ANESTH ANALG 1990;71:2W3

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The Antinociceptive Role of p- and &Opiate Receptors and Their Interactions in the Spinal Dorsal Horn of Cats Keiichi Omote, MD, Luke M. Kitahata, and Itsuo Nakagawa, MD

MD, PhD,

OMOTE K, KITAHATA LM, COLLINS JG, NAKATANI K, NAKAGAWA I. The antinociceptive role of p- and 6 opiate receptors and their interactions in the spinal dorsal horn of cats. Anesth Analg 1990;71:23-8.

This study was undertaken to examine the antinociceptive roles of different subtypes of opiate receptors and their interactions at the level of the spinal cord. We recorded extracellularly the activity of the single wide dynamic range neurons evoked by noxious radiant heat (51°C) in decerebrute, spinally transecfed cats. The separate intrathecal administration of DAGO (selective p-agonist, n = 28), morphine (less selective p-agonist, n = 22), DPDPE (selective Gagonist, n = 25), and DADL (less selective Gagonist, n = 17) produced statistically significant suppression of noxiously evoked activity in a time- and dosedependent manner. In addition, intravenously administered naloxone (nonselective opiate antagonist) reversed the suppressive efects of all opiates studied. Intravenously administered ICI 174,864 (selective &antagonist) reversed the effects of DPDPE. These results, based on relative selectivity for opiate receptors, indicate thaf both p- and

Spinal analgesia utilizing opiate agonists is the result of specific agonist-opiate receptor interactions in the spinal cord. Clinical application of spinal opiates has proven to be an important means of controlling acute and chronic pain in humans. Martin et al. (1) suggested three opiate receptor subtypes (p, K, and a) in the chronically spinalized dog preparation. Lord et al. (2) postulated the presence of a distinct receptor selective for the enkephalins that was termed the &receptor. Thereafter, binding assays confirmed the existence of p-, &, and Presented in part at the 1989 meeting of the International Anesthesia Research Society, New Orleans, Louisiana, May 1989. Supported by National Institutes of Health grant NS-09871. Received from the Department of Anesthesiology, Yale University, New Haven, Connecticut. Accepted for publication February 28, 1990. Address correspondence to Dr. Collins, Department of Anesthesiology, Yale University School of Medicine, 333 Cedar Street, New Haven. CT 06510. 61990 by the International Anesthesia Research Society

J. G. Collins,

PhD,

Keio Nakatani,

MD,

Sopiate receptors can modulate the input of nociceptive information in the spinal dorsal horn. To study interactions between p- and &receptors at the level of the spinal cord, a combination of the above agonists was injected intrathecally-namely, an ineffective or slightly effective dose of DAGO (1 or 1.5 pg, respectively) that was combined with an ineffective dose of DPDPE (30 pg). The intrathecal combination of DAGO and DPDPE produced significant synergistic suppressive effects of noxiously evoked activity. These findings, again based on relative selectivity, suggest that drug combinations that include both selective p- and Sagonists may be a useful method of lowering the total amount of any one drzig, thus decreasing the likelihood of side efects, while at the same time producing significant analgesia. Clearly, combinations of other p- and Sagonists should be studied to confirm the utility of such combinations. Key Words: ANALGESICS, RECEPTORSSpinal cord. RECEPTORS, OPIATES-p, 6. SPINAL CORD, OPIATE RECEPTORS.

K-receptors (3-5). The status of the a-receptor is unclear, but it is unlikely that it is involved in analgesia. Initial opinion was that analgesia was mediated by p-receptors but not &receptors in the central nervous system (6-8).However, considerable evidence for 6-receptor-mediated analgesia, particularly at the spinal level, has been accumulating (9-13). The development of highly selective opiate agonists has been needed to substantiate the specific opiate subtypes involved in spinal opiate analgesia. A great deal of effort to develop highly selective agents has produced [ D-Ala2, MePhe4, Gly5-011 enkephalin (DAGO), and [D-Pen2,D-Pen5]enkephalin (DPDPE), which have been found to exhibit a high degree of selectivity for p- and &receptors, respectively (14,15). One of the purposes of this study was to compare the ability of intrathecally administered opiate prototypes (selective p-agonist DAGO, less selective pagonist morphine sulfate [MS], selective Gagonist

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1990;71:2%3

DPDPE, less selective Sagonist [D-Ala2-D-Leu5]enkephalin [DADL]) to suppress noxiously evoked activity of wide dynamic range (WDR) neurons in the spinal dorsal horn of decerebrate, spinal cats. Furthermore, the relative selectivity of these agents was evaluated by comparing the reversibility of neuronal suppression by the nonselective opiate antagonist naloxone with that produced by the selective 6 antagonist, N,N-diallyl-Tyr-Aib-Phe-Leu-OH (ICI 174,864) (16). The other purpose of this investigation was to examine the potential synergistic effects of the selective agonists. The complicated pharmacology associated with modulation of sensory information at the level of the spinal cord provides an opportunity for the development of more effective ways to block the processing of pain information. We are interested in determining if there is an interaction between selective p- and Sopiate agonists at the spinal level. If the combination of p- and &receptor agonists at doses that alone produce no or only minimal effects shows significant suppression of noxiously evoked activity of WDR neurons, then the clinical use of such combinations may help to avoid complications including respiratory depression, physical dependency, and/or tolerance.

