Effects of β-endorphin, morphine and naloxone on arginine vasopressin secretion and the electroencephalogram

0306-4522 79!,201-1895$02.00,0

Nuuroscirn<~~ Vol.4.pp,1895m 1902 PergamonPress Ltd1979. Printed m GreatBritam OIBRO

EFFECTS OF /3-ENDORPHIN, MORPHINE AND NALOXONE ON ARGININE VASOPRESSIN SECRETION AND THE ELECTROENCEPHALOGRAM H. M. FIREMARKand R. E. WEITZMAN Departments of Neurology and Medicine, Harbor-UCLA Medical Center, Torrance. CA 90509, U.S.A. Abstract-fi-Endorphin injected into the third ventricle of conscious rabbits resulted in a sustained elevation of the concentration of arginine vasopressin in blood plasma. Tenfold greater doses of morphine were required to produce a comparable response. The secretion of arginine vasopressin appears not to be a consequence of respiratory depression or hemodynamic alterations induced by fl-endorphin or morphine. Most pharmacological effects of ~~ndorphin and morphine were reversed by naloxone but the effects on the secretion of arginine vasopressin were not. Furthermore. pretreatment with naloxone failed to prevent the rise in plasma levels of arginine vasopressin after administration of morphine. Paroxysmal seizure discharges were recorded on the electroencephalogram after injection of j3-endorphin into the lateral ventricle, but not after injection into the third ventricle, Frequency analysis revealed slowing and reduced power in all frequency bands of the electroencephalogram. Both morphine and the endogenous peptide P-endorphin have similar effects on the release of arginine vasopressin and these effects appear to be independent of naloxone-sensitive opiate receptors.

been recognized since the work of DE BODO (1944) that morphine causes an antidiucesis that is largely dependent on the integrity of the neucohypophysis and intact h~othalamiconeurohy~physiai connections (DUKE, PICKFORD & WATT, 1951). The identification in brain and pituitary of fl-endocphin, a peptide with opiate-like activity, and the presence of opiate receptors in the neucohypophysis (SIMANTOV& SNYDER 1977) led us to study the effect of /J-endocphin on the secretion of acginine vasopcessin (AVP) and to compare its effects with those of morphine. In addition we have studied the effect of the narcotic antagonist naloxone on basal AVP secretion and on AVP secretion stimulated by /3-endorphin or morphine. The effects of #-endocphin on the electcoencephalogram (EEG) have been analyzed. IT HAS

EXPERIMENTAL

PROCEDURES

Surgery Adult male New Zealand white rabbits weighing approx. 2.5 kg were used in these studies. Under pentobarbital anesthesia a jugular vein was cannulated with Silastic tubing. The catheter was advanced into the vena cava and was brought out of the animal at the back of the neck. Catheters were maintained patent by frequent flushing with heparinized saline. Stainless steel cannulas (David Kopf Instruments) were placed into the third ventricle using standard techniques and were secured to the skull with acrylic cement. Stereotaxic coordinates of the cannula tip were AP(O), H(-3), in the mid-sagittal plane using the positioning described by SAWYER,EVERETT & GREEN (1954). Skull electrodes were implanted at the Following locations: frontal - 2mm to right of the sagittal suture

Abbreviations: AVP, arginine

troencephalogram. NSC.4112-F

vasopressin;

EEG, elec-

and 12 mm anterior to the coronal suture, parietal - 8 mm to right of the sagittal suture at the coronal suture, occipital - 2 mm to the right and to the left of the sagittal suture 1mm anterior to the lambdoidal suture. The animals were permitted to recover for several days prior to study. Adminisrrotion

