JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2002, 53, 4, 741750
www.jpp.krakow.pl
a b b M. ZUBRZYCKA , J. FICHNA , A. JANECKA *
EFFECT OF CEREBRAL VENTRICLES PERFUSION WITH
MORPHICEPTIN AND MET-ENKEPHALIN ON TRIGEMINO-
HYPOGLOSSAL REFLEX IN RATS
a b Department of Experimental and Clinical Physiology and Department of Medicinal Chemistry,
Institute of Physiology and Biochemistry, Medical University of Lodz, Poland
Opioids administered by intracerebroventricular injections produce analgesic
responses in rats. The present study was undertaken to investigate the effects of a
highly selective µ-opioid receptor ligand morphiceptin on trigemino-hypoglossal
reflex in rats. The analgesic effect of morphiceptin was compared with another
opioid peptide, Met-enkephalin. With the experimental settings used in this study, we
have demonstrated that both morphiceptin and Met-enkephalin show significant
dose-dependent analgesic effects after i.c.v. administration in rats as assayed by
trigemino-hypoglossal reflex test. The antinociceptive response to Met-enkephalin
was short lasting and was observed 10 to 15 min after i.c.v. perfusion. Morphiceptin
had a relatively longer duration of antinociceptive action, the effect was observed 20-
50 min after i.c.v. perfusion. Neither morphiceptin nor Met-enkephalin produced
antinociception after peripheral injections. The results of the present study indicate
that both tested peptides act at µ-opioid receptors situated in the central nervous system. They also suggest that µ-opioid receptors present in the central nervous
system are an important element of the trigemino-hypoglossal reflex arc. For that
reason selective µ-opioid receptor ligands, like morphiceptin, inhibit the reflex more
significantly.
Keywords: opioid receptors, evoked tongue jerks, analgesic effects 742
INTRODUCTION
Morphine is an alkaloid which relieves pain and acts in the central nervous system (CNS). Morphine and its derivatives are the most potent class of analgesics used clinically. The high potency and specificity of morphine suggested that it may bind to some receptors in the CNS to induce its biological effects.
In the early 1970s specific opioid receptors were identified in brain and peripheral tissues (1, 2). Since morphine is not an endogenous ligand for these receptors the search for the endogenous neurotransmitters at opioid receptors had begun. This led to identifying the enkephalins (3, 4), dynorphins (5) and β- endorphin (6) as natural peptide ligands for these receptors. Enkephalins and dynorphins are widely distributed in the CNS and are considered the predominant central opioid peptide neurotransmitters (7, 8).
Extensive physiological and pharmacological studies have defined at least three major types of opioid receptors; µ, δ and κ (9). Each of these receptors maintains a unique pattern of expression while displaying characteristic binding affinities for various subtype-selective ligands. Named after morphine, the
β-opioid receptor is the physiological target of such potent analgesics as morphine and fentanyl, as well as the endogenous opioid peptides: β-endorphin,
Met-enkephalin (Met-Enk), dynorphins (10) and endomorphins (11).
Another class of opioid peptides that show some preference for the -opioid receptor are pronase-resistant peptides, which are present in body fluids for example β-casomorphins (β-CMs) in bovine or human milk (12, 13). β-CMs are obtained from the milk protein, β-casein, by proteolytic fragmentation. A
tetrapeptide amide, Tyr-Pro-Phe-Pro-NH2 (morphiceptin), was originally synthesised as an analogue possessing the N-terminal tetrapeptide fragment of bovine β-CM-7 (14) and then was isolated from an enzymatic digest of bovine β- casein (15). Morphiceptin is of particular interest, because it was found to have morphine-like physiological activity, to bind with fairly high affinity and to be extremely selective for the µ-opioid receptor (15).
The trigemino-hypoglossal reflex may serve as a model to test the effects of various neuropeptides present in the cerebrospinal fluid and brainstem centers
(16). The sensory and motor center of the trigemino-hypoglossal reflex is located in the vicinity of the fourth cerebral ventricle. Since the hypoglossal nucleus is located superficially under the floor of the fourth ventricle, it is possible that the chemical compounds may penetrate into this nucleus from the lumen of the fourth ventricle faster than into the sensory nucleus of the trigeminal nerve, which lies further from the lumen of the fourth ventricle. The excitation or inhibition of the trigemino-hypoglossal reflex may be evaluated according to the mean amplitude of recorded evoked tongue jerks. 743
In the present study we investigated the effects of intracerebroventricular
(i.c.v.) and peripheral (intravenous, i.v. and intraperitoneal, i.p.) administration of morphiceptin and Met-Enk on trigemino-hypoglossal reflex in rats.
