold higher); however, it had low potency for both. By several criteria, the ISO response was mediated by the alpha(2A)-AR, supporting the hypothesis of agonist-directed trafficking of receptor signaling or agonist-specific G protein selectivity. In contrast, the apparent G(i) pathway selectivity of oxymetazoline appears to be mediated by an endogenous serotonergic receptor. It is intriguing that a classic beta-AR agonist that activates G(s) through beta(2)-ARs also appears to produce a G(s)-selective conformation of the G(i)-coupled alpha(2A)-AR. (+info)
Analgesic mechanisms of ketamine in the presence and absence of peripheral inflammation.
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BACKGROUND: The studies on the mechanisms of ketamine antinociception have led to conflicting results. In this study, the authors investigated the contribution of supraspinal monoaminergic descending inhibitory system to ketamine analgesia for acute nociception and inflammation-induced hyperalgesia. METHODS: Male Sprague-Dawley rats were used. The paw withdrawal latencies to radiant heat stimuli were measured to assess the thermal nociceptive threshold. The analgesic effects of intrathecal or intraperitoneal ketamine were examined in the rats that received unilateral intraplantar carrageenan and in those that were untreated. In addition, it was examined whether pretreatment with intrathecal yohimbine or methysergide inhibited the analgesic effects of ketamine. Using an intrathecal microdialysis method, noradrenaline and 5-hydroxytryptamine concentrations in lumbar cerebrospinal fluid were measured after intraperitoneal ketamine in both saline- and carrageenan-treated rats. RESULTS: In the untreated rats, intraperitoneal but not intrathecal ketamine produced antinociceptive effects in a dose-dependent manner. Pretreatment with intrathecal yohimbine or methysergide inhibited these antinociceptive effects. Intraplantar carrageenan significantly reduced paw withdrawal latencies on the injected paw but not on the contralateral paw. Both intraperitoneal and intrathecal ketamine reversed the shortened paw withdrawal latencies on the injected side in a dose-dependent manner without any effects on the contralateral side. Neither yohimbine nor methysergide inhibited these antihyperalgesic effects. In analyses of monoamines, the magnitude of increase in monoamines after intraperitoneal ketamine was significantly smaller in the carrageenan-treated rats than in the saline-treated rats. CONCLUSION: These results demonstrated that ketamine produced antinociceptive effects through an activation of the monoaminergic descending inhibitory system, whereas, in a unilateral peripheral inflammation-induced hyperalgesic state, the monoaminergic system did not contribute to the antihyperalgesic effects of ketamine. The mechanisms of the antinociceptive and antihyperalgesic properties of ketamine are different. (+info)
We previously reported that nicotine-induced nitric oxide (NO)-mediated cerebral neurogenic vasodilation was dependent on intact sympathetic innervation. We hypothesized that nicotine acted on sympathetic nerve terminals to release norepinephrine (NE), which then acted on adrenoceptors located on the neighboring nitric oxidergic (NOergic) nerve terminals to release NO, resulting in vasodilation. The adrenoceptor subtype in mediating nicotine-induced vasodilation in isolated porcine basilar arterial rings denuded of endothelium was therefore examined pharmacologically and immunohistochemically. Results from using an in vitro tissue bath technique indicated that propranolol and preferential beta(2)-adrenoceptor antagonists (ICI-118,551 and butoxamine), in a concentration-dependent manner, blocked the relaxation induced by nicotine (100 microM) without affecting the relaxation elicited by transmural nerve stimulation (TNS, 8 Hz). In contrast, preferential beta(1)-adrenoceptor antagonists (atenolol and CGP-20712A) did not affect either nicotine- or TNS-induced relaxation. Results of double-labeling studies indicated that beta(2)-adrenoceptor immunoreactivities and NADPH diaphorase reactivities were colocalized in the same nerve fibers in basilar and middle cerebral arteries. These findings suggest that NE, which is released from sympathetic nerves upon application of nicotine, acts on presynaptic beta(2)-adrenoceptors located on the NOergic nerve terminals to release NO, resulting in vasodilation. In addition, nicotine-induced relaxation was enhanced by yohimbine, an alpha(2)-adrenoceptor antagonist, which, however, did not affect the relaxation elicited by TNS. Prazosin, an alpha(1)-adrenoceptor antagonist, on the other hand, did not have any effect on relaxation induced by either nicotine or TNS. The predominant facilitatory effect of beta(2)-adrenoceptors in releasing NO may be compromised by presynaptic alpha(2)-adrenoceptors. (+info)
Dual interaction of agmatine with the rat alpha(2D)-adrenoceptor: competitive antagonism and allosteric activation.
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In segments of rat vena cava preincubated with [(3)H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [(3)H]-noradrenaline release, the EP(3) prostaglandin receptor-mediated and the alpha(2D)-adrenoceptor-mediated inhibition of evoked [(3)H]-noradrenaline release was investigated. Agmatine (0.1-10 microM) by itself was without effect on evoked [(3)H]-noradrenaline release. In the presence of 10 microM agmatine, the prostaglandin E(2)(PGE(2))-induced EP(3)-receptor-mediated inhibition of [(3)H]-noradrenaline release was not modified, whereas the alpha(2D)-adrenoceptor-mediated inhibition of [(3)H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [(3)H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [(3)H]-clonidine and [(3)H]-rauwolscine to rat brain cortex membranes (K(i) values 6 microM and 12 microM, respectively). In addition, 30 and 100 microM agmatine increased the rate of association and decreased the rate of dissociation of [(3)H]-clonidine resulting in an increased affinity of the radioligand for the alpha(2D)-adrenoceptors. [(14)C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [(14)C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the alpha(2D)-adrenoceptor and enhances the effects of alpha(2)-adrenoceptor agonists probably by binding to an allosteric binding site of the alpha(2D)-adrenoceptor which seems to be labelled by [(14)C]-agmatine. (+info)