of drugsand

sampling

ofblood

~-Endorphin (60 nmol), kindly supplied by Dr R. Guillemin of the Salk Institute, or morphine sulfate (60 or 600nmol) were dissolved in artificial cerebrospinal fluid (MERLIS, 1940) and volumes of 10-25 ~1 were injected into the ventricular cannula. The cannula was then flushed with an additional lO$ of artificial cerebrospinal fluid. The cannula dead space was approx. 10~1. Three ml blood samples were withdrawn from the vena cava catheter and the volume removed was replaced with a 50% solution of rabbit plasma in saline containing heparin (50 pU/ml). Blood samples were withdrawn 1, 5 and 10 min before injection and 10, 20, 40, 60, 80, 100, 120 and 140 min after injection. Naloxone [0.2 mg (approx. 0.08 mg/kg) i.v.j was then given and blood samples were withdrawn 3, 10 and 20 min thereafter. The blood samples were transferred to tubes containing 20 ~1 of 15% dipotassium ethylenediamine tetra-acetate and were kept in an ice bath until the conclusion of each experiment, at which time they were centrifuged and plasma was withdrawn for radioimmunoassay of AVP (WEITZMAN,FISHER,MENICK,LINC & GUILLEMIN,1977). Pretreatment with intraventricular naloxone was used in an effort to block the effects of intraventricular morphine. Three dosage and sampling schedules were used: (A) 60nmol naloxone followed after 20min by morphine 6OUnmol. Blood samples were taken 0.5, 5 and 10min before naloxone (baseline), 5, 10 and 20 min after naloxone and 10 and 20 min after morphine. (B) 120 nmol naloxone followed immediately by morphine 6OQnmol. Blood samples as for (A) for baseline and 5, 10 and 20min after naloxone and morphine. (C) 600nmol naloxone followed after 10 min by 600 nmol morphine. Blood samples as for (A) for baseline and after morphine; 5, 7.5 and 10 min after nafoxone.

1895

1896

FIREMARK and R. E.

H.M.

The EEG was recorded from bipolar leads; one occipital electrode was used as a ground. A Grass model P5 EEG amplifier was used and the signal was recorded on magnetic tape using a Honeywell model 5600 tape recorder. Frequency analysis of the EEG was carried out using a Nicolet Med 80 averaging computer. Four bands of EEG (O-5, 5-8, 8- 12 and 12-20 Hzi were analyzed. Typically eight periods of 5.12s of EEG were averaged for each power spectrum and signals Larger than -f 100 I_‘V were rejected as an artifact. Statistical treatment of results The AVP data were iog~ithmically transformed to correct for heterogeneity of variance. Statistical comparisons of the transformed mean plasma AVP levels prior to and following injection of test substances were performed using Student’s t-test for paired data. The arithmetic means and standard errors are presented in the text and tabies afthough they were not used in the statistical evaluations. RESULTS The position of the ventricular cannulas was confirmed by the injection of bromphenol blue solution into the cannula immediately prior to the killing of the animal. After fixation in lO~/i,formaiin for several weeks the brains were sectioned and the area of blue staining examined. In all instances the staining was most intense in tissues surrounding the 3rd ventricle. On occasion light staining of the walls of lateral ventricles indicated diffusion of the dye from the 3rd ventricle. Effects oj drug treatments on plasma levels ~asopressi~r

of arginine

Control. Injection of 50 ~1 of artificial cerebrospinal fluid and withdrawal of blood samples did not alter plasma AVP levels. The mean AVP level was 0.8 k 0.1 $J/ml (mean & s.E.M.,II= 1 I) during the 10 min baseline period and 0.7 k 0.1 yU/ml during the 140 min period following the injection (Table 1). /?-Endorphin. The intraventricular injection of 60 nmol ~-endorphin (200 pg) produced a prompt increase in plasma AVP levels. The increase was biphasic with an initial elevation 10 and 20 min after

