The spinal antinociceptive actions of morphine metabolites morphine-6-glucuronide and normorphine in the rat

The profound and prolonged effects of morphine in patients with renal dysfunction have been associated with high plasma levels of the opiate metabolites morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) rather than an increased concentration of morphine. We present here electrophysiological evidence to suggest that potent spinal antinociception can be produced by both M6G and normorphine, another metabolite of morphine. Extracellular recordings of Aβ- and C-fibre-evoked responses of convergent dorsal horn neuroneswere made in the halothane anaesthetised rat. M6G elicited dose-dependent, naloxone-reversible inhibitions of C-fibre-evoked responses which were completely suppressed (8% of control) by 2 μg M6G whereas Aβ-fibre-evoked responses were only reduced to 57% of controls. The ED₅₀ for the effects of M6G on C-fibre-evoked activity was calculated to be 0.53 μg. Systematic administration of M6G (2 mg/kg) also profoundly reduced noxious evoked neuronal activity. intrathecal normorphine was less potent than M6G but complete selective inhibitions of C-fibre-evoked responses could be elicited by 25 μg and the ED₅₀ was calculated to be 2.68 μg. No such inhibitions were observed following administration of M3G. A comparison with intrathecal morphine in the same preparation reveals that normorphine is equipotent with morphine whereas M6G is 13-fold more potent. These results therefore confirm that M6g and normorphine might be significant contributers to opiate analgesia after administration of morphine.     

Depression of C fiber-evoked activity by intrathecally administered reactive red 2 in rat thalamic neurons

To investigate the possible role of spinal purinoceptors in nociception, the potent P₂-purinoceptor antagonist reactive red 2 was studied in rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intrathecal (i.t.) application of reactive red 2 (6–200 μg) caused a dose-dependent reduction of the evoked activity in thalamic neurons. The estimated ED₅₀ was 30 μg, and the maximum depression of nociceptive activity amounted to about 70% of the control activity at a dose of 100 μg. Morphine, administered i.t. at a maximally effective dose (80 μg), inhibited the evoked nociceptive activity by only up to 55% of the control activity. An i.t. co-injection of reactive red 2 (100 μg) and morphine (80 μg) caused a maximum reduction of the evoked thalamic activity by up to 85% of the control activity, thus, exceeding significantly the effect elicited by either drug alone. Similarly, i.t. co-injection of almost equipotent dosages of reactive red 2 (30 μg) and morphine (30 μg) caused a maximum reduction of the evoked activity by up to 72% of the control activity, which again exceeded significantly the effect of either drug alone. The results suggest that in rats reactive red 2 exerts antinociception by blockade of P₂-purinoceptors in the spinal cord and, hence, support the idea that ATP may play an important role in spinal transmission of nociceptive signals. An activation of the spinal opioid system does not seem to contribute to the effect of reactive red 2 but might act additive or even synergistically with its antinociceptive action.     

The peripheral nociceptive actions of intravenously administered 5-HT in the rat requires dual activation of both 5-HT2 and 5-HT3 receptor subtypes

In 16-week-old Sprague-Dawley rats lightly anesthetized with pentobarbital, 5-HT (3–96 μg/kg, i.v.;n = 6) produced distinct pseudaffective responses and a dose-dependent (slope= 17.2 ± 6.8s/log₁₀dose) inhibition of the tail-flick (TF) reflex (ED₅₀ = 32.6 ± 9.2 μg/kg). In the same rats, a 1:1 combination of α-methyl 5-HT (a 5-HT₂ receptor selective agonist) and 2-methyl 5-HT (a 5-HT₃ receptor selective agonist) (3–192 μg/kg, i.v.), produced the same profile of pseudaffective responses and also resulted in a dose-dependent (slope= 34.0± 7.0s/log₂dose) inhibition of the TF reflex (ED₅₀ = 88.4 ± 20.5 μg/kg). In contrast, administration of α-methyl 5-HT (3–192 μg/kg, i.v.) or 2-methyl 5-HT (3–192 μg/kg, i.v.) alone did not produce any pseudaffective responses or any change in TF latency from baseline. In conscious 16-week-old male Sprague-Dawley rats, administration of 5-HT (48 μg/kg, i.v.;n = 5), or a 1:1 combination of α-methyl 5-HT and 2-methyl 5-HT (total dose= 120 μg/kg, i.v.;mn = 5), resulted in a passive avoidance behavior assessed in a step-down paradigm (slopes= 139.7 ± 58.2and154.9 ± 63.9s/trial, respectively), and the same profile of distinct pseudaffective responses exhibited by the lightly pentobarbital-anesthetized rats. However, administration of either α-methyl 5-HT (96 μg/kg, i.v.;n = 4) or 2-methyl 5-HT (96 μg/kg, i.v.;n = 4), while producing significant 5-HT receptor-mediated cardiovascular responses, produced a learned behavior not different from saline (0.25 ml, i.v.;n = 6) (slopes= 7.6 ± 2.5, 6.3 ± 1.8and7.4 ± 3.6s/trial, respectively). These results are consistent with the hypothesis that the peripheral nociceptive responses to i.v. 5-HT requires dual activation of 5-HT₂ and 5-HT₃ receptor subtypes.     