MATERIALS AND METHODS
Experimental animals and anaesthesia
The experimental protocol in the present study was approved by the Local Ethical Committee for Animal Research and it complies with the European Community guidelines for the use of experimental animals.
Mail Wistar rats weighing 350-380 g were used for the experiments. The animals were kept
o under standard conditions: temperature 22 C, a 12 h light-dark cycle, and allowed tap water and rodent chow ad libitum. The rats were anaesthetised with a single i.p. injection of chloralose solution in a dose of 150 mg/kg body weight.
Perfusion of cerebral ventricles in rats
The rats head was immobilised by introduction of ear bars into the external auditory meati and fixing the maxilla with jaw clamps in a stereotaxic instrument specially adapted for perfusion of the cerebral ventricles (Fig. 1). The skin of the animals head, anaesthetised with 2% polocaine solution, was incised in the midline and the skull bones were exposed. On the basis of modified co-
Fig. 1. Position of a rat skull in a stereotaxic instrument adapted for perfusion of cerebral ventricles.
A inflow cannula for lateral ventricle, B outflow cannula for cerebellomedullar cistern. 744
ordinates given by De Groots stereotaxic atlas (17), the sites for drilling holes in the skull bones were determined: to the lateral ventricles - 9 mm anterior to the frontal interaural zero plane and 3 mm lateral to the sagittal zero plane.
The system of cerebral ventricles was perfused by inserting stainless steel cannulae into both lateral ventricles and to the cerebellomedullary cistern (Fig. 2). The container with perfusion fluid was positioned 20 cm above the animals head. McIlwain-Rodnights solution, prepared according to Daniel and Lederis (18) was used for perfusion. The outflow cannula inserted into the cerebellomedullary cistern was connected to a polyethylene tube ca 100 cm long which provided
D
@
ETJ[%] (7->
7 LPH > PL Q @
QPROPO QPROPO QPROPO QPROPO
E
@
(7->
ETJ[%]
7LPH > PL Q@
QPROPO QPROPO QPROPO QPROPO
Fig. 2. Time-course of inhibition of the evoked tongue jerks (ETJ) induced by tooth pulp stimulation in rats during i.c.v. perfusion with morphiceptin (a) and Met-Enk (b) (10, 25, 50 and 100 nmol/ml). Values illustrated in the graph represent means ± standard deviation (x ± SD). 745
the outflow for the perfusion fluid. The flow rate at the end of the tubing in the course of perfusion was 0.5-0.7 ml/10 min.
After control perfusion with McIlwain-Rodnights solution, the cerebral ventricles were perfused with solutions of morphiceptin or Met-Enk added to McIlwain-Rodnights solution to obtain desired concentrations.
At the end of each experiment the cerebral ventricles were perfused with 1% trypan blue solution till the stain appeared in the outflow tubing leading out of the cerebellomedullary cistern.
Peripheral infusion in rats
The i.v. infusion in rats was performed by inserting polyethylene cannula filled with McIlwain-
Rodnights solution to femoral vein and infusing morphiceptin and Met-Enk solutions with the average speed 0,5 ml/min. Morphiceptin and Met-Enk were also administered by i.p. injections.
Tooth pulp stimulation
After placing the animals head in a stereotaxic instrument, the tips of incisors were cut off with a dental separator and stainless steel wire electrodes were inserted into the pulp and fixed with dental cement. Bipolar stimulation was delivered 6 times per minute, with a train of 4 impulses of
200 Hz frequency, 3 ms single impulse duration and 4-5 V amplitude, using a programmed stimulator.
The amplitudes of electrical impulses stimulating the incisor pulp were adjusted individually for each animal. At the beginning of each experiment the intensity of stimulus inducing maximum tongue jerks was determined. Then, the amplitude of impulses was reduced to obtain the amplitude of evoked tongue jerks (ETJ) equal to half of the maximum values. The amplitude of stimulating impulses adjusted in this way, as well as the other parameters, remained unchanged till the end of the experiment.
