Pharmacology di Toxicology 1992, 70, 347401.
Interactions between Substance P and Norepinephrine in the Regulation of Nociception in Mouse Spinal Cord Per Kristian Eide and KjeU Hole Department of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway (Received June 6, 1991; Accepted November 27, 1991) Abstract: This study examined interactions between effects of the undecapeptide substance P and norepinephrine and the a-adrenoceptor agonist clonidine in the mouse spinal cord. All compounds were injected into the lumbar subarachnoid space, and effects on the tail-flick reflex and the behavioural response to intrathecal substance P were evaluated. The tailflick response latencies were markedly increased 5-20 min. after intrathecal application of norepinephrine (0.0125-0.1 pg) or clonidine (0.0125-0.1 pg). The actions of both intrathecal norepinephrine (0.025 pg) and intrathecal clonidine (0.025 pg) were significantly attenuated when substance P (5 pg) was given intrathecally 55 and 45 min. before the agonists. There was a significant relationship between the tail-flick response latencies and the tail skin temperature. However, the tail-flick results were not due to changes in the skin temperature. Intrathecally applied substance P (10 ng) produced a response consisting of biting of the caudal part of the body and a few hindlimb scratches. The number of bites was significantly reduced 5 min. after injection of norepinephrine (0.1 pg) or clonidine (0.05-0.1 pg), but the number of scratches was unchanged. The data show that increased stimulation of spinal u-adrenoceptors inhibits a spinal nociceptive reflex as well as the action of substance P in the spinal cord. Substance P modulates the action of a-adrenoceptor agonists on the tail-flick reflex, which may tentatively be explained by downregulation of a-adrenoceptors by substance P.
Descending noradrenaline neurones are involved in the control of nociceptive responsiveness (Proudfit 1988). The tailflick reflex, which is an extensively used test of nociception in rats and mice, is inhibited by intrathecal application of noradrenaline or the a,-adrenoceptor agonist clonidine (Howe & Yaksh 1982; Post et al. 1985; Reddy et al. 1980), and facilitated by intrathecal a-adrenoceptor blockers (Proudfit & Hammond 1981; Sagen & Proudfit 1984). Recently it has been reported that the tail-flick test poses a methodological problem since the tail-flick latencies depend on the skin temperature of the tail in rats (Tjerlsen et al. 1988) and mice (Eide & Tjerlsen 1988). In the rat, changes in the tail-flick latencies produced by intrathecal clonidine and the a-adrenoceptor blocker yohimbine resulted from changes in the skin temperature of the tail (Tjerlsen et al. 1990). Thus, it seems necessary to reevaluate results of tailflick testing after manipulation of spinal noradrenaline systems. The mechanisms of action underlying the effects of noradrenaline on nociception is unknown, but some data indicate interactions with substance P, which is a putative transmitter of primary afferent nociceptive neurones (Pernow 1983). The release of substance P from rat spinal cord slices is inhibited by noradrenaline (Pang & Vasko 1986). In the rat intrathecal injection of substance P changed the effect of intrathecal noradrenaline on the tail-flick reflex (Nance & Sawynok 1987), but the role of changes in the skin temperature was not taken into consideration. This study examined in mice whether pretreatment with substance P can modify the effects of norephinephrine and the a-adrenergic agonist clonidine on the tail-flick reflex, and whether changes in the reflex resulted from alterations
in the tail skin temperature. We also examined whether aadrenergic agonists could reduce the behavioural response to intrathecal substance P. Materials and Methods Animals. Male albino mice (Bom:NMRI, Bom-mice, Ry, Denmark) weighing 20-30 g were used. The animals were housed 16 to a cage (15x22 x 38 cm) with free access to food and water and were maintained in climate- and light-controlled rooms (21-24", 12/ 12 hr dark-light cycle with lights on at 7 a.m.). Drugs and administration route. (-)-Norepinephrine hydrochloride [( -)-Artereno1 HCI, Sigma Chemical Co.], clonidine hydrochloride (Sigma) and substance P (Sigma) were all dissolved in 0.9% NaCI. The solution of substance P also contained 0.02 N acetic acid (pH 4 5 ) . All drugs were given intrathecally in a volume of 5 pl. The intrathecal injection technique was adapted from the method described by Hylden & Wilcox (1980). A 30-gauge needle connected to a microsyringe with polyethylene tubing was inserted between L5 and L6. Tests of nociception. The animals were placed individually in standard macrolone cages 1 hr before testing. The observer was unaware of the drug treatment of the animals. Tail-flick testing was performed by means of an IITC Inc. Mod. 33 Analgesiameter. Each animal was restrained in a tube and radiant heat focused on the dorsal surface about 1.0 cm from the tip of the tail. Tail-flick latency was recorded as the time from the onset of stimulation to the withdrawal of the tail from the beam. Cut-off latency was 8 sec. The tail skin temperature was measured on the dorsal surface approximately 1.0 cm proximal to the centre of the heated area by means of copper-constantan thermocouple probes (0.05 mm copperconstantan wire, the junction measuring 0.1 x 0.6 mm). The tail skin temperature at the start of stimulation and the tail-flick latency were recorded simultaneously by an IBM Personal Computer.
398
PER KRISTIAN EIDE AND KJELL HOLE
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Intrathecal SP produces a behavioural response consisting of bites directed towards the caudal part of the body and a few reciprocal hindlimb scratches. The number of caudally directed bites was counted for the first 1 min. after injection of substance P.
