serum prolactin and growth hormone responses to ...

Serum prolactin and growth hormone responses to naloxone and intracerebral ventricle morphine administration in heifers L. S. Leshin, L. A. Rund, F. N. Thompson, M. B. Mahaffey, W. J. Chang, D. J. Byerley and T. E. Kiser J ANIM SCI 1990, 68:1656-1665.

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SERUM PROLACTIN AND GROWTH HORMONE RESPONSES TO NALOXONE AND INTRACEREBRAL VENTRICLE MORPHINE ADMINISTRATION IN HEIFERS'**J L. S. Leshin4, L. A. Rund5, F. N. Thompson6, M. B. Mahaffey7. W. J. Chang5, D. J. Byerley5 and T. E. Kiser5 University of Georgia, Athens 30602 ABSTRACT

These studies examined responses of serum prolactin (PRL) and growth hormone (GH) to opioid agonist and antagonist administration in heifers. To minimize nonspecific and behavioral effects and to facilitate future studies with specific opioid receptor agonists, a cannula was placed within the third cerebral ventricle of the brain of 4- to 10-mo-old heifers to directly access hypothalamic regions involved in the regulation of PRL and GH secretion. Increasing doses of morphine (M) from 2 to 1,500 pg injected into the third cerebral ventricle increased ( P < .00l) serum PRL concentrations in a dose-related manner. Growth hormone responses were variable, resulting in elevated ( P < .05) serum concentrations following morphine, but no dose-related effects were apparent. Both PRL and GH responses to 700 pg M were absent when an intracerebral venmcle injection of an equimolar dose of naloxone, an opioid receptor antagonist, was administered prior to M. In a replicated 4 x 4 latin square, the effects of intravenous naloxone on PRL and GH responses was tested in young (86 f 11 d) and older (234 k 6 d) heifers. Naloxone at doses of 1. 2 and 4 mgkg reduced ( P c .05) serum concentrations of PRL for 45 to 60 min. Mean concentrations of GH tended to be higher (P < .07) in older heifers. All doses of naloxone decreased (P < .05) serum GH concentrations in older heifers but proved ineffective in younger heifers. There were no differences between doses of naloxone on either PRL or GH. These data suggest that endogenous opioids are involved in the regulation of PRL and GH secretion in heifers. (Key Words: Heifers, Naloxone, Morphine, Prolactin, Somatotropin.) J. h i m . Sci. 1990. 68:1656-1665

Introduction

Studies with rats (Dupont et al., 1977; Rivier et al., 1977), sheep (Schillo et al., 1985;

'This research was supported by funds from the Georgia Vet. Exp. Sta., Georgia Agric. Exp. Sta. and USDA Grant (t85-CRCR-1- 1854. %he authors appreciate the use of facilities and equipment in the Anirn. Physiol. Unit of Russell Res. Center. USDA-ARS, Athens, GA. 3The authors acknowledge the skilled technical assislance of C. E. Eslienne and C. K. Smith. 4Present address: Anirn. Physiol. Res. Unit, USDA, ARS, Richard B. Russell Agnc. Res. Center, Athens, GA 30613. 'Anirn. and Dairy Sci. Dept., College of Agric. 6Dept. of Physiol. and Phmacol.. College of Vet. Med. 'Dept. of Anill. and Radiol., College of Vet. Med. Received June 15, 1989. Accepted September 22, 1989.