Recording tongue jerks
The tip of the animals tongue was attached with a silk thread to an isotonic rotating tensometric transducer. The amplitude of tongue jerks was recorded by a Line Recorder TZ-
4620 (Laboratorni Pristroje Praha, Czech Republic). The tongue was stretched with the same force, ca. 5.8 G throughout the experiment, the amplification of the recorder also remained unchanged.
For each animal during the first 10 min of perfusion or within 10 min after injection the amplitude of tongue jerks evoked by tooth pulp stimulation was recorded. The mean amplitude of ETJ was regarded as an indicator of magnitude of the trigemino-hypoglossal reflex. Mean amplitudes of ETJ, induced by pulp stimulation during perfusion or after peripheral administration of McIlwain-Rodnights solution and investigated solutions, were compared separately.
Data processing and statistical analysis
The amplitude of ETJ recorded on a tape was measured in millimetres. The arithmetical means were calculated from 30 ETJ obtained in the 5 min course of perfusion with the investigated solution or adequate 5 min time interval after peripheral injection. Statistical comparison of the results was performed by using a one-way analysis of variance (Anova). A P value of less than 0.01 was considered to be statistically significant. All values were presented as means ± standard deviation (x ± SD). 746
RESULTS
Experiments were carried out on ten groups of rats, n=6 animals in each group. In all tested groups the nociception was measured using ETJ and the baseline was correlated with control infusion or injection with McIlwain-
Rodnights solution.
I.c.v. perfusion with morphiceptin
Morphiceptin in the concentrations 10, 25, 50 and 100 nmol/ml showed significant inhibition of the amplitude of ETJ (87,08±1,95%, 53,24±1,62%,
35,00±2,92%, 21,38±2,00%, respectively, p<0.01), with the lowest value observed after 20 min of perfusion (Fig. 3). In higher concentrations (200 and 300 nmol/ml) morphiceptin induced convulsions and generated respiratory system failure.
D D (a)
@
@
(7->
(7->
&RQFHQWUDWLRQ>QPROPO@ &RQFHQWUDWLRQ>QPROPO@
E (b) E
@
@
(7->
(7->
Fig. 3. Dose-response correlation for &RQFHQWUDWLRQ>QPROPO@ i.c.v. perfusion with morphiceptin (a) &RQFHQWUDWLRQ>QPROPO@ and Met-Enk (b). 747
I.c.v. perfusion with Met-Enk
Met-Enk in the concentrations 10, 25, 50 and 100 nmol/ml showed inhibition of the amplitude of ETJ (94,85±4,22%, 87,62±3,33%, 60,25±5,13%,
46,50±3,33%, respectively, p<0.01), with the lowest value observed after 10 to 15 min of perfusion (Fig. 4).
Peripheral injections of morphiceptin
The i.v. and i.p. administration of morphiceptin or Met-Enk in the concentrations 10, 25, 50, 100, 200 and 300 nmol/ml showed no significant change in ETJ.
Calculating EC50 values
For both peptides, morphiceptin and Met-Enk, the median effective
concentration (EC50) was calculated. EC50 is a concentration at which 50% of rats
exhibit the analgesic effect (19). EC50 values for morphiceptin and Met-Enk after i.c.v. administration are 31,95±2,17 nmol/ml and 90,56±4,22 nmol/ml, respectively (p<0.01).
DISCUSSION
In the experiments performed in this study, the effect of neuropeptides on neural structures was measured by the magnitude of tongue refractory movements evoked by stimulation of a trigeminal nerve branch. The mechanisms of nociception in the region supplied by the V nerve have been investigated for over ten years using trigemino-hypoglossal reflex (20, 21, 22). This reflex can serve as a good model to test the effects of various neuropeptides perfused through the cerebral ventricles on the brain stem nuclei. Stretching the tongue does not alter the excitability of motoneurons of the XII nerve nucleus because no intrafusal spindle has been detected in the muscles of the tongue (23). The excitation or inhibition of the trigemino-hypoglossal reflex may be evaluated according to the amplitude of the ETJ and may be an objective indicator of the intensity of nociceptive impulses (21, 22).
Morphiceptin is a potent opioid peptide from the group of β-casomorphins, acting with high selectivity and affinity at µ-opioid receptors. Met-Enk is known to have affinity at both µ- and δ- opioid receptors with preference to δ-opioid receptor.