Role of the nucleus raphe magnus in opiate analgesia as studied by the microinjection technique in the rat

The analgesic effects of morphine (5 μg, 0.2 μl) microinjected into the nucleus raphémagnus (NRM) and the surrounding reticular formation of the rat were tested using vocalization after electric shock to the tail as the test for analgesia. Only sites in the NRM produced powerful analgesic effects, strongest analgesia being equivalent to 3 mg/kg i.v. morphine. The analgesia produced by the microinjection was reversed by systemic naloxone. Pretreatment with systemic cinanserin, a blocker of serotonergic receptors, led to a pronounced diminution of the analgesic effects of the morphine. The effects of microinjection of naloxone (5 μg 0.2 μl) were studied for their effect on analgesia produced by systemic morphine. The analgesia following 3 mg/kg i.v. morphine was diminished by the microinjection of naloxone but the naloxone almost completely reversed the analgesic effects of 1.5 mg/kg i.v. morphine. These results further substantiate the role of the NRM in analgesic mechanisms.     

Potency ratios of morphine and morphine-6β-glucuronide analgesia elicited from the periaqueductal gray, locus coeruleus or rostral ventromedial medulla of rats

The present study examined whether morphine and morphine-6β-glucuronide (M6G) analgesia on the tail-flick and jump tests differed in potency in the periaqueductal gray, the locus coeruleus or the rostral ventromedial medulla. Morphine and M6G significantly and dose-dependently elicited analgesia on both nociceptive tests from each site. Site-specific differences were observed in the potency of M6G, but not morphine analgesia on both tests. Periaqueductal gray placements displayed analgesic ED_50s on the tail-flick (morphine: 2.1 μg, M6G: 0.2 μg) and jump (morphine: 2.2 μg, M6G: 0.4 μg) tests with respective potency ratios of 12.9 and 6.5. Locus coeruleus placements displayed analgesic ED_50s on the tail-flick (morphine: 1.7 μg, M6G: 0.1 μg) and jump (morphine: 3.4 μg, M6G: 0.2 μg) tests with respective potency ratios of 15.9 and 15.1. Rostral ventromedial placements displayed analgesic ED_50s on the tail-flick (morphine: 1.4 μg, M6G: 0.06 μg) and jump (morphine: 1.9 μg, M6G: 0.08 μg) tests with potency ratios of 21.9 on both tests. The greater analgesic sensitivity of the rostral ventromedial medulla to M6G may be due to either pharmacodynamic (splice variants of the MOR-1 gene) and/or pharmacokinetic (lipid solubility) factors.     

Electrophysiological studies on the spinal effects of dermorphin, an endogenous μ-opioid agonist

The intrathecal administration of dermorphin, an endogenous heptopeptide first discovered in amphibia, produces dose-dependent selective inhibitions of C fibre-evoked responses in rat dorsal horn nociceptive neurones (ED₅₀ 0.11 μg). Naloxone (10 μg) but not ICI 174,864 (125 μg) antagonised the effects of the peptide. Aβ-fibre-evoked activity was relatively unaffected. Thus dermorphin can profoundly inhibit nociceptive afferent input in the spinal cord, and in this preparation is more potent (approximately 40X) than morphine.     