Leshin and Jackson 1987), monkeys (Gold et al., 1979), pigs (Trudeau et al.,-1988) and humans (Foley et al., 1979) demonstrated that opioids enhance secretion of prolactin (PRL) and growth hormone (GH).Opioid peptides are found within the hypothalamus of cattle in the vicinity of hypophysiotropic neurons that regulate anterior pituitary hormone secretion (Malven et al., 1986; Leshin et al., 1988). Morphine (M) is used commonly to mimic the effects of endogenous opioids involved with endocrine regulation of metabolic and reproductive functions. However, intravenous M administered to steers was associated with pronounced behavioral effects (Peck et al., 1988b). Thus, central administration of small doses of opioid agonists might minimize nonspecific or toxic effects of the drug. Our objectives were to establish a reliable method of directly accessing hypothalamic

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PRL AND GH RESPONSES TO NALOXONE AND MORPHINE

regions involved in regulation of pituitary hormone secretion, to examine the effects of intracerebral opioid agonist administration on serum concentrations of PRL and GH with regard to dose dependency and specificity, and to examine whether endogenous opioids modulate PRL and GH serum concentrations by interrupting their actions with the opioid receptor antagonist naloxone (Nal). Because responses to peripherally administered M were established previously by our laboratory, we initially used this opioid agonist in cerebral ventricle cannula studies. Methods

Installation of Third Cerebral Ventricle Cannulas. Heifers were housed in pens and had ad libitum access to hay with 1 to 2 kg of grain supplement daily. Six heifer calves averaging 206 f 28 (;T f SE) d of age (range 125 to 302 d) were subjected to third cerebral ventricle cannulation. Following 24 h of feed deprivation, calves were tranquilized with xylazine* (.02 m@g) and ketamine9 (4 mg/ kg). Following intubation, they were maintained in surgical anesthesia with 3 to 4% halothane. The head was placed within a head holder similar to that described by Stewart and Bailey (1973). The head was secured by ear bars in the auditory meatus and spacer blocks on both sides of the jaw and nose. Ear bars and spacer blocks were adjusted so that the dorsal aspect of the head was parallel to a raised crossbar upon which the stereotaxic cannula holder was positioned. The cannula holder was positioned perpendicular to the dorsal aspect of the skull in both the coronal and sagittal planes. After the head was properly placed and secured, a position along the dorsal sagittal midline of the head, three quarters of the distance extending from a transverse line between the caudal margins of the eye orbits to the top of the poll, was located and marked by a small pointed punch through the skin and into the underlying bone. A 10- to 15-cm midline incision was made over the frontal bones. Skin, muscles and overlying periosteum were retracted, exposing the frontal

[email protected] Laboratories,Shawnee,KS. %etaset@. Bristol-Myers Co., Syracuse, NY. '%inhpBreon Lab., New York. ''Brisiol Labs, Syracuse, NY.

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bones, sagittal suture and punch marking. A cannula hole, centered over the sagittal suture, was drilled through the skull 10 mm posterior to these punch marks. The cannula with occluding stylet was lowered through the sagittal sinus and into the brain 60 to 68 mm from the top of the skull. During the last few milliliters of depth, the stylet was removed and replaced by an inner cannula the same length as the outer cannula. If cerebrospinal fluid (CSF)did not freely flow out the cannula, very brief gentle suction was applied. If CSF did not freely flow, the stylet was replaced and lowered another 1.0 mm. These steps were repeated until CSF could be easily withdrawn. To verify placement within the third ventricle, .5 ml CSF was withdrawn and replaced with .5 ml radioopaque dye (.5 ml Iohexollo). Lateral radiographs were obtained with a Bowie portable x-ray apparatus. Upon verification of proper placement, bone screws were placed into adjacently drilled holes and the cannula was secured to these screws with dental acrylic. Postoperative treatment included ampicillin" i.m. twice daily for 3 d. One week prior to experiments, calves were adapted to standing in stanchions in which they were free to lie or stand but they could not turn around. In the stanchions, they had free access to hay and water. Morphine Administration. Each heifer was used in two or three experiments that examined the PRL and GH responses to M administration. Experiment 1 and 2 examined the dose-response relationship between doses of cerebral ventricle M administration and serum PRL and GH concentrations. Experiment 3 examined whether the M-induced responses of PRL and GH observed in Exp. 1 and 2 were mediated through opioid receptors by administering the opioid receptor antagonist, Nal, just prior to M administration. Experiment 1 occurred 10 to 27 d following surgery; Exp. 2 occurred 3 to 14 d following Exp. 1. Indwelling jugular vein catheters were installed the day prior to all experiments. On experimental days, calves were placed in stanchions and had access to feed and water. Blood samples were obtained at 15-min intervals throughout the experiment. After 60 to 90 min of sampling, the head was gently held and the stylette was removed and replaced with an inner injection cannula assembly filled with artificial CSF (aCSF). This assembly consisted of an inner injection cannula the