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Fig. 2. The effect of intrathecal clonidine (0.0125,0.025, 0.05 or 0.1 pg) on tail-flick latency and tail skin temperature. Testing took place 5 min. before injection (“Pre”) and 5, 10 and 20 min. after injection (n = 8 in each group). Ambient temperature was 21.5-22.2”. * P
Interactions between Substance P and Norepinephrine in the Regulation of Nociception in Mouse Spinal Cord Per Kristian Eide and KjeU Hole Department of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway (Received June 6, 1991; Accepted November 27, 1991) Abstract: This study examined interactions between effects of the undecapeptide substance P and norepinephrine and the a-adrenoceptor agonist clonidine in the mouse spinal cord. All compounds were injected into the lumbar subarachnoid space, and effects on the tail-flick reflex and the behavioural response to intrathecal substance P were evaluated. The tailflick response latencies were markedly increased 5-20 min. after intrathecal application of norepinephrine (0.0125-0.1 pg) or clonidine (0.0125-0.1 pg). The actions of both intrathecal norepinephrine (0.025 pg) and intrathecal clonidine (0.025 pg) were significantly attenuated when substance P (5 pg) was given intrathecally 55 and 45 min. before the agonists. There was a significant relationship between the tail-flick response latencies and the tail skin temperature. However, the tail-flick results were not due to changes in the skin temperature. Intrathecally applied substance P (10 ng) produced a response consisting of biting of the caudal part of the body and a few hindlimb scratches. The number of bites was significantly reduced 5 min. after injection of norepinephrine (0.1 pg) or clonidine (0.05-0.1 pg), but the number of scratches was unchanged. The data show that increased stimulation of spinal u-adrenoceptors inhibits a spinal nociceptive reflex as well as the action of substance P in the spinal cord. Substance P modulates the action of a-adrenoceptor agonists on the tail-flick reflex, which may tentatively be explained by downregulation of a-adrenoceptors by substance P.
Descending noradrenaline neurones are involved in the control of nociceptive responsiveness (Proudfit 1988). The tailflick reflex, which is an extensively used test of nociception in rats and mice, is inhibited by intrathecal application of noradrenaline or the a,-adrenoceptor agonist clonidine (Howe & Yaksh 1982; Post et al. 1985; Reddy et al. 1980), and facilitated by intrathecal a-adrenoceptor blockers (Proudfit & Hammond 1981; Sagen & Proudfit 1984). Recently it has been reported that the tail-flick test poses a methodological problem since the tail-flick latencies depend on the skin temperature of the tail in rats (Tjerlsen et al. 1988) and mice (Eide & Tjerlsen 1988). In the rat, changes in the tail-flick latencies produced by intrathecal clonidine and the a-adrenoceptor blocker yohimbine resulted from changes in the skin temperature of the tail (Tjerlsen et al. 1990). Thus, it seems necessary to reevaluate results of tailflick testing after manipulation of spinal noradrenaline systems. The mechanisms of action underlying the effects of noradrenaline on nociception is unknown, but some data indicate interactions with substance P, which is a putative transmitter of primary afferent nociceptive neurones (Pernow 1983). The release of substance P from rat spinal cord slices is inhibited by noradrenaline (Pang & Vasko 1986). In the rat intrathecal injection of substance P changed the effect of intrathecal noradrenaline on the tail-flick reflex (Nance & Sawynok 1987), but the role of changes in the skin temperature was not taken into consideration. This study examined in mice whether pretreatment with substance P can modify the effects of norephinephrine and the a-adrenergic agonist clonidine on the tail-flick reflex, and whether changes in the reflex resulted from alterations
in the tail skin temperature. We also examined whether aadrenergic agonists could reduce the behavioural response to intrathecal substance P. Materials and Methods Animals. Male albino mice (Bom:NMRI, Bom-mice, Ry, Denmark) weighing 20-30 g were used. The animals were housed 16 to a cage (15x22 x 38 cm) with free access to food and water and were maintained in climate- and light-controlled rooms (21-24", 12/ 12 hr dark-light cycle with lights on at 7 a.m.). Drugs and administration route. (-)-Norepinephrine hydrochloride [( -)-Artereno1 HCI, Sigma Chemical Co.], clonidine hydrochloride (Sigma) and substance P (Sigma) were all dissolved in 0.9% NaCI. The solution of substance P also contained 0.02 N acetic acid (pH 4 5 ) . All drugs were given intrathecally in a volume of 5 pl. The intrathecal injection technique was adapted from the method described by Hylden & Wilcox (1980). A 30-gauge needle connected to a microsyringe with polyethylene tubing was inserted between L5 and L6. Tests of nociception. The animals were placed individually in standard macrolone cages 1 hr before testing. The observer was unaware of the drug treatment of the animals. Tail-flick testing was performed by means of an IITC Inc. Mod. 33 Analgesiameter. Each animal was restrained in a tube and radiant heat focused on the dorsal surface about 1.0 cm from the tip of the tail. Tail-flick latency was recorded as the time from the onset of stimulation to the withdrawal of the tail from the beam. Cut-off latency was 8 sec. The tail skin temperature was measured on the dorsal surface approximately 1.0 cm proximal to the centre of the heated area by means of copper-constantan thermocouple probes (0.05 mm copperconstantan wire, the junction measuring 0.1 x 0.6 mm). The tail skin temperature at the start of stimulation and the tail-flick latency were recorded simultaneously by an IBM Personal Computer.
398
PER KRISTIAN EIDE AND KJELL HOLE
r
Intrathecal SP produces a behavioural response consisting of bites directed towards the caudal part of the body and a few reciprocal hindlimb scratches. The number of caudally directed bites was counted for the first 1 min. after injection of substance P.
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Minutes after Clonidine injection 44
Fig. 2. The effect of intrathecal clonidine (0.0125,0.025, 0.05 or 0.1 pg) on tail-flick latency and tail skin temperature. Testing took place 5 min. before injection (“Pre”) and 5, 10 and 20 min. after injection (n = 8 in each group). Ambient temperature was 21.5-22.2”. * P