Morphine-3-glucuronide: hyperglycemic and neuroendocrine potentiating effects

The greater potency of morphine-6-glucuronide (M6G) as well as the inactivity of morphine-3-glucuronide (M3G) with respect to the antinociceptive effects of the parent molecule, morphine (MOR), have been well established. It has been suggested that M3G is an antagonist of MOR's antinociceptive and respiratory depressive effects. The present study addressed the central nervous system (CNS) interaction of these opiate metabolites on their metabolic and hormonal effects. Whole body glucose kinetics were assessed on conscious, chronically catheterized, unrestrained rats. M3G (5 μg) or H₂O (5 μl) was injected intracerebroventricularly (i.c.v.) 15 min prior to the bolus administration of H₂O (5 μl), M6G (1 μg), or MOR (80 μg). i.c.v. M3G (5 μg) resulted in behavioral excitation, hyperglycemia (+50%), stimulation of glucose rate of appearance (R_a; +100%), glucose rate of disappeaance (R_d; +70%), and metabolic clearance rate (MCR; +33%) within 30 min after injection with no alterations in hormone concentrations. i.c.v. M6G and MOR produced progressive hyperglycemia with significantly high catecholamine and corticosterone levels. M3G pretreatment resulted in enhanced elevations in plasma glucose levels (+52% and +18%), plasma lactate (+138% and +108%), norepinephrine (+96% and +30%), and epinephrine (+62% and +67%) in response to both i.c.v. MOR and M6G administration. These findings suggest a non-opiate and non-hormonal mechanism for M3G-induced hyperglycemia. In contrast, the metabolic and hormonal responses to i.c.v. M6G and MOR are associated with elevations in catecholamine and corticosterone levels, which are remarkably enhanced by M3G pretreatment, most likely through accelerated catecholamine release. Our findings suggest a modulatory role for MOR glucuronidation, not only by rendering it inactive, as in the case of M3G, but by an interplay of the metabolic effects of the parent molecule and its metabolite     

Differential effects of specific δ and κ opioid receptor antagonists on the bidirectional dose-dependent effect of systemic naloxone in arthritic rats, an experimental model of persistent pain

In an attempt to determine the opioid receptor class(es) which underly the two opposing effects of naloxone in models of persistent pain, we tested the action of the selective δ antagonist naltrindole, and that of the κ antagonist MR-2266 on the bidirectional effect of systemic naloxone in arthritic rats. As a nociceptive test, we used the measure of the vocalization thresholds to paw pressure. The antagonists were administered at a dose (1 mg/kg i.v. naltrindole, 0.2 mg/kg i.v. MR-2266), without action per se, but which prevents the analgesic effect of the δ agonist DTLET (3 mg/kg, i.v.) or the κ agonist U-69, 593 (1.5 mg/kg, i.v.) respectively, and does not influence the effect of morphine (1 mg/kg i.v.) or the μ agonist DAMGO (2 mg/kg, i.v.) in these animals. In arthritic rats injected with the δ antagonist, the paradoxical antinociceptive effect produced by 3 μg/kg i.v. naloxone was not significantly modified (maximal vocalization thresholds (% of control) were 146 ± 9% versus 161 ± 7% in the control group). By contrast, the hyperalgesic effect produced by 1 mg/kg i.v. naloxone was significantly reduced (maximal vocalization thresholds were87 ± 4% versus 69 ± 5% in the control group). In rats injected with the κ antagonist, the antinociceptive effect of the low dose of naloxone was almost abolished (mean vocalization thresholds were 115 ± 3% versuss 169 ± 7%) whereas the hyperalgesic effect of naloxone 1 mg/kg i.v. was not significantly modified (mean vocalization thresholds =70 ± 3% and 65 ± 3%, respectively). Based on these results, the possible role of each receptor subtype in the putative control exerted by endogenous opioid substances on nociceptive messages in pathological conditions is discussed.     

Antinociceptive effect of ketanserin in mice: involvement of supraspinal 5-HT2 receptors in nociceptive transmission