WEITZMAN

lo-endorph~n injection and a second progressive rise in AVP levels between 60 and 140 min after the injection. The mean plasma AVP level rose from I.0 + 0.1 $J/ml during the baseline period to 4.5 I 0.8 pU/ml during the 140 minute period following injection. The increase was significant (P < O.oOI. II = 9) (Table 1). The respiratory rate slowed progressively and marked alterations of posture, activity and reflex behaviors were observed (Table 2). Morphine. Equivalent doses (60 nmol) of morphine produced slight elevations of the plasma AVP level which were not significantly different from baseline. Behavioral changes were minor and animals remained quiet throughout the experimental period. The rate of respiration slowed to a degree comparable to that observed with fl-endorphin (Table 2). A ten-fold greater dose of morphine (~nmol) injected intraventricularly produced a marked increase in plasma AVP levels; this was most prominent during the first 60min after injection. The mean plasma AVP rose from a baseline level of 0.6 t 0.1 pU!rnl to 4.0 & 1.8 $._J/ml during the entire 140 min period following the injection (P < 0.05) (Table 1). The major behavioral change seen after 6OOnM morphine was excitation and increased activity during the first 20 min after the injection. Respiratory depression was equivalent to that seen with P-endorphin and 60 nmol morphine. Nuloxone. Naloxone (0.2 mg) given intravenously I40 min after ~-endorphin produced a prompt reversal of postural, respiratory and reflex effects of /3-endorphin. Within a few seconds rigidity disappeared and the respiratory rate rose to approach the baseline rate. However, 20min later mild respiratory depression and extensor posturing recurred, reflecting the short biological effect of naloxone. Despite the reversal of these effects by naloxone, the plasma AVP level was not significantly different in the 20 min after naioxone as compared to the 20 min before naloxone (Table 3). Similarly naloxone did not significantly alter plasma AVP levels in the animals previously injected with morphine or controls injected with artificial cerebrospinal fluid alone. Pretreatment with naloxone intraventricularly did

TABLE 1. EFFECT OF I~~A~N~~c~L~ &ENWRPHIN AND MORPHINE ON THF CONCENTRATlON OF ARGININE VASOPRESSIN IN BLOOD PLASMA

Plasma

Control &Endorphin (60 nmol) Morphine sulfate (60 nmol) Morphine sulfate (600 nmol)

AVP (pU/ml) mean & S.E.M. Experimental ‘Baseline’ 0.8 + 1.0 4 1.4 + 0.6 +

0.1 0.1 0.2 0.1

0.7 f 4.5 + 2.2 + 3.9 +

0.1 0.8 0.1 1.8

II

PC

11 9 5 8

N.S.
* Mean AVP values for each animal during each period were log,, transformed and compared using Student’s paired t-test. The group arithmetic means and standard errors are presented although they were not used in the statistical evaluation. The ‘baseline’ txriod renresents mean of values I. 5 and 10 min prior to intraven-

tricuiar injection. ’ The experimentai period represents 140 min after intraventricular

injection.

mean of values IO. 20,40.60,80.

100. 120 and

Rigid Rigid

Tail down normal Normal Slight extension of head, hind legs

42 + 10.0 41 k 13.8

169 k 22.6

142 & 14.6 136 f 18.1

120 140 Minutes after naloxone 3

10 20

Moving Exploring Less active

Nil Nil

Nil Nil

Cornea1 normal

reflex

Extensor jerk with sound stimulus

40 k 8.9 52 f 9.0

80 100

Opisthotonus Less neck extension

53 f 9.0

60 Nil

65 f 7.1

Cornea1 reflex decreased, nystagmus Righting response lost Cornea1 reflex absent

40

Extension of neck and hind legs Rigid, tail up Opisthotonus

80 f 5.2

20

I38 f 23.4 132 f 26.3

131 + 36.5

45 * 9.7 47 f 4.9

57 + 4.7 51 f 5.2

64 f 7.8

83 f 21.1

108 f 23.2

15.7

117 f

Exploring Sniffing Licking Clonic extensor jerking Nil

1 I4 + 9.9

Other

158 + 20.1 I54 + 19.0 141 * 17.9

Activity

Morphine Respirations per minute

135 + 14.9 I28 k 16.4 126 f 17.9

Posture

/I-Endorphin 60 nmol

-10 -5 -1 Minutes after injection 10

Respirations per minute

Alert, active

Nil

Nil

Activity

sulfate 60 nmol

17.1

13.4

132 f 21.1 116 f 23.3

157 k 18.0

53 + 8.9 45 f 5.3

57 f 8.1 52 f 6.5

60 f 5.6

75 f

97 f 23.1

134 f

186 f. 9.7 I78 * 10.4 178 + 9.1

Morphine Respirations per minute

TABLE 2. EFFECTSOF INTRAVENTRICULAR /3-ENDORPHIN AND MORPHINE ON RESPIRATION, POSTUREAND ACTIVITY

Active, exploring

Nil Nil

Nil Nil

Nil

Exploring Circling Jumping Excited Scratching Sound sensitive Quiet

Activity

sulfate 600 nmol

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H. M. FIREMARKand R. E. WEITZMAN TABLE 3. EFFECTOF INTRAvFNOtiS NALOXOKL OK TH!:(‘ON(‘ENTRATIONOF

