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Respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem
Brain Research, 138 (1977) 585-590 © Elsevier/North-HollandBiomedicalPress
585
Respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem
JESOS FLOREZand AFRICA MEDIAVILLA Department of Pharmacology, Faculty of Medicine, University o]" Santander, National Med. Center 'M. de Valdecilla', Santander (Spain)
(Accepted August 3rd, 1977)
Increasing evidence is being accumulated that several endogenous peptides may serve as the natural ligands for opiate receptors. Some opiate effects on the central nervous system have been reported to follow the cerebroventricular and brain administration of enkephalins and endorphins, such as analgesia and other behavioral effects1,2,5,1a,16,17,19, depression of spontaneous and induced firing of individual neurons 4,11,12,15, and appearance of physical dependence22. Since depression of the respiratory center is one of the most characteristic action of opiates, it seems pertinent to investigate the respiratory actions of endorphins and other endogenous peptides that might present opiate activities. It has been shown that, of all cerebrospinal fluid spaces, the ventral surface of the brain stem is the most sensitive site to the respiratory depressant action of morphine 9. In the present study we report the respiratory action of met-enkephalin injected into the ventral surface of the brain stem. The study was performed on 16 cats lightly anesthetized with a mixture of pentobarbital (25 mg/kg) and urethane (500 mg/kg). Although these anesthetics depress the respiratory center 10, the respiratory activity remains fairly steady for about 4 h. The trachea was cannulated and a carotid artery was catheterized for the purpose of blood pressure and cardiotachogram recordings through appropriate transducers. An indwelling catheter in the cephalic vein was used for i.v. administrations. For injection at the ventral surface of the brain stem, a stainless steel hypodermic needle was introduced stereotaxically following the method described by Feldberg and GuptaS; the angle, however, was 22-24 °C directed posteriorly. The spread of the metenkephalin solution was assessed by injecting a similar volume of dye at the end of the experiment; the cat was killed 5 min later. In 14 cats, the staining was extended on both sides, 5-6 mm from the mid line, and dorsocaudally from the trapezoid bodies down to the caudal part of the medulla; no staining was observed in the fourth ventricle. Two experiments were discarded because the spread of the dye was only restricted to one side of the brain stem. Respiration was continuously monitored by using a pneumotachograph adapted to a Nihon RJF-5 integrator to yield the full respiratory wave. End-expiratory COs was monitored with a Godart capnograph. Respiratory function was assessed by
586 measuring the spontaneous resting respiratory parameters (frequency, tidal volume and minute volume), as well as their responses to stimulation with 5 ~ COz in 02. Body temperature was measured with a rectal thermometer and was maintained at 37 0.5 °C with a heating pad. Saline solutions of met-enkephalin (Serva Feinbiochemica) were prepared daily and injected in a volume of 50/~l. In a first group of 7 cats, the effects of metenkephalin were observed for 1 h; in 4 of these, a second dose was injected subsequently and the effects were followed for another hour. In a second group of 5 cats, naloxone HCl (Endo Laboratories) dissolved in 0.5-1 ml of saline was injected
A 30[ 'llUl ..............~ ' , ' , ; ; ' , ' : . . ' . 0 t "'''' ............................................
VT ml.
'i."~ ..............................
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200I
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" -
0
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1 min
|
1601 Met- ~. e l~alin,.1.65, pnlo.I ventral aurmce Drain stem
t Naloxone 0.1mg/kg i.v.
O'
B
M~-Enk
Fig. 1. Effectsof met-enkephalin applied to the ventral surface of the brain stem on respiration, blood pressure and cardiac frequency. From top to bottom: integrated recording of respiratory movements (inspiration downwards), pneumotachogram, blood pressure and heart rate. A: effects of metenkephalin and complete reversal by naloxone. B: effects of a second dose of met-enkephalin administered 15 min after naloxone; the antagonist still prevents a full effect of met-enkephalin.
587 t
I I
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l,Y,, 01
3
5
15
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II
r
,
I II 1
5
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,,, 30
60
rain.