The involvement of 5-HT₂ receptors in pain transmission was investigated in mice. Subcutaneous administration of the selective 5-HT₂ receptor antagonist ketanserin produced dose-dependent antinociception in the hot-plate and acetic acid-induced writhing tests withED₅₀ values (95% confidence limit) of 1.51 (1.13–1.89) and 0.62 (0.10–1.40) mg/kg, respectively, but was without any significant effect on the tail-flick test. Pretreatment with the catecholamine depletors 6-hydroxydopamine (2.5 μg, i.c.v.) orα-methyl-p-tyrosine (200 mg/kg, s.c.), or the serotonin synthesis inhibitorp-chlorophenylalanine methylester (200 mg/kg, s.c.), resulted in a significant decrease in the antinociceptive effect of ketanserin. Likewise, intrathecal (i.t.) administration of 1 μg/mouse of idazoxan (anα₂-antagonist), methysergide (mixed 5-HT₁, and 5-HT₂ antagonist) or ketanserin also reversed the antinociceptive effect of s.c. administered ketanserin. The results of this work indicate that 5-HT₂ receptors located supraspinally may inhibit descending nociceptive neurotransmission. In addition, these studies suggest that 5-HT₂ receptors located at the spinal level modulate nociception.     

κ-opioid receptor mediated antinociception in rats is dependent on the functional state of dihydropyridine-sensitive calcium channels

The modulatory effect of the dihydropyridine Ca²⁺ channel antagonist nimodipine on the analgesic action of the κ-opioid receptor agonist U-69,593 was analyzed using the tail-flick test in rats. The antinociceptive effect of U-69,593 (0.25–4 mg/kg) was antagonized by L-type Ca²⁺ channel blockade with nimodipine (200 μg/kg, i.p.), the ED₅₀ being increased from 1.4 to 7.3 mg/kg. On the contrary, when an increase in the density of these channels was induced by means of chronic and simultaneous treatment with nimodipine (1 μg/h, 7 days) and sufentanil (2 μg/h, 8 days), the analgesic effect of U-69,593 was potentiated by 5-fold. Our results suggest a functional coupling between κ-opioid receptors and L-type Ca²⁺ channels in nociception.     

Beneficial effect of i.c.v. naloxone in anaphylactic shock is mediated through peripheral β-adrenoceptive mechanisms

In mice, fatal shock induced by release of endogenous histamine by compound 48/80 was reversed by the intracerebroventricular (i.c.v.) administration of the opiate antagonist naloxone (10–25 μg) but not by the systemic administration of the selective peripherally acting antagonist, naltrexone methyl bromide (2–5 mg/kg). Moreover, systemac or i.c.v. administration of morphine (25 mg/kg and 25 μg, respectively) exacerbated shock induced by compound 40/80. This effect was blocked by i.c.v. naloxone (10 μg) or naltrexone methyl bromide (10 μg) but not by systemic naltrexone methyl bromide (5 mg/kg). The pathogenic effect of i.c.v. morphine was blocked by the systemic administration of the opiate antagonist Win 44,441-3 (5 mg/kg) but not by its inactive (+) isomer, Win 44,441-2. The results suggest possible involvement of central opiate (endorphin) mechanisms in the pathophysiology of fatal histamine shock in mice.     

Modification of morphine-induced locomotor activity by pertussis toxin: biochemical and behavioral studies in mice

The effect of pertussis toxin (PTX) on the locomotor-enhancing action of systemic and intracerebroventricular (i.c.v.) morphine was investigated in mice. Mice were i.c.v. injected with either PTX (0.25 and 0.5 μg) or saline as a control. The s.c. (5–20 mg/kg) and i.c.v. (7–30 nmol) administration of morphine produced a dose-related locomotor-enhancing action in control mice. The peak effect of morphine (30 nmol, i.c.v.)-induced hyperlocomotion was observed 90 min after the morphine injection. At the same time, morphine significantly increased dopamine (DA) metabolism in the limbic forebrain (nucleus accumbens and olfactory tubercle). Similarly, the selective μ-opioid receptor agonist[d-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAGO, 4 nmol, i.c.v.) also significantly increased locomotor activity and DA metabolism in the limbic forebrain. Both morphine- and DAGO-induced hyperlocomotion and elevation of DA turnover were antagonized by pretreatment with the μ antagonist β-funaltrexamine (β-FNA). These results suggest that the locomotor-enhancing action of morphine results from the activation of central μ-opioid receptors, and that the activation of the mesolimbic DA system may be involved in the expression of morphine-induced hyperlocomotion in mice. Furthermore, pretreatment with PTX (0.5 μg, i.c.v., 6 days prior to the testing) significantly reduced hyperlocomotion and elevation of DA turnover in the limbic forebrain which had been induced by administrations of morphine (30 nmol, i.c.v.) and DAGO (4 nmol, i.c.v.). These findings suggest that the central PTX-sensitive GTP-binding protein (G-protein) mechanism may play an important role in opioids-induced locomotor-enhancing action. Furthermore, the activation of mesolimbic DA transmission by μ-opioid agonists may also be mediated by a PTX-sensitive G-protein mechanism in mice.     