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same length as the guide cannula, attached to a short length of flexible Silastic tubing terminating in a rubber injection septum (total void volume = 100 pl). This facilitated repeated injections during each experiment. The first injection of aCSF (200 N) was followed at 2-h intervals by increasing doses of morphine sulfate12 in aCSF to provide injection dosages of 2, 22 and 220 pg. Injections occurred over a 15- to 20-s period. Experiments ended 2 h after the last injection. Experiment 2 was similar in design to Exp. 1. Following the initial injection of aCSF in Exp. 2, M was infused to produce injection dosages of 200, 700 and 1,500 pg. Experiment 3 was performed 2 to 11 d following Exp. 2 in four heifers. Following 60 min of blood sampling, the inner ventricle cannula was inserted and 200 pl of aCSF was infused; sampling continued for 2 h, then Nal13 (1.84 p o l ) was injected, followed 3 to 5 min Later by 700 pg M (1.84 pmol) and sampling continued another 2 h. Naloxone Administration. Two age groups of Angus heifer calves (n = 8 in each group) were habituated to stanchions for 2 wk prior to Exp. 4. On the day of the experiment, one group averaged 86 f 11 d of age and 98 f 5 kg BW, and the other group averaged 234 f 6 d of age and 195 f 5 kg BW. A jugular vein catheter was placed in each calf the day prior to the experiment by percutaneous puncture. On the following four consecutive days, doses of 0, 1, 2 and 4 mg NaJ/kg BW were rapidly given (i.v.) to heifers in a replicated 4 x 4 latin square arrangement composed of days, calves and doses. Blood samples were obtained at 15-min intervals for 60 rnin prior to Nal administration and for 90 rnin afterward. Radioimmunoarsays. Blood samples were allowed to clot overnight at 4°C. Serum was harvested after cenmfugation and stored at -2O'C until it was assayed. Prolactin. The RIA for the quantification of prolactin in bovine serum was described previously (Wallner et al., 1983). Ovine PRL (LER-860-2)14 was used as radioiodinated tracer and NIH-P-B3 wa5 used as bovine standard. The linear range of the standard

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curve was from .05 to 2.0 ng/tube. Intra- and interassay coefficients of variation were 10.2 and 15.4%, respectively. Cross-reactivity with bovine GH @GH-1-1) was less than .001%. Growth Hormone. Bovine GH (USDAbGH-1-1, AFP-6500) was used for both radiolabeled antigen and as standard. Bovine GH was radioiodinated by the chloramine T method. Sequential additions to a vial containing 2.5 pg bGW.5 p1 .01 M NaHCO3 were 20 pl .5 M phosphate buffer, pH 7.0; .5 mCi Na1251 (10 pl) and 30 p a 0 p1 Chloramine T15. After addition of Chloramine T the mixture was allowed to react for 12 s then was stopped by addition of excess sodium metabisulfite (250 pg/lOO pl). The reaction mixture was diluted with 100 p1 of 1.0% bovine serum albumin barbital saline (BBS) at pH 8.6 and transferred to a .8- x 17-cm Sephadex G-75 column previously equilibrated with BBS. Elution into 1.0-ml fractions with BBS yielded two peaks, the first of which was usable for assay tracer, the second probably consisting of free IZ5I. Antiserum to ovine GH (NIADDK-antioGH-2) was diluted to a working concentration of 1:50,000 in 1:400 normal rabbit serum. Assay buffer consisted of 1% bovine serum albumin in phosphate buffered saline, pH 7.0. Following incubation (24 h, 4°C) of antiserum (200 pl) with standards or serum samples in assay buffer (500 pl), 10,000 cpm (100 kl) of radiolabeled tracer diluted in assay buffer was added and the mixture was allowed to incubate an additional 48 h. Separation of bound from free radiolabeled GH occurred by addition of a mixture of 200 p1 of sheep anti-rabbit gamma globulin serum (1:25) and 500 p1 of 6% polyethylene glycol (molecular weight 8,000) per tube, followed immediately by centrifugation (1,500 x g, 30 min). Binding in 0 standard tubes ranged from 25 to 36%, with nonspecific binding of 1.4 to 2.9% of total count tubes. The linear portion of the standard curve ranged from .04 to 2 ns/ tube. Increasing volumes (50 to 400 pl) of a pool of bovine serum depressed binding parallel to the standard curve. Recovery of added bGH (.l to 2 ng) to this serum pool was 99 f 8%. Cross-reactivity with bPRL (NIH-PB3) was less than .001%. Intra- and interassay coefficients of variation were 3.7% and 9.570, respectively. Statislical Analysis in Experiments I , 2, and 3. To satisfy the criterion of variance homogeneity, PRL values were log-transformed before