ARGININI

vASOPRESSIN IN 131001~PI.ASMA

Plasma

AVP (/rU ‘ml) mean 2 s.L..M. Before* lld0X0lW _______

li ~__

/J-Endorphin 60 nmol Mprphine Morphine Control

(6) 171 (51 (71

sulfate 600 nmol sulfate 60 nmol

6.1 2.2 I.8 0.0

+ + T f

AlIter+ ndoxonc

3.0 0.8 0.4 0.2

x.2 3.5 1. 0.Y ~~

+- 3.2 :z 1.3 + 0.5 _: 0.3

* Mean of plasma AVP levels I20 and I40 min after Injection. t Mean of plasma

AVP levels 3, 10 and 20 min after 0.2 mg naloxonc

i.r,

TABLE 4. EFFECT OF INTRAVENTKIU LAR NALOXOW PRL'TREATMENT ON MORPHINE-INDLi(‘FI> SFCRFTION OF ARGININk \'AWPRFSSIN - ..-

Plasma

Baseline After naloxone After morphine

AVP ($_.ml) mean 2 s.I..M. A(H = 5) B(rl = 6) Clf? = 4) _.____~~_____

I .I * 0.4 I.0 & 0.4 2.5 f 0.‘)

(I.7 * 0.3 1.x + 0.5t

0.0 + 0.5 0.‘) + 0.3 4.9 * 1.7*

Protocol A: 60 nmol naloxone followed after 20 min by 600 nmol morphine. Blood samples were taken 0.5. 5 and tOmin before naloxone (baseline). 5. IO and 2Omin after naloxone and IO and 20 min after morphine. Protocol B: I20 nmol naloxone followed immediately by 600 nmol morphine. Blood samples as in A for baseline and 5, IO and 20 min after naloxone and morphine. Protocol C: 600 nmol naloxone followed after 10 min by 600 nmol morphine. Blood samples as in A for baseline and after morphine; 5. 7.5 and IO mm after naloxone. Mean AVP values for each animal during each period were log,, transformed and compared using Student’s paired t-test. The group arithmetic means and standard errors arc presented although they were not used in the statistical evaluation. * P < 0.05 baseline I’.$after morphine. t P < 0.01.

not prevent or significantly reduce AVP secretion induced by morphine (Table 4). The mean rise of plasma AVP above baseline 10 and 20 min after 600 nmol morphine without naloxone pretreatment was 5.1 &- 2.6 pU/ml (mean t_ s.E.M.). The comparable mean increase of AVP levels 10 and 20 min after morphine in naloxone treated groups A, B and C were 2.2 & 1.0, 1.0 + 0.7 and 4.0 + 1.4 respectively. One way analysis of variance of the log,, transformed data indicated no significant difference between the groups (F = 0.81, degrees of freedom 3, 19). Respiratory depression was usually nearly completely prevented by treatment B and C. In all groups excitation and exploring typical of that seen after morphine administration was essentially unchanged by naloxone pretreatment. Electroencephalogram

The baseline electroencephalogram in the rabbits studied had a predominant background rhythm of 6-7 Hz (Fig. 1) with superimposed beta activity (> 13 Hz). Sound stimuli elicited brief, 2-3 s periods of desynchronization. Ten minutes after the injection of

/I-endorphin the predominant rhythm became slower, typically 3-5 Hz, and the voltage decreased. Thereafter the desynchronization produced by sound stimuli was prolonged, often lasting 6-8 s. Occasional bursts of high-voltage, sharp 56 Hz activity appeared but spike discharges were not seen. The intrinsic variability of the EEG signal limits quantitative comparisons but frequency analysis permits comparison of the amount of EEG activity occurring in each frequency band with time. A marked decrease in mean EEG power occurred between 10 and 20min after /I-endorphin injection (Table 5). EEG power returned towards baseline values after 90 min. The change with time was significant in each frequency band. DISCUSSION The postural, analgesic and reflex effects produced by intraventricular /I-endorphin in rabbits are similar to those described for rats and mice (BLOOM, SEGAL, LIN(; & GUILLEMIN, 1976: WEI. TSENG, Lou & LI. l977), however ‘wet dog shakes’ were not observed in