Activation of central μ-opioid receptors is involved in clonidine analgesia in rats

The analgesic effect of clonidine in spontaneously hypertensive rats (SHR) and in normotensive Sprague-Dawley (SD) rats was assessed by using the formalin pain test. The analgesic response of SD rats to low doses (15–60 μg/kg i.p.) but not to a high dose (150 μg/kg i.p.) of clonidine was inhibited by naloxone, 2 mg/kg i.p., and a similar interaction was noted in SHR. In both rat strains, the analgesic response to low i.p. doses of clonidine was also inhibited by injection of 5 μg of naloxone or 7 μg of β-funaltrexamine, a μ-receptor antagonist, into the lateral cerebral ventricle. I.c.v. injection of 5 μg of ICI 174864, a δ-receptor antagonist, potentiated or did not influence clonidine analgesia in SD rats and SHR, respectively. It is concluded that the analgesic response to clonidine involves activation of central μ-opioid receptors in both SHR and SD rats, possibly by an endogenous opioid released by clonidine.     

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla

In urethane anesthetized rats, iontophoretic application of morphine or α-methylnoradrenaline (α-MNE) inhibited (80–100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of α-MNE was blocked selectively by theα₂-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75–100%) by low i.v. doses of clonidine (10–15 μg/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 ± 0.9 spikes/s to 12.3 ± 1.5 spikes/s;n = 8). During withdrawal these cells could still be inhibited (80–100%) by i.v. injection of clonidine (15 μg/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenregic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive forα₂A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate andα₂-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal, Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.     

Differential modulation by baclofen on antinociception induced by morphine and β -endorphin administered intracerebroventricularly in the formalin test

Our previous studies have demonstrated that supraspinal GABAergic receptors are differentially involved in the antinociception induced by morphine and β-endorphin given intracerebroventricularly (i.c.v.) in the tail-flick and hot-plate tests. These two models employed a phasic, thermal nociceptive stimulus. The present study was designed to examine the possible involvement of supraspinal GABAergic receptors in opioid-induced antinociception in the formalin test. Morphine (1 μg) and β-endorphin (1 μg) given i.c.v. displayed the almost complete inhibitory effects against the hyperalgesic response in both phases. Muscimol (75–100 ng) and baclofen (5–10 ng) injected i.c.v. produced the hypoalgesic response in the both phases. The hypoalgesic response induced by muscimol and baclofen observed during the second phase was more pronounced than that observed during the second phase. Baclofen (2.5 ng), at the dose which did not affect the hyperalgesic response, resulted in a significant reversal of the i.c.v. administered β-endorphin-induced hypoalgesic response observed during the second, but not the first, phase. However, the hypoalgesic response induced by i.c.v. administered morphine was not changed by the same dose of muscimol or baclofen injected i.c.v. Our results indicate that, at the supraspinal level, GABA_Breceptors appear to be involved in the modulation of antinociception induced by supraspinally administered β-endorphin, but not morphine, in the formalin test model.     

Priming enhances endotoxin-induced thermal hyperalgesia and mechanical allodynia in rats

Central inflammation is an integral component and contributor of the pathology of many debilitating diseases and has been shown to produce spontaneous pain and hyperalgesia. Recently, administration of lipopolysaccharide (LPS) into the lateral ventricle of rats was shown to elicit both thermal hyperalgesia and tactile allodynia [K. Walker, A. Dray, M. Perkins, Hyperalgesia in rats following intracerebroventricular administration of endotoxin: effect of bradykinin B₁ and B₂ receptor antagonist treatment, Pain 65 (1996) 211–219]. In this study, we have replicated the LPS model with some adaptations and correlated the nociceptive behaviors with an increased expression of activated macrophages in the central nervous system. We also examined the effects of priming on LPS-induced decreases in thermal nociceptive thresholds and mechanical response thresholds following either central or peripheral administration. Intracerebroventricular (i.c.v.) administration of LPS (0.2 μg/rat) did not alter either thermal (hot plate) or mechanical (von Frey filaments) thresholds compared to baseline values in the first few hours after injection. However, priming rats by pretreating with i.c.v. LPS (0.2 μg) 24 h prior to testing with i.c.v. LPS (0.2 μg) produced significant mechanical allodynia and thermal hyperalgesia. The mechanical allodynia had an onset of 80 min after injection and a duration of 5 h. A similar time course was observed for thermal hyperalgesia, although its expression was less pronounced. Immunohistochemical studies indicated an increased expression of activated macrophages in the brain parenchyma of primed rats but not in unprimed rats. Intraperitoneal (i.p., 2 mg/kg) administration of LPS had no significant effect on either thermal or mechanical thresholds in the first few hours after injection; however, priming rats via i.p. (0.2 mg/kg) or i.c.v. (0.2 μg) LPS produced a reduction in both thermal nociceptive thresholds and mechanical response thresholds in rats given a subsequent i.p. injection of LPS. This study demonstrates that priming is an effective protocol for the induction of central inflammation and increases the duration of these behaviors after i.c.v. administration.     