1
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, , , ,
3
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MINUTE VOLUME
•
BLOOD PRESSURE
O
TIDAL VOLUME
Z~
HEART RATE
r-I
RESPIRATORY FREQUENCY
30
60
mln.
Fig. 2. Time course of the effects of met-enkephalin applied to the ventral surface of brain stem, on respiratory parameters, blood pressure and cardiac frequency. Values are expressed as the per cent change calculated for each cat in relation to the control value. Symbols represent the mean of 7 cats 4- S.E.M. Control values: frequency 37.5 ± 2.5 bpm, tidal vol. 20.3 4- 1.1 ml, minute vol. 760.2 4- 23.8 ml, blood pressure 132.7 ± 3.9 mmHg, cardiac rate 229.1 ± 6.5 bpm. Inset: respiratory response to stimulation with COs 5 ~, for a constant end-expiratory CO2 level of 6.2%, in the control period and 15 min after met-enkephalin. i.v. between the two doses of met-enkephalin, 5 min prior to the second dose of the peptide. In a further two cats, naloxone was injected at the time of the peak respiratory depression. Met-enkephalin, at the dose of 1.6/zmole directly applied to the ventral surface of the brain stem, consistently depressed respiration (Fig. 1). The response was characterized by an immediate reduction of tidal volume (--35.8 ~o, range: --19.5 to --57), whereas frequency was less affected ( - - 1 9 . 7 ~ , range + 1 4 to --66.4). The time-response curves for every parameter are shown in Fig. 2. A distinctive feature was the short latency and duration of the effect: the depression started a few seconds after injection and the peak effect on minute volume was attained in about 50 sec ~---47.3 ~o, range: --22.0 to --80), but at 3 min the magnitude of the depression had been reduced substantially (--15.0~o, range: 0 to --27.0). At 15 min the values of resting minute volume were similar to those of control, but the ventilatory response to COs stimulation was still significantly lower than the control response (Fig. 2 inset, paired t-test P < 0.05). The assessment of tidal volume was critical since, by the time of maximal depression, frequency was minimally affected and thereafter it was slightly increased. End-expiratory CO2 values remained above control for about 15 min; indeed, the low tidal volume accounted for a reduced alveolar ventilation.
588 Coincident with the respiratory effects, met-enkephalin caused a reduction in blood pressure and cardiac frequency (Figs. 1 and 2). The intensity of the depression was smaller than that induced on respiration, and the time course of the responses also differed slightly: the peak effect appeared between 1 and 2 min after injection (for blood pressure,--29.7/0, r a n g e - - 1 3 . 8 t o - - 5 2 . 0 ; for cardiac frequency, - - 1 6 6"/ /o~ range: --6.6 to --33.8), and the recovery was more gradual and delayed, so that at 5 min the depression showed a value that corresponded to more than half the peak effect. Naloxone was able to antagonize all of the depressant effects. When it was injected i.v. at the time of the peak depression, a complete and abrupt reversal of both, the respiratory and cardiovascular effects was obtained (Fig. 1). In another set of experiments, the ability of naloxone to prevent the respiratory and cardiovascular actions of a second dose of met-enkephalin was determined. In 4 control cats, the responses to a second dose of met-enkephalin injected 60 min after the first one was similar to those induced by the first dose, in quality as well as intensity (Table I, A). In a second group of 5 cats, naloxone (0.2 mg/kg) was administered i.v. 5 min before injecting the second dose of met-enkephalin: a partial antagonism of the respiratory and cardiovascular effects was clearly obtained (Table I, B). The respiratory and cardiovascular responses to met-enkephalin seem to have the characteristics of an opioid action: ventilation, blood pressure and heart rate were depressed, and these effects were consistently antagonized by naloxone. Indeed, the difference between the time course of the respiratory effect of met-enkephalin and that reported for morphine 9 is evident, but it can be attributed to the kinetic peculiarities of each compound. It has been proposed that the short duration of the analgesic action of met-enkephalin observed in rodents is the consequence of enzymatic cleavage 14. In this respect, the analysis of the effects of less labile endorphins will provide further insight into the action of these compounds at the brain stem level. Respiratory depression o/
. . . .