/I-endorphin, morphine, naloxone and vasopressin

FIG. 1. Electroencephalogram recorded from skull electrodes in an unanesthetized rabbit. Bipolar leads from (top to bottom) R frontal-R parietal, R parietal-R occipital, R frontal-R occipital, R occipital-L occipital. Time marker: 1 s intervals; calibration marker: 5OpV. (A) Baseline EEG before injection. Theta activity (5-7 Hz) and superimposed beta activity (> 13 Hz) are prominent. Calibration marker indicates injection of /I-endorphin. (B) One minute after injection of 60 nmol fi-endorphin into the third ventricle. (C) Ten minutes after injection of b-endorphin. Note the decrease in voltage. (D) 130 minutes after injection of )%endorphin. Sound stimulus at the arrow is followed by an artifact due to extensor jerk, then desynchronization of EEG. (E) Thirty seconds after intravenous injection of 0.2 mg naloxone.

rabbits. Respiratory depression has not been commented upon in prior descriptions of the pharmacological effects of intraventricular p-endorphin, but in man intravenous P-endorphin produced prolonged respiratory slowing (CATLIN, HUI, LOH & Lx, 1978).

Effects on the secretion ofarginine

vasopressin

/?-Endorphin (60 nmol) injected into the third ventricle produced a marked increase in plasma AVP, and this increase appeared to be biphasic, an early

1900

H. M. FIREMARKand R. E. WEITZMAN TARLE5. ELECTROENCEPHALOGRAM AFTERINTRAVENTRICULAR ADMINISTRATION OF /I-ENWRPHIN

Picowatts of EEG power

in each

frequency

band

during

successive

time periods

after

/I-endorphin.

Mean k S.E.M. Time Baseline I@-30 min 60 90min 120 140min P*

No. of animals 5 5 3 3

@5 Hz

5 8Hz

8m12 Hz

13.20Hz

444 + 109.9 383 k 100.9 345 + 117.3 438 f 214.2 <0.025

170 + 42.8 94 * 33.1 56 + 11.5 I93 + 68.6 < 0.025

61 * 18.4 41 ) 18.2 23 _t 6.1 87 + 26.6 iO.01

55 & 13.6 3x + 12.0 24 + 3.8 45 ) 10.8 < 0.025

/I-Endorphin (60 nmol) dissolved in artificial cerebrospinal fluid was administered opened into the third ventricle of the rabbits. * One way analysis of variance for differences with time with Greenhouse-Geisser & POLAND,1976.)

rise corresponding to a period of excitatory activity and a prolonged later rise corresponding to the period of rigidity and opisthotonus. A similar but shorter rise in AVP has been reported after intravenous administration of larger doses of P-endorphin without obvious effects on posture or activity (WEITZMAN et a/., 1977). The stimulation of AVP secretion by P-endorphin does not appear to be a consequence of respiratory depression since an equimolar dose of morphine produced an equivalent respiratory slowing but no significant rise in AVP. SKOWSKY,SMITH & SWAN (1978) reported that enkephalins produced a sustained fall in blood pressure beginning l--2 min after lateral ventricular injection in cats, and suggested that this might mediate the release of AVP. However LAUBIE, SCHMITT, VINCENT & REMOND (1977) reported that cisternal injection of p-endorphin and synthetic methionine-enkephalin analogues, but not methionineenkephalin, produced a transient (10 min) increase in blood pressure followed by delayed hypotension. The initial increase in AVP in our studies occurred during the period when blood pressure is reported to be transiently elevated (LAUBIE et al., 1977). This suggests that the hypertension may have been a consequence of the pressor effect of secreted AVP. Naloxone reversed the late hypotension induced by /%endorphin in the study of LAUBIE et al. (1977) but did not eliminate the rise in AVP in our animals. Furthermore KANJANAPOTH~(1975) showed that morphine can produce an antidiuretic action and an increased excretion of vasopressin without hypotension. These observations suggest that hemodynamic alterations are not the primary mechanism for P-endorphin-induced AVP secretion. The site of action and mechanism by which /?-endorphin promotes AVP secretion is not known at present. WEITZMAN et al. (1977) found that neither fl-endorphin nor naloxone altered the spontaneous release of AVP from rat neurohypophysis in vitro and concluded that the site of /?-endorphin stimulation of AVP release was not at the neural lobe. A direct action upon hypothalamic neurosecretory cells is possible although actions elsewhere in brain with in-

via a cannula correction.