Fos-like immunoreactivity induced by intraplantar injection of endotoxin and its reduction by morphine

C-Fos-like immunoreactivity (FLI) in the central nervous system, has been associated with the processing of nociceptive information in acute and chronic pain animal models. The aim of this study was to investigate whether intraplantar (i.pl.) injections of endotoxin (ET, 1.25 μg) can induce FLI in the lumbar spinal cord of rats and to assess the effects of morphine injection on c-fos expression. FLI was studied in various groups of rats at 2, 3, 4, 6, 9 and 24 h following ET injections. Labeled neurons were mainly detected in the lumbar segments ipsilateral to the ET-injected leg, with a major peak (71.01±4.79 positive neurons) at 4 h and a second peak (29.87±5.97 positive neurons) at 9 h followed by a recovery to the baseline at 24 h after ET injections. Within the laminae, the majority of positive neurons was observed at 2–3 h in laminae I and II and in deep laminae (V and VI mainly) starting at 4 h after ET injections. Rostrocaudally, labeled neurons were observed initially in L₄–L₅ segments (2–3 h post-ET) after which they extended to L₂–L₆ segments at 4 h after ET. Morphine injections either i.p. (1 or 2 mg/kg) or i.pl. (50 μg) significantly reduced ET-induced hyperalgesia and simultaneously the FLI. The maximum effect was observed on labeled neurons in the deep laminae (V and VI mainly). We conclude that local injections of ET can induce FLI in the lumbar spinal cord with a temporal and spatial patterns comparable to the described hyperalgesia, and that both FLI and hyperalgesia are reduced by morphine in a dose-dependent manner with a maximal effect shown by the local i.pl. morphine injections.     

Differential hemodynamic, metabolic and hormonal effects of morphine and morphine-6-glucuronide

Although the hyperglycemic effect of morphine has been previously described, it is not clear whether this is the result of increased glucose production and/or decreased glucose utilization and if this metabolic effect is lost with glucuronidation. This study assessed the hemodynamic (heart rate; HR and mean arterial blood pressure; MABP), hormonal and whole body glucose metabolic effects of morphine (MOR) and its metabolite morphine 6-glucuronide (MOR-6G) in conscious unrestrained chronically catheterized rats. Whole body glucose kinetics were assessed with a primed constant intravenous infusion of [3-³H]gluccose in rats infused i.c.v. with H₂O (Con; 5 μl/h), MOR (80 μg/h) or MOR-6G (1 μg/h) for a total of 4 h. MOR administration resulted in a significant 20% elevation in HR and no change in MABP. MOR-6G produced a 14% increase in HR and no change in MABP. A significant rise in plasma glucose (+23%), hepatic glucose production (R_a; +27–61%) and whole body glucose utilization (R_d; +31–61%) was also observed within 60 min of MOR administration. I.c.v. MOR-6G resulted in hemodynamic, metabolic and hormonal parameters of H₂O infused rats. I.c.v. MOR resulted in a significant increases in epinephrine (2-fold), norepinephrine (50%), corticosterone (97%) with no alterations in plasma insulin and glucagon. I.c.v. MOR-6G resulted in more marked elevations in norepinephrine (5-fold), epinephrine (7-fold) and similar elevation in corticosterone (99%) and modest elevation of glucagon (40%). These results indicate that (i) MOR-induced hyperglycemia is the result of direct central (CNS) mechanisms that result in increased hepatic glucose production, (ii) MOR-induced stress response is enhanced at least 80-fold with glucuronidation, and (iii) MOR inhibits the pancreatic glucose-stimulated insulin release.     