TABLE I Antagonism of naloxone to the respiratory and cardiovascular depressant effects of met-enkephalin
Two doses of met-enkephalin, 60 rain apart, were given to two groups of cats. In the group B, naloxone was administered i.v. 5 rain before the second dose of the peptide. Values are expressed as the mean ± S.E.M. Asterisks indicate statistically significant difference related to the effect of the first dose at a P < 0.05 (Student's paired t-test). Peak depression (%) Met-enkephalin 1st dose
Met-enkephalin 2nd dose
--44.2 :t: 8.8 --30.8 ± 8.9 --13.3 i 4.0
---49.0 ± 10.6 --28.7 :k 7.0 --12.2 ± 1.7
4 5 . 3 ± 9.4 --33.1 £- 2.5 --19.2 5_ 4.5
--21.4 :t: 8.3* --15.7 ± 3.7* - - 5.6 ± 1.9"
(A) Control (n - 4)
Respiratory minute volume Blood pressure Cardiac rate (B) Naloxone (n = 5)
Respiratory minute volume Blood pressure Cardiac rate
589 following the intraventricular administration of a pituitary peptide in rats has been described, but neither the magnitude nor the time course of the effects was reportedlL It must be pointed out that met-enkephalin was administered into anesthetized animals, and that some synergism of the peptide with the anesthetics can be responsible for the full effect. In any case, when naloxone was injected at the time of peak depression, the effects of the peptide were completely reversed. The dose of met-enkephalin used in the present study was about twice that reported by other investigators, who found negative results in analgesic and behavioral studies also conducted in cats s,17, and it was approximately 10 times the equieffective dose of morphine. It reflects the consequence of the small potency of the compound as observed in the opiate displacement and guinea pig iluem assaysS, 6, when it is compared with larger fragments of the lipotropin molecule and other endorphins. It is, therefore, necessary to analyze the activity of these endorphins at the level of the brain stem and compare their potency ratios with those obtained from other in vitro as well as in vivo studies. The short latency of the responses suggests that met-enkephalin is acting on some of the ventral surface areas in the brain stem involved in respiratory and cardiovascular regulation 20,21, rather than on the pontomedullary centers. The exact localization of the affected area will require a more discrete administration of the drug, but, since met-enkephalin presents such a short duration of action, it serves as a good candidate for this type of studies. It will be important to elucidate the existence of opioid receptors on those surface areas, which can contribute to the consistent respiratory depression induced by the narcotic analgesics. This work was supported by a research grant from the Spanish Health Service. Naloxone was kindly supplied by Dr. M. J. Ferster (Endo Lab., Europe). The technical assistance of J. M. Shnchez is gratefully acknowledged.
1 Belluzzi, J. D., Grant, N., Garsky, V., Sarantakis, D., Wise, C. D. and Stein, L., Analgesia induced in vivo by central administration of ertkephalin in rat, Nature (Lond.), 260 (1976) 625-626. 2 Bloom, F., Segal, D., Ling, N. and Guillemin, R., Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness, Science, 194 (1976) 6304532. 3 Bradbury, A. F., Smyth, D. G., Snell, C. R., Birdsall, N. J. M. and Hulme, E. C., C-fragment of lipotropin has a high affinity for brain opiate receptors, Nature (Lond.), 260 (1976) 793-795. 4 Bradley, P. B., Briggs, I., Gayton, R. J. and Lambert, L. A., Effects ofmicroiontophoretically applied methionine-enkephalin on single neurones in rat brainstem, Nature (Lond.) ,261 (1976) 425--426. 5 Bfischer, H. H., Hill, R. C., R/~mer,D., Cardinaux, F., Closse, A., Hauser, D. and Pless, J., Evidence for analgesic activity of enkephalin in the mouse, Nature (Lond.), 261 (1976) 423-425. 6 Cox, B. M., Goldstein, A. and Li, C. H., Opioid activity ofa peptide, fl-lipoprotin-(61-91), derived from fl-lipotropin, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 1821-1823. 7 Feldberg, W. and Gupta, K. P., Morphine hyperglycaemia, J. PhysioL (Lond.), 238 (1974) 487-502. 8 Feldberg, W. and Smyth, D. G., The C-fragment of lipotropin - - a potent analgesic, J. PhysioL (Lond.), 260 (1976) 30-31P. 9 Fl6rez, J., McCarthy, L. E. and Borison, H. L., A comparative study in the cat of the respiratory effects of morphine injected intravenously and into the cerebrospinal fluid, J. PharmacoL exp. Ther., 163 (1968) 448--455.