Analgesic activities of morphiceptin and Met-Enk were examined by intracerebroventricular administration in rats. The antinociceptive response to
Met-Enk was short lasting and was observed 10 to 15 min after i.c.v. perfusion.
Morphiceptin had a relatively longer duration of antinociceptive action, the effect was observed 20-50 min after i.c.v. perfusion and was dose dependent. The 748
results indicate that both peptides act at µ-opioid receptors, situated in the CNS and that they produce analgesia and attenuate the effects evoked by nociceptive stimuli through secondary neuromediators activated after the reaction between the ligand and the receptors located in the brain structures (24, 25).
The higher concentrations of morphiceptin and Met-Enk, 200 and 300 nmol/ml, were found to affect the respiratory centre in medulla oblongata, causing deep depression of the activity of the respiratory system and eventually leading to total apnoea. Morphiceptin in those toxic concentrations was also responsible for strong convulsive response of the rat. Both, clonic and respiratory effects produced by high concentrations of morphiceptin resemble those of morphine origin (24, 26).
Neither morphiceptin nor Met-Enk produced antinociception after peripheral injections. It suggests that the peptides were not able to react at opioid receptors situated in the brain structures, involved in the trigemino-hypoglossal reflex arc evoked by tooth pulp stimulation. A possible explanation is that morphiceptin and
Met-Enk administered peripherally are inactivated by specific blood enzymes, peptidases (27), or that they cannot cross the blood-brain barrier and reach the
CNS due to their pharmacological properties (28-30).
The good correlation between analgesic activities and receptor binding affinities of morphiceptin and Met-Enk confirms the previous observations (25) that µ-opioid receptor mediates the major analgesic activity of opioid peptides.
In summary, we have demonstrated that the trigemino-hypoglossal reflex can be inhibited by i.c.v. administration of µ-opioid receptor ligands. It suggests that
µ-opioid receptors present in the CNS are an important element of the trigemino- hypoglossal reflex arc. Morphiceptin, as a more selective µ-opioid receptor ligand, inhibits the trigemino-hypoglossal reflex in rats more significantly than
Met-Enk.
Acknowledgements: The study was supported by grants of Medical University of Lodz (No.
502-11-812 and 502-11-728).
REFERENCES
1. Pert CB, Snyder SN. Opiate receptor: Demonstartion in nervous tissue. Science 1973; 179:
1011-1014.
2. Snyder SH, Pasternak GW, Pert CB. Opiate receptor mechanisms. In Handbook of
Psychopharmacology Synaptic Modulators. LL Iversen, SH Snyder (eds). New York
London, Plenum Press, 1971, pp. 329-360.
3. Hughes J, Smith T, Kosterlitz H, Fothergill L, Morgan B, Morris H. Identification of two
pentapeptides from the brain with potent opiate agonist activity. Nature 1975; 258: 577-579.
4. Simantov R, Snyder SH. Morphine-like peptides, leucine enkephalin and methionine
enkephalin: interactions with the opiate receptor. Mol Pharmacol 1976; 12: 987-998.
5. Goldstein A. Binding selectivity profiles for ligands of multiple receptor types: Focus on opioid
receptors. Trends Pharmacol. Sci 1987; 8: 456-459. 749
6. Loh HH, Tseng LF, Wei E, Li CH. beta-Endorphin is a potent analgesic agent. Proc Natl Acad
Sci 1976; 73: 2895-2898.
7. Akil H, Richardson DE, Hughes J, Barchas D. Enkephalin-like material elevated in ventricular
cerebrospinal fluid of pain patients after analgetic focal stimulation. Science 1978; 201: 463-465.
8. Khachaturian H, Lewis M, Watson SJ. Enkephalin systems in diencephalon and brainstem of
rat. J Comp Neurol 1983; 220: 310-320.
9. Goldstein A, Tachibana S, Lowney L, Hunkapiller M, Hood L. Dynorphin (1-13),
anextraordinary potent opioid peptide. Proc Natl Acad Sci USA 1979; 76: 6666-6670.
10. Wood PL, Iyengar S. Central actions of opiates and opioid peptides: In vivo evidence for opioid
receptor multiplicity. In The Opiate Receptors. GW Pasternak (ed). New Yersey, Humana Press,
1988.
11. Zadina JE, Hackler L, Ge LJ, Kastin AJ. A potent and selective endogenous agonist for the mu
opiate receptor. Nature 1997; 386; 499-502.