Methods The protocol of this study was approved by the Yale Animal Care and Use Committee, and institutional, state, and federal guidelines for the humane care and use of laboratory animals were observed during all aspects of this study. Details of the experimental methods have been previously described (17). Ninety-six cats of both sexes, weighing between 3 and 4.2 kg, were prepared for electrophysiologic experiments. General anesthesia (halothane, nitrous oxide, and oxygen) was used for the surgical preparation, which included tracheostomy and catheterization of a jugular vein and a carotid artery. Animals were rendered decerebrate by bilateral electrolytic lesions in the midbrain reticular formation. Decerebration rendered the animals unconscious and enabled general anesthesia to be discontinued. A laminectomy was performed from L-4 to L-6, exposing the lumbar spinal cord. The dura was cut and reflected and physiologic saline at 37°C bathed the spinal cord. The lungs were mechanically ventilated with 100% oxygen and physiologic variables (blood pressure, body temperature, and end-tidal CO,) were monitored and maintained within normal limits. For each experiment, a tungsten microelectrode

was inserted by hydraulic micromanipulator into the lumbar enlargement of the spinal dorsal horn. While the electrode was advanced into the spinal cord, the receptive fields on the ipsilateral hind leg were stimulated. After isolation of a single neuron, the neuron type was identified by the characteristic response to the following stimuli: air puff, light touch with a camel hair brush, pinch with forceps, cooling with ethyl chloride spray, and noxious radiant heat (51°C). A neuron was classified as a WDR neuron based on its response profile to increasing intensities of receptive field stimulation. After identification of a WDR neuron, as a control study, noxious radiant heat (51"C, 8 s) was focused on the center of the receptive field, and the noxiously evoked activity of the neuron was recorded. A thermocouple, which provided feedback for stimulus control, was positioned adjacent to the center of the receptive field to maintain the stimulus temperature constant. After control studies, the physiologic saline that had been bathing the spinal cord was carefully removed and gently replaced with 0.5 mL of drugsaline solution. The drugs used in the experiment were DAGO and DPDPE (Peninsula Laboratories, Belmont, Calif.), DADL and ICI 174,864 (Cambridge Research Biochemicals, Valley Stream, N.Y .), morphine sulfate (Mallinckrodt, St. Louis, Mo.), and naloxone hydrochloride (Manati, Puerto Rico). Intrathecally administered agonists consisted of 1, 1.5, 10, and 20 pg of DAGO ( n = 6, 6, 10, and 6, respectively) or 25, 200, and 400 pg of MS ( n = 8, 7, and 7, respectively), 20, 100, and 250 pg of DADL ( n = 6, 5, and 6, respectively), or 30, 100, and 200 pg of DPDPE ( n = 6, 6, and 13, respectively). Subsequent to spinal agonist administration, noxiously evoked activity of the individual WDR neurons by radiant heat was recorded every 3 min for periods up to 30 min. In most experiments, the ability of naloxone or ICI 174,864 to reverse the effects of agonists was examined by administering 100 pg of naloxone or 200 pg of ICI 174,864 intravenously 31 min after agonist administration. In combination studies, in order to examine the interaction between DAGO and DPDPE, an ineffective or slightly effective dose (1 or 1.5 pg, respectively) of DAGO was combined with an ineffective dose of DPDPE (30 pg). After the control study, the combination of 1 pg ( n = 6) or 1.5 pg ( n = 6) of DAGO and 30 pg of DPDPE was gently applied to the spinal cord. Naloxone (100 pg) was intravenously administered at 31 min after the application of the combined agonists. All data were collected on line and analyzed on line as well as off line by a PDP 11/40 computer. The

p- AND &INTERACTIONS

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ANESTH ANALG 1990;71:2>8

CONTROL

15 MIN

30 MIN

CONTROL

3 MIN

15 MIN

3 MIN

30 MIN

NALOXONE 1 OOug

MS 200pg

.-. P v

a

p I

3

K

1 %

2

n

10 s

Y

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Figure 1. Effects of DAGO on a single WDR neuron. Intrathecal DAGO (10 pg) suppressed the evoked activity to 23% of control at 30 min and intravenous naloxone (100 pg) produced reversal to 81% of control.

integrated neuronal activity and the analog skin temperature were recorded on a polygraph. The variables were expressed as percent of control values or as percent suppression, plus or minus standard error of the mean. The dose-dependent suppression of opiates was analyzed using analysis of variance followed by Student's t-test. The reversal effects of antagonists and the suppressive effects of combined agonists were analyzed using paired and unpaired Student's f-test, respectively. A two-tailed P value of

The antinociceptive role of mu- and delta-opiate receptors and their interactions in the spinal dorsal horn of cats.

This study was undertaken to examine the antinociceptive roles of different subtypes of opiate receptors and their interactions at the level of the sp...
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