analysis. In Exp. 1, 2 and 3, means of the 2 h following treatments were calculated for each animal then analyzed by repeated measures analysis of variance using the General Linear Model procedure of SAS (1985) to examine treatment effects. Treatments common to all experimental days were analyzed separately to examine for possible treatment x experimental day interactions. The Bonferroni-t-test was used to test differences between doses. Amplitudes of secretory episodes of GH were defined as the difference between nadir and peak values with a peak value being at least twofold greater than the nadir. Mean GH amplitudes were compared between pre-M and post-M periods by t-test for unequal variances. Experiment 4. Prolactin and GH concentrations were analyzed for effects of replication, heifer, day, dose, age and time and the interactions of age and time with heifer, day and dose using the General Linear Model procedure of SAS (1985) for this repeated measures replicated latin square arrangement. Differences between doses were identified from Tukey’s test. Age x dose interactions were identified from appropriate contrasts of least square means obtained from SAS. Results

Experimenfs I and 2. Mean serum PRL and GH concentrations for the 2 h following administration of different doses of M in both Exp. 1 and 2 were combined and summarized in Figure 1. Mean serum PRL concentrations before and after infusion of 200 pl aCSF averaged 20.2 f 1.6 nglml. Increasing doses of M increased (P < .001) serum PRL concentrations (Figure 1). Doses of 2 and 22 pg M elevated serum PRL to 27.2 f 3.9 and 36.0 8.2 ng/ml, respectively. However, M did not significantly raise mean serum PRL concentrations from pre-M concentrations until 220 pg was administered. elevating PRL to 51 f 7.3 ngfml (f < .05). The response to 700 and 1,500 pg M doses increased (f < .05)PRL above the 2 and 22 pg M dose; however, the response to 700 and 1,500 pg doses were not significantly different from the response to the 220 pg dose. Small pulses of PRL sometimes were observed before and after infusion of aCSF; however, prolonged (>30 min) increases in PRL concentrations during or immediately following these procedures did not occur. Following M administration, PRL increased