590 10 Fl6rez, J. and Borison, H. L., Effects of central depressant drugs on respiratory regulation in the decerebrate cat, Resp. Physiol., 6 (1969) 318-329. 11 Frederickson, R. C. A. and Norris, F. H., Enkephalin-induced depression of single neurons in brain areas with opiate receptors - - antagonism by naloxone, Science, 194 (1976) 440-442. 12 Gent, J. P. and Wolstencroft, J. H., Effects of methionine-enkephalin and leucine-enkephalin compared with those of morphine on brain stem neurones in cat, Nature (Lond.), 261 (1976) 426427. 13 Gr~tf, L., Sz6kely, J. I., R6nai, A. Z., Dunai-Kov~ics, Z. and Bajusz, S., Comparative study on analgesic effect of Meta-enkephalin and related lipotropin fragments, Nature (Lond.), 263 (1976) 240-242. 14 Hambrook, J. M., Morgan, B. A., Rance, M. J. and Smith, C. F. C., Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates, Nature (Lond.), 252 (1976) 782-783. 15 Hill, R. G., Pepper, C. M. and Mitchell, J. F., Depression of nociceptive and other neurones in the brain by iontophoretically applied met-enkephalin, Nature (Land.), 262 (1976) 604-606. 16 Jacquet, Y. F. and Marks, N., The C-fragment of/%lipotropin: an endogenous neuroleptic or antipsychotogen?, Science, 194 (1976) 632-635. 17 Meglio, M., Hosobuchi, Y., Loh, H. H., Adams, J. E. and Li, C. H.,/~-endorphin: behavioral and analgesic activity in cats, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 774-776. 18 R6nai, A. Z., Sz6kely, J. I., Gr~tf, L., Dunai-Kov~cs, Z. and Bajusz, S., Morphine-like analgesic effect of a pituitary hormone, /3-1ipotropin, Life Sci., 19 (1976) 733-738. 19 Teschemacher, H., Bl~issig, J. and Kromer, W., Porcine pituitary peptides with opiate-like activity: partial purification and effects in the rat after intraventricular injection, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 294 (1976) 293-295. 20 Trouth, C. O., Loeschcke, H. H. and Berndt, J., Topography of the circulatory responses to electrical stimulation in the medulla oblongata. Relationship to respiratory responses, Pfliigers Arch. ges. Physiol., 339 (1973) 185-201. 21 Trouth, C. O., Loeschcke, H. H. and Berndt, J., A superficial substrate on the ventral surface of the medulla oblongata influencing respiration, Pfliigers Arch. ges. Physiol., 339 (1973) 135-152. 22 Wei, E. and Loh, H. H., Physical dependence on opiate-like peptides, Science, 193 (1976) 12621263.