12. Brantl V, Teschemacher H, Henschen A, Lottspeich F. Novel opioid peptides derived from
casein (beta-casomorphins). I. Isolation from bovine casein peptone. Hoppe-Seylers Z Physiol
Chem 1979; 360: 1211-1216.
13. Brantl V, Pfeiffer A, Herz A, Henschen A, Lottspeich F. Antinociceptive potencies of beta-
casomorphin analogs as compared to their affinities towards mu and delta opiate receptor sites
in brain and periphery. Peptides 1982; 3: 793-797.
14. Chang K-J, Killian A, Hazum E, Cuatrecasas P, Chang J-K. Morphiceptin (Tyr-Pro-Phe-Pro-
NH2): A potent and specific agonist for morphine (µ) receptors. Science 1981; 121: 75-77.
15. Chang K-J, Su IF, Brent D A, Chang J-K. Isolation of a specific mu-opiate receptor peptide,
morphiceptin, from an enzymatic digest of milk proteins. J Biol Chem 1985; 260: 9706-9712.
16. Luczynska M, Traczyk WZ. Influence of cerebral ventricle perfusion with hexapeptide
derivatives of substance P on evoked tongue jerks in rats. Brain Res 1980; 198: 403-410.
17. De Groot J. The rat forebrain in Stereotaxic Coordinates. Amsterdam 1963.
18. Daniel AR, Lederis K. Release of neurohypophysial hormones in vitro. J Physiol 1967; 190:
171-187.
19. Hendershot LC, Forsaith J. Antagonism of the frequency of phenylquinone-induced writhing in
the mouse by weak analgesics and nonanalgesics. J Pharmacol Exp Ther 1959; 125: 237-240.
20. Lesnik H, Traczyk WZ. Effect of increased concentrations of Ca++ and Mg++ in the fluid
perfusing the cerebral ventricles, and hypoxia on evoked tongue jerks. Acta Physiol Pol 1978;
29:27-35.
21. Luczynska M, Traczyk WZ. Influence of cerebral ventricle perfusion with hexapeptide
derivatives of substance P on evoked tongue jerks in rats. Brain Res 1980; 198:403-410.
22. Zubrzycka M, Janecka A, Koziolkiewicz W, Traczyk WZ. Inhibition of tongue reflex in rats by
tooth pulp stimulation during cerebral ventricle perfusion with (6-11) substance P analogs.
Brain Res 1997; 753:128-132.
23. Green JD, Negishi K. Membrane potentials in hypoglossal motoneurons. J Neurophysiol 1963;
26:835-856.
24. Brantl V, Teschemacher H, Bläsig J, Henschen A, Lottspeich F. Opioid activities of β-
casomorphins. Life Sci 1981; 28: 1903-1909.
25. Chang KJ, Cuatrecasas P, Wei ET, Chang JK. Analgesic activity of intracerebroventricular
administration of morphiceptin and beta-casomorphins: correlation with the morphine (mu)
receptor binding affinity. Life Sci 1982; 30: 1547-1551.
26. Schug SA, Zech D, Grond S. Adverse effects of systemic opioid analgesics. Drug Saf 1992;
7: 200-213.
27. Taniguchi T, Fan XT, Kitamura K, Oka T. Effects of peptidase inhibitors on the enkephalin-
induced anti-nociception in rats. Jpn J Pharmacol 1998; 78: 487-492. 750
28. Adler MW, Rowan CH, Geller EB. Intracerebroventricular vs. Subcutaneous drug
administration: apples and oranges? Neuropeptides 1984; 5: 73-76.
29. Egleton RD, Abbruscato TJ, Thomas SA, Davis TP. Transport of opioid peptides into the central
nervous system. J Pharm. Sci 1998; 87: 1433-1439.
30. Vujic-Redzic V, Dimitrijevic M, Stanojevic S, Kovacevic-Jovanovic V, Miletic T, Radulovic J.
Peripheral effects of methionine-enkephalin on inflammatory reactions and behavior in rat.
Neuroimmunomodulation 2000; 8: 70-77.
Received: June 12, 2002
Accepted: October 29, 2002
Authors address: Dr. Anna Janecka, Department of Medicinal Chemistry, Medical University of
Lodz, ul. Lindleya 6, 90-131 Lodz, Poland, Tel./fax: (4842) 6784277
E-mail: [email protected]