*

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within 15 or 30 min and often remained elevated near peak concentrations for almost the entire 2-h period. Serum concentrations of GH were not affected by insertion of the injection cannula and infusion of aCSF, averaging 2.4 f .3 ndml (Figure 1). There was no dose-dependent increase of GH following M administration; however, mean GH concentrations throughout the times of M administration increased (f < .05) to 5.4 f .8 ng/ml. Growth hormone secretory episodes attributable to the injection of increasing doses of M were difficult to ascertain because they sometimes were delayed in onset beyond 30 min or were absent. In some cases M was administered during an endogenous increase in GH. Although amplitude of GH increases following M were of variable size, mean amplitude was greater (P c . O l ) than endogenous GH increases occurring prior to M administration (Pre-M 6.8 5 1.4, range 3.8 to 14.8 ng/ml, n = 8; Post-M 12.6 k 2.2, range 3.4 to 37.0 ng/ml, n = 26). Hormone concentrations might be expected to increase during sequential doses of M. To examine whether there were carryover effects from previous smaller dose injections, the third dose (220 pg) of Exp. 1 was given as the first dose (200 pg) in Exp. 2. The 209.41 difference in dosage is due to the 20 pg in the void volume of the cannula from the previous injection in Exp. 1. For PRL. the response to the 200-pg dose of Exp. 2 (50.8 f 10.1 ngfml) was similar to the response to the 220-pg dose of Exp. 1 (53.6 f 11.4 ngml). Comparison of other treatments common to both Exp 1 and 2 revealed no differences in PRL or GH responses prior to replacement of the stylet with the inner cannula or after aCSF injection. However, for GH, the response to 200 pg morphine (first dose of Exp. 2; 9.8 f 2.0 ng/ml) was greater (f c .OS) than the response to the 220-pg dose (third dose of Exp. 1; 3.1 f 1.0 ng/ml). Experiment 3. In the third experiment, equimolar concentrations of Nal and M were administered. Naloxone was administered 3 to 5 min prior to M. Comparison of the PRL response to 700 pg M (78.2 rt 1.8 ndml, Figure 2A, data of Exp. 2) clearly shows that pretreatment with Nal attenuated the PRL response to M (17.0 6.2 ng/ml, Nal/M, Figure 2B, Exp. 3). There was no difference between b a d concentrations of PRL before inner cannula insertion. after aCSF administra-


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Figure 1. Two-hour mean serum prolactin (PRL)and growth hormone (GH) responses to increasing doses of morphine. The results of Exp. 1 and 2 were combined. Different letters overlying PRL response bars indicate differences ( P < .05) beiween doses. For GH,the dose-response relationship was not significant. although overall morphine elevated (P< .05) GH concenuations.

tion or after Nal/M administration (Figure 2B). In all calves, brief increases of GH occurred within 30 min following aCSF infusion, increasing (P .c .05) the 2-h mean concenuation of GH above the preinjection concentration of 2.2 f .7 ng/ml (Figure 2B). Concentrations of GH returned to preinjection values 30 to 45 min prior to injection of Nal/M. The GH response following Nd/M was not different from that of basal (preinjection of aCSF) GH secretion (Figure 2B). Mean GH concentrations following NaUM were not significantly different from the response to 700 pg M (4.0f 1.0 ng/ml, Exp. 2, Figure 2A). but there were no episodic increases in GH during the 2-h interval following Nal/M administration. Experiment 4. Mean concentrations of PRL varied prior to Nal administration (Figure 3). There was no effect of heifer age on pre-Nal PRL concentrations. which averaged 8.4 f .2 ng/ml. Saline (0 mg Nalkg BW) did not affect mean PRL concentrations. Following 1, 2 or 4 mg Nalkg BW, mean serum PRL was lower (P e .05) compared with the post-treatment saline mean (Table 1). Concentrations of PRL were lowest 45 to 60 min following administration of Nal, averaging 5.7 f .2 ng/ml; then they tended to return to pretreatment concentrations (Figure 3). Concentrations of GH prior to treatment tended to be greater (P = .07)in the older heifers (86 d, 1.8 f .I ng/ml; 234 d, 3.7 f .2

ng/mi). Following Nal treatment there was a small but nonsignificant decrease in mean GH concentrations in younger heifers (Table 1;

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Figure 2. Two-hour mean serum prolactin (PRL) and growth hormone (GH) responses to 700 pg morphine (M) alone (A) and to 700 pg M given 3 to 5 min after an equimolar dose of naloxone (N)was administered (B).Data in A and

B were taken from Exp. 2 and 3, respectively. Basal represents samples obtained prior to insenion of the cerebral ventricle injection cannula;aCSF represents the period followingcannula insation and injection of artificial cerebrospinal fluid. M represents the period following M administration; N/M represents the period following the administration of naloxone 3 to 5 min before M. Asterisks indicate differences (P< .OS) from basal concenuations.