585
Respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem
JESOS FLOREZand AFRICA MEDIAVILLA Department of Pharmacology, Faculty of Medicine, University o]" Santander, National Med. Center 'M. de Valdecilla', Santander (Spain)
(Accepted August 3rd, 1977)
Increasing evidence is being accumulated that several endogenous peptides may serve as the natural ligands for opiate receptors. Some opiate effects on the central nervous system have been reported to follow the cerebroventricular and brain administration of enkephalins and endorphins, such as analgesia and other behavioral effects1,2,5,1a,16,17,19, depression of spontaneous and induced firing of individual neurons 4,11,12,15, and appearance of physical dependence22. Since depression of the respiratory center is one of the most characteristic action of opiates, it seems pertinent to investigate the respiratory actions of endorphins and other endogenous peptides that might present opiate activities. It has been shown that, of all cerebrospinal fluid spaces, the ventral surface of the brain stem is the most sensitive site to the respiratory depressant action of morphine 9. In the present study we report the respiratory action of met-enkephalin injected into the ventral surface of the brain stem. The study was performed on 16 cats lightly anesthetized with a mixture of pentobarbital (25 mg/kg) and urethane (500 mg/kg). Although these anesthetics depress the respiratory center 10, the respiratory activity remains fairly steady for about 4 h. The trachea was cannulated and a carotid artery was catheterized for the purpose of blood pressure and cardiotachogram recordings through appropriate transducers. An indwelling catheter in the cephalic vein was used for i.v. administrations. For injection at the ventral surface of the brain stem, a stainless steel hypodermic needle was introduced stereotaxically following the method described by Feldberg and GuptaS; the angle, however, was 22-24 °C directed posteriorly. The spread of the metenkephalin solution was assessed by injecting a similar volume of dye at the end of the experiment; the cat was killed 5 min later. In 14 cats, the staining was extended on both sides, 5-6 mm from the mid line, and dorsocaudally from the trapezoid bodies down to the caudal part of the medulla; no staining was observed in the fourth ventricle. Two experiments were discarded because the spread of the dye was only restricted to one side of the brain stem. Respiration was continuously monitored by using a pneumotachograph adapted to a Nihon RJF-5 integrator to yield the full respiratory wave. End-expiratory COs was monitored with a Godart capnograph. Respiratory function was assessed by
586 measuring the spontaneous resting respiratory parameters (frequency, tidal volume and minute volume), as well as their responses to stimulation with 5 ~ COz in 02. Body temperature was measured with a rectal thermometer and was maintained at 37 0.5 °C with a heating pad. Saline solutions of met-enkephalin (Serva Feinbiochemica) were prepared daily and injected in a volume of 50/~l. In a first group of 7 cats, the effects of metenkephalin were observed for 1 h; in 4 of these, a second dose was injected subsequently and the effects were followed for another hour. In a second group of 5 cats, naloxone HCl (Endo Laboratories) dissolved in 0.5-1 ml of saline was injected
A 30[ 'llUl ..............~ ' , ' , ; ; ' , ' : . . ' . 0 t "'''' ............................................
VT ml.
'i."~ ..............................
NTG
200I
m .mB.HPg.lOO
" -
0
H R 320[
1 min
|
1601 Met- ~. e l~alin,.1.65, pnlo.I ventral aurmce Drain stem
t Naloxone 0.1mg/kg i.v.
O'
B
M~-Enk
Fig. 1. Effectsof met-enkephalin applied to the ventral surface of the brain stem on respiration, blood pressure and cardiac frequency. From top to bottom: integrated recording of respiratory movements (inspiration downwards), pneumotachogram, blood pressure and heart rate. A: effects of metenkephalin and complete reversal by naloxone. B: effects of a second dose of met-enkephalin administered 15 min after naloxone; the antagonist still prevents a full effect of met-enkephalin.
587 t
I I
I
I
I Ill
l,Y,, 01
3
5
15
t
II
r
,
I II 1
5
15
,,, 30
60
rain.
1
I
, , , ,
3
•
MINUTE VOLUME
•
BLOOD PRESSURE
O
TIDAL VOLUME
Z~
HEART RATE
r-I
RESPIRATORY FREQUENCY
30
60
mln.