Vagal afferent-mediated inhibition of a nociceptive reflex by intravenous serotonin i the rat. I. Characterization

The effect of intravenous (i.v.) serotonin (5-HT) on nociception and blood pressure in male Sprague-Dawley rats. Intravenous 5-HT produced a dose-dependent (6–192 μg/kg, i.v.) inhibition of the nociceptive tail-flick (TF) reflex in lightly pentobarbital-anesthetized (ED50 = 40 μg/kg) and conscious rats (ED50 = 44 μg/kg). In the lightly pentobarbital-anesthetized rat, the blood pressure response to i.v. 5-HT was typically a triphasic response with a marked Bezold-Jarisch reflex-induced decrease in pressure (associated with a brief period of apnea) followed by a pressor phase and a subsequent delayed hypotension. In the conscious rat, the response was typically biphasic with the late hypotensive phase absent. A variety of anatomical and pharmacological manipulations were to characterize the 5-HT-induced inhibition of the TF reflex and associated changes in blood pressure. Prevention of 5-HT-induced reflex apnea by artificial ventilation did not affect inhibition of the TF reflex produced by 5-HT. Pharmacological manipulations were performed to mimic, as closely as possible, the acute increases and decreases in blood pressure associated with i.v. 5-HT. Nitroprusside (8 μg/kg, i.v.) produced a decrease in blood pressure of similar magnitude and rate as that associated with the Bezold-Jarish reflec-induced decrease in pressure produced by 72 μg/kg 5-HT, but did not change TF latency from baseline. Similarly, acute increases in pressure produced by phenylphrine (8 μg/kg, i.v.), intended to mimic the secondary pressor effect of 5-HT, did not change TF latency. The short-acting ganglion blocker trimethaphan (5 mg/kg, i.v.) closely mimicked the late hypotensive phase produced by 5-HT but again resulted in no change in TF latency. Pretreatment with the ganglion blocker chlorisondamine (2.5 mg/kg) abolished all depressor responses to 72 μg/kg 5-HT, but did not significantly affect the TF reflex. These results indicate that acute changes in blood pressure and respiration associated with i.v. 5-HT do not contribute to inhibition of the TF reflex. This conclusion was confirmed in experiments in which bilateral vagotomy abolished approximately 70% of the 5-HT-induced inhibition of the TF reflex (and all depressor responses), and resulted in a significantly greater pressor response. Finally, low thoracic spinal cord transection (T_9–10) abolished the inhibition of the TF reflex produced by i.v. 5-HT. Therefore, 5-HT stimulates vagal afferents and inhibits the TF reflex by activating descending inhibitory system from the brainstem. Taken together with the behavioral responses observed, these results support the notion that 5-HT, when administered i.v., is a noxious stimulus.     

Analgesic effect of intrathecal morphine demonstrated in ascending nociceptive activity in the rat spinal cord and ineffectiveness of caerulein and cholecystokinin octapeptide

The effect of intrathecal injections of morphine and the two peptides, caeruelin and cholecystokinin octapeptide (CCK-8), on the activity in ascending axons of the spinal cord evoked by electrical stimulation of primary nociceptive afferents was studied in spinal rats with decerebration. Morphine (20 μg) depressed the spontaneous activity and the activity evoked from either Aδ- or C-fibres. The co-activation by Aδ-fibre stimulation of ascending axons activated by stimulation of C-fibres and the activity in ascending axons activated by stimulation of afferent Aβ-fibres were not influenced by morphine. C-Fibre-evoked ascending activity was also depressed by morphine (10 μg and 5 μg). Ascending nociceptive activity was not changed by caerulein (30 ng) and CCK-8 300 ng, but it was depressed by a subsequent injection of morphine (20 μg). The depressant effects of morphine were abolished by an intravenous injection of naloxone which, when given alone, facilitated the ascending nociceptive activity. It is concluded that: (1) an intrathecal injection of morphine selectivity depressed the ascending nociceptive activity; (ii) the depression produced by morphine is an equivalent for spinal analgesia following intrathecal injection of morphine to man; and (iii) the two components of the spinal nociceptive system, the motor and the sensory path, can independently be influenced by drugs.