PRL AND GH RESPONSES TO NALOXONE AND MORPHINE TABLE 1. MEAN (fSEM)* SERUM PROLACTIN AND GROWTH HORMONE (nglrnl) FOLLOWING THE ADMINISTRATION OF D[FFERENT DOSES OF NALOXONE TO ANGUS HEIFERS

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Figure 4). However, in the older group, both 2 and 4 mg Nal/kg BW decreased (P .05) mean GH concentrations (Table 1). The 1, 2 and 4 mg Nal/kg BW doses lowered serum GH concentrations by 30 min; however, GH concentrations in the 1 m a g group returned earlier than the 2 or 4 mg/kg group to control values (Figure 4). Saline treatment did not significantly alter mean GH concentrations. Behavioral Responses to Morphine and Naloxone. There were no obvious behavioral responses to 2 or 20 pg M. Sometimes the 200 pg dose resulted in a small degree of hyperactivity. After administration of 700- and 1,500-pg M doses, vocalizations occurred, sometimes persistently. With the 1,500-pg dose, salivation was evident and all calves were hyperactive, shifting weight on their legs and swaying their heads back and forth. Only occasional vocalizations were evident from calves following Nal/M together in Exp. 3. There were no obvious behavioral responses following i.v. doses of Nal in Exp. 4.

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These data indicate that intracerebral venmcle administration of M, an opioid receptor agonist, increases serum PRL and to a lesser extent GH via an opioid receptor mechanism. Administration of M intravenously to steers and postpartum cows increases PRL secretion (Peck et al., 1988a,b). Intravenously administered opioids also increase GH concentrations

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in heifers (Armstrong and Johnson, 1989). The hormonal responses to M and the absence of these responses when Nal was administered prior to M suggests that opioid receptors are involved in modulating GH and PRL secretion. Because the effects of M on GH concentrations were to enhance the amplitude of episodic serum GH increases but were not dosedependent, different opioid receptor subtypes may modulate GH and PRL. The effects of Nal, an opioid receptor antagonist, is dependent on the physiological state of the animal. Endogenous opioids must be functionally active to reveal Nal antagonism of opioid modulated PRL and GH response. In the present experiments, changes of PRL and GH concentrations following Nal indicates that an endogenous opioid was involved in maintaining or elevating PRL and GH concentrations. Ineffectiveness of Nal to influence GH in young heifers suggests that endogenous opioid regulation of GH either was not yet developed or it was not active at this age. The use of Nal does not provide information about which specific opioid receptor was involved because Nal binds with only 10-fold greater affinity to p receptors than to 6 and K receptors in brain tissue (Chang, 1984). The dose-response relationship for M-induced increases of PRL concentrations agrees with possible p receptor modulation, whereas the lack of a dose-response relationship for GH may reflect the absence of p receptor modulation. In rats and humans, M increased PRL at lower doses than those required to increase GH (Spiegel et al., 1982a,b; Tolis et al., 1975). Evidence in the rat also suggests that PRL secretion was mediated through p and K opioid receptors (Panerai et al., 1985; Krulich et al., 1986), whereas GH secretion was mediated through 6 receptors (Koenig et al., 1984). In sheep, both 6 and K receptors may be involved in GH secretion (Della-Fera et al., 1984). Use of highly specific opioid agonists and antagonists in similar experiments would provide more conclusive evidence for the types of opioid receptors regulating PRL and GH in the heifer. Administration of M centrally and Nal intravenously was consistent with evidence suggesting that the site of action of endogenous opioids and exogenously administered opioid agonists was within the central nervous system. Morphine and Nal derivatives that do not pass through the blood-brain barrier, such