Fig. 2. Time course of the effects of met-enkephalin applied to the ventral surface of brain stem, on respiratory parameters, blood pressure and cardiac frequency. Values are expressed as the per cent change calculated for each cat in relation to the control value. Symbols represent the mean of 7 cats 4- S.E.M. Control values: frequency 37.5 ± 2.5 bpm, tidal vol. 20.3 4- 1.1 ml, minute vol. 760.2 4- 23.8 ml, blood pressure 132.7 ± 3.9 mmHg, cardiac rate 229.1 ± 6.5 bpm. Inset: respiratory response to stimulation with COs 5 ~, for a constant end-expiratory CO2 level of 6.2%, in the control period and 15 min after met-enkephalin. i.v. between the two doses of met-enkephalin, 5 min prior to the second dose of the peptide. In a further two cats, naloxone was injected at the time of the peak respiratory depression. Met-enkephalin, at the dose of 1.6/zmole directly applied to the ventral surface of the brain stem, consistently depressed respiration (Fig. 1). The response was characterized by an immediate reduction of tidal volume (--35.8 ~o, range: --19.5 to --57), whereas frequency was less affected ( - - 1 9 . 7 ~ , range + 1 4 to --66.4). The time-response curves for every parameter are shown in Fig. 2. A distinctive feature was the short latency and duration of the effect: the depression started a few seconds after injection and the peak effect on minute volume was attained in about 50 sec ~---47.3 ~o, range: --22.0 to --80), but at 3 min the magnitude of the depression had been reduced substantially (--15.0~o, range: 0 to --27.0). At 15 min the values of resting minute volume were similar to those of control, but the ventilatory response to COs stimulation was still significantly lower than the control response (Fig. 2 inset, paired t-test P < 0.05). The assessment of tidal volume was critical since, by the time of maximal depression, frequency was minimally affected and thereafter it was slightly increased. End-expiratory CO2 values remained above control for about 15 min; indeed, the low tidal volume accounted for a reduced alveolar ventilation.
588 Coincident with the respiratory effects, met-enkephalin caused a reduction in blood pressure and cardiac frequency (Figs. 1 and 2). The intensity of the depression was smaller than that induced on respiration, and the time course of the responses also differed slightly: the peak effect appeared between 1 and 2 min after injection (for blood pressure,--29.7/0, r a n g e - - 1 3 . 8 t o - - 5 2 . 0 ; for cardiac frequency, - - 1 6 6"/ /o~ range: --6.6 to --33.8), and the recovery was more gradual and delayed, so that at 5 min the depression showed a value that corresponded to more than half the peak effect. Naloxone was able to antagonize all of the depressant effects. When it was injected i.v. at the time of the peak depression, a complete and abrupt reversal of both, the respiratory and cardiovascular effects was obtained (Fig. 1). In another set of experiments, the ability of naloxone to prevent the respiratory and cardiovascular actions of a second dose of met-enkephalin was determined. In 4 control cats, the responses to a second dose of met-enkephalin injected 60 min after the first one was similar to those induced by the first dose, in quality as well as intensity (Table I, A). In a second group of 5 cats, naloxone (0.2 mg/kg) was administered i.v. 5 min before injecting the second dose of met-enkephalin: a partial antagonism of the respiratory and cardiovascular effects was clearly obtained (Table I, B). The respiratory and cardiovascular responses to met-enkephalin seem to have the characteristics of an opioid action: ventilation, blood pressure and heart rate were depressed, and these effects were consistently antagonized by naloxone. Indeed, the difference between the time course of the respiratory effect of met-enkephalin and that reported for morphine 9 is evident, but it can be attributed to the kinetic peculiarities of each compound. It has been proposed that the short duration of the analgesic action of met-enkephalin observed in rodents is the consequence of enzymatic cleavage 14. In this respect, the analysis of the effects of less labile endorphins will provide further insight into the action of these compounds at the brain stem level. Respiratory depression o/
. . . .