that

(RURIN

direct effects on the neurosecretory cells cannot be excluded. The observation that naloxone administered to animals pretreated with either endorphin or morphine did not reverse the elevation of plasma AVP levels is at variance with the observation of BISSET,CHOWDREY 8~ FELDBERG (1978) that the antidiuretic responses (measured by bioassay) to intravenous enkephalin and C-fragment were reversed by naloxone. A possible explanation for the difference between their results and ours may lie in the different species, doses and routes of administration used. Lack of action of naloxone. The inability of naloxone in the present study to prevent or reverse the secretion of AVP provoked by b-endorphin or morphine suggests that opiate receptors sensitive to naloxone are not directly involved in AVP release in the rabbit. Several authors have presented evidence for the existence of multiple opiate receptors with varying naloxone responsiveness. and several effects of opioid peptides and morphine have been resistant to naloxone. BRADLEY, BRIGGS, GAYTON & LAMBERT (1976) found that excitatory actions of morphine on rat brain stem neurones were not antagonized by naloxone although depressant actions were. In the cat GENT & WOLSTENCROFT(1976) reported that naloxone did not block effects of either methionine-enkephalin or morphine on brain stem neurones. BUSCHER,HILL, ROMER, CARDINAUX, CLOSSE, HAUSER & PLESS (1976) noted that doses of naloxone that blocked the analgesic effect of morphine and methionine-enkephalin did not inhibit the analgesic effect of leucine-enkephalin in mice. Similarly naloxone did not block or reverse rotation behavior induced by morphine injected into the midbrain reticular formation (JACQUET, KLEE, RICE, IIJIMA & MINAMIKAWA. 1977) and in mouse vas deferens, naloxone was approximately ten times less effective than expected in antagonizing the effect of opioid peptides (LORD, WAI~RFIELD, HUGHES & KOSTERLITZ, 1977). Directly comparable to our findings is the report by COCCHI, SANTAGOSTINO, GIL-AD, FERRI & MUELLER(1977) that the release of prolactin induced by leucine-enkephalin is not antagonized by naloxone. Our data support

/?-endorphin, morphine, naloxone and vasopressin

these observations

and provide another opiate effect not blocked by naloxone.

example of an

Effects on the electroencephalogram

HENRIKSEN, BLOOM, LING & GUDLLEMIN(1977); HAVLICEK, LEYB~, REZEK & PINSKY (1977) and UREA, FRENK, L~EBESKIND& TAYLOR (1977) have reported the production of seizures following intraventricular or intracerebral application of fi-endorphin or enkephalins. We have not observed seizure discharges following injection of /I-endorphin into the third ventricle, but in a single experiment 31 pg (9 nmol) of ~~ndorphin injected into the lateral ventricle produced clear paroxysmal high-voltage discharges in the surface EEG beginning about lf min after the injection. These discharges were unaccompanied by convulsive activity. NICOLL, SIGGINS, LING, BLOOM & GUILLEMIN (1977) found that hippocampal neurones, unlike cells in other regions, were excited by opioid peptides, and FRENK, MCCARTY & LESBESKIND (1978) concluded that enkephalins produced seizures only when injected in or near the dorsome-

dial nucleus of the thalamus. In the rabbit the lateral ventricle is in proximity to the hippocampus and the dorsal thalamus, whereas the injection site we used in the third ventricle is quite remote from these regions.

1901

Thus we have been able quantitatively to assess the effects of fl-endorphin on surface EEG activity undistorted by paroxysmal seizure events. Our finding of decreased EEG power, most marked in the 5-8, 8-12 and 12-20 Hz frequency bands, is directly opposite to the finding of HAVLICEK et al. (1977) who reported a significant increase in power in all frequency bands

following intraventricular injection of 50,~4g of /?-endorphin to an unspecified species and via an unreported site. These suggestions of apparent regional, dose and species related differences in the effects of /I-endorphin and other opioid peptides emphasize the hazards of generalization about the physiologic~ functions of these substances based on a limited series of studies.

Acknowledgements-This work was supported by grant NS 12089 from the National Institute of Neurological and Communicative Disorders and Stroke and grant MG 2472 from the Greater Los Angeles Affihate of the American Heart Association. We wish to thank CHARLESNELSONfor technical assistance, MIGUELH. GOKEZ for help with the electrophysiological studies and ANITA REV~CZKYand RUBYELAWRENCEfor skilful performance of the radioimmunoassays. ALAN FORSYTH,Ph.D., Department of Biomathematics, UCLA, provided statistical advice.

REFERENCES Bismr G. W., CHOWDREY H. S. & FELDBERG W. (1978) Release of vasopressin by enkephalin. Brt. J. Pharmac. 65370-371. BLOOMF., SEGALD., LING N. & GUILLEMINR. (1976) Endorphins: Profound behavioral effects in rats suggest new etiological factors in mental illness. Science, N.Z 194, 630-632. BRADLEYP. B., BRIGGSI., GAYT~NR. J. & LAMBERTL. A. (1976) Effects of microiontophoretically applied methionine enkephalin on single neurones in rat brainstem. Nature, Lond. 261,465426. BUSCHERH. H., HILL R. C., ROMERD., CARDINAUXF., CLOSSEA., HAUSERD. & PLESSJ. (1976) Evidence for analgesic activity of enkephalin in the mouse. Nature, Lond. 261,423-425. CAI-LIND. H., HLJI K. K., LOH H. H. & LI C. H. (1978) ~-~ndo~hin: Subjective and objective effects during acute narcotic abstinence in man. Adu. Biochem. Psyc~~harm. l&341-350. C’~XCH~ D., SANTAGC~STINO A., GIL-AD A., Foam S. & MILLER E. E. (1977) Leu-enkephaiin-stimulated growth hormone and prolactin release in the rat: Comparison with the effect of morphine. Life Sci. 20, 2041-2046. DE Bono R. C. (1944)The antidiuretic action of morphine and its mechanism. J. Pharmac. exp. Ther. 82, 74-85. DUKE H. N., PICKFORDM. & WATI J. A. (1951) The antidiuretic action of morphine: its site and mode of action in the hypothalamus of the dog. Q. J. exp. Physiol. 36, 149-158. FRANKH., MCCARTYB. C. & LIEBESKIND J. C. (1978) Different brain areas mediate analgesic and epileptic properties of enkephalin. Science, N.Y. 2BO,335-337. GENTJ. P. & WOLSTENCROFT J. H. (1976) Effects of methionine enkephalin and leucine enkephalin compared with those of morphine on brainstem neurones in cat. Nature, Lond. 261,42&427. HA~LIC~KV., LEYBINL., REZEKM. & PINSKYC. (1977) The opposite behavioral and EEG effects of low and high doses of /I-endorphin. Abstracts of the 59th Annual Meeting of the Endocrine Society, p. 178. HENRIKSENS. J., BLOOMF. E., LING N. & GUILLEMINR. (1977) Induction of limbic seizures by endorphins and opiate alkaloids: Electrophysioiogical and behavioral correlates. Neuro~jence A&s 3, 293. JACQUETY., KLEEW., RICE K. C., IUIMAI. & MINAMIKAWA J. (1977) Stereospecific and non stereospecific effects of (+) and (-)-morphine: Evidence for a new class of receptors? Science, N. Y. 198, 842-845. KANJANAP~THID. (1975) The release of vasopressin by hypotensive drugs. Ph.D. Thesis, University of London. St. Thomas’s Hospital Medical School (quoted by BILLETet al. [ 19781). LAIJBIEM., SCHMITTH., VINCENTM. 8~ REMONDG. (1977) Central cardiovascular effects of morphinomimetic peptides in dogs. Eur. J. Pharmac. 46.67-71. LoRDJ. A. H., WA~FIELD A. A., HUGHESJ. L KOSTERLITZ H. W. (1977) Endogenous opioid peptides: multipte agonists and receptors. Nature, Lond. 267,495-499. MERLISJ. K. (1940) The effect of changes in the calcium content of the cerebrospinal fluid on spinal reflex activity in the dog. Am. J. PhysioI. 131, 67-72.

1902

H. M. FIREMARKand R. E. WEKZMAN

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(Accrpted 9 July 1979)