TABLE I Antagonism of naloxone to the respiratory and cardiovascular depressant effects of met-enkephalin
Two doses of met-enkephalin, 60 rain apart, were given to two groups of cats. In the group B, naloxone was administered i.v. 5 rain before the second dose of the peptide. Values are expressed as the mean ± S.E.M. Asterisks indicate statistically significant difference related to the effect of the first dose at a P < 0.05 (Student's paired t-test). Peak depression (%) Met-enkephalin 1st dose
Met-enkephalin 2nd dose
--44.2 :t: 8.8 --30.8 ± 8.9 --13.3 i 4.0
---49.0 ± 10.6 --28.7 :k 7.0 --12.2 ± 1.7
4 5 . 3 ± 9.4 --33.1 £- 2.5 --19.2 5_ 4.5
--21.4 :t: 8.3* --15.7 ± 3.7* - - 5.6 ± 1.9"
(A) Control (n - 4)
Respiratory minute volume Blood pressure Cardiac rate (B) Naloxone (n = 5)
Respiratory minute volume Blood pressure Cardiac rate
589 following the intraventricular administration of a pituitary peptide in rats has been described, but neither the magnitude nor the time course of the effects was reportedlL It must be pointed out that met-enkephalin was administered into anesthetized animals, and that some synergism of the peptide with the anesthetics can be responsible for the full effect. In any case, when naloxone was injected at the time of peak depression, the effects of the peptide were completely reversed. The dose of met-enkephalin used in the present study was about twice that reported by other investigators, who found negative results in analgesic and behavioral studies also conducted in cats s,17, and it was approximately 10 times the equieffective dose of morphine. It reflects the consequence of the small potency of the compound as observed in the opiate displacement and guinea pig iluem assaysS, 6, when it is compared with larger fragments of the lipotropin molecule and other endorphins. It is, therefore, necessary to analyze the activity of these endorphins at the level of the brain stem and compare their potency ratios with those obtained from other in vitro as well as in vivo studies. The short latency of the responses suggests that met-enkephalin is acting on some of the ventral surface areas in the brain stem involved in respiratory and cardiovascular regulation 20,21, rather than on the pontomedullary centers. The exact localization of the affected area will require a more discrete administration of the drug, but, since met-enkephalin presents such a short duration of action, it serves as a good candidate for this type of studies. It will be important to elucidate the existence of opioid receptors on those surface areas, which can contribute to the consistent respiratory depression induced by the narcotic analgesics. This work was supported by a research grant from the Spanish Health Service. Naloxone was kindly supplied by Dr. M. J. Ferster (Endo Lab., Europe). The technical assistance of J. M. Shnchez is gratefully acknowledged.
1 Belluzzi, J. D., Grant, N., Garsky, V., Sarantakis, D., Wise, C. D. and Stein, L., Analgesia induced in vivo by central administration of ertkephalin in rat, Nature (Lond.), 260 (1976) 625-626. 2 Bloom, F., Segal, D., Ling, N. and Guillemin, R., Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness, Science, 194 (1976) 6304532. 3 Bradbury, A. F., Smyth, D. G., Snell, C. R., Birdsall, N. J. M. and Hulme, E. C., C-fragment of lipotropin has a high affinity for brain opiate receptors, Nature (Lond.), 260 (1976) 793-795. 4 Bradley, P. B., Briggs, I., Gayton, R. J. and Lambert, L. A., Effects ofmicroiontophoretically applied methionine-enkephalin on single neurones in rat brainstem, Nature (Lond.) ,261 (1976) 425--426. 5 Bfischer, H. H., Hill, R. C., R/~mer,D., Cardinaux, F., Closse, A., Hauser, D. and Pless, J., Evidence for analgesic activity of enkephalin in the mouse, Nature (Lond.), 261 (1976) 423-425. 6 Cox, B. M., Goldstein, A. and Li, C. H., Opioid activity ofa peptide, fl-lipoprotin-(61-91), derived from fl-lipotropin, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 1821-1823. 7 Feldberg, W. and Gupta, K. P., Morphine hyperglycaemia, J. PhysioL (Lond.), 238 (1974) 487-502. 8 Feldberg, W. and Smyth, D. G., The C-fragment of lipotropin - - a potent analgesic, J. PhysioL (Lond.), 260 (1976) 30-31P. 9 Fl6rez, J., McCarthy, L. E. and Borison, H. L., A comparative study in the cat of the respiratory effects of morphine injected intravenously and into the cerebrospinal fluid, J. PharmacoL exp. Ther., 163 (1968) 448--455.
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