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MINUTES OF SAMPLING Figure 3. Serum prolactin (PRL)responsesto 0.1.2 or 4 mg naloxone& BW administered i.v. at time 60 min, indicated by the arrow. Data from young and old heifers were. combined because there was no effect of age on serum PRL concentrations. Pooled SEM = 2.3 ng/ml.

as morphine methyliodide and naloxone methylbromide, were effective in altering PRL and GH concentrations when given via the intracerebral venmcle but not systemically (Panerai et al., 1981). Similarly, Ragavan and Frantz (1981a,b) reported that intravenmcular, but not intravenous, administration of anti-pendorphin sera reduced basal and stressinduced blood PRL concentrations. Inhibition of PRL secretion in rodents is mediated by the tuberoinfundibular dopaminergic neurons projecting from the arcuate nucleus to the median eminence (Moore, 1987). Opioids raise PRL concentrations by inhibiting activity of these dopaminergic neurons (Gudelsky and Porter, 1979; Van Vugt et al., 1979; Haskins et al., 1981). In heifers, dopaminergic regulation of PRL was demonstrated by Benoit et al. (1989). Furthermore, in cattle, immunoreactive P-endorphin and tyrosine hydroxylase (one of the enzymes for dopamine synthesis) have been localized in neurons within the arcuate nucleus with extensive projections to the median eminence (Leshin et al., 1988, 1989). Content studies revealed the presence of other opioid peptides, namely met-enkphalin and dynorphin, also within the hypothalamus of cattle (Malven et al.. 1986). Besides

inhibition of dopamine secretion, opioids may also act on systems that stimulate PRL secretion, such as thyrotropin-releasing hormone (Leshin and Jackson, 1987), because Arita and Porter (1984) suggest that dopamine was not sufficient to account for the increase of PRL after opioid administration. In cattle, thyrotropin-releasing hormone administration provokes secretion of both PRL and GH (Convey et al., 1973; Johke, 1978). Although both young and old heifers secreted GH episodically, mean serum GH concentrations tended to be lower in young heifers. Increases of serum GH concentrations following M administration were not as consistent as increases of PRL concentrations. In addition, GH but not PRL increased occasionally following aCSF administration. These GH increases could be due to time relative to feeding or the start of experimentation, or to the repetitive infusions into the venmcle. In the present study, the magnitude of GH responses to M was variable in individual animals, similar to the variability previously reported for bull calves and dairy cows administered exogenous growth hormone releasing hormone (GHRH;Enright et al., 1986, 1987). This may be attributed to the animals'



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mg/kg Naloxone

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MINUTES OF SAMPLING Figure 4. Serum growth hormone (GH)responses in heifers of 86 (top) or 234 (bottom) days of age to 0, 1 , 2 or 4 mg naloxonefig BW admin~steredi.v. at time 60 min indicated by the arrow.Pooled SEM = .9nghnl.

endogenous episodic secretion of GHRH and somatostatin (SRIF). However, mean amplitude of these GH increases were greater following M administration than prior to M administration. Although M may not initiate GH secretion in heifers, opioid receptors probably are functionally linked to GH regulatory systems to modulate the amplitude of serum GH concentrations. Chihara et al. (1978) demonstrated that j3-endorphin induced release of GH was not mediated through SRIF. However, Drouva et al. (1981) demonstrated that P-endorphin was effective in inhibiting K+-stimulated release of SRIF. In rats, the elevation of GH caused by opioid administration was inhibited by passive immunization against GHRH (Miki et al., 1984; Wehrenberg, et al., 1985). Thus, opioids may influence the timing of GHRH and SRIF secretion and thereby influence GH secretion. In summary, third cerebral venmcle administration of M resulted in a dose-related increase in serum PRL concentrations in

heifers suggesting mediation through specific opioid receptors. Morphine-induced release of serum GH concentrations was more variable and likely dependent on endogenous episodic secretion of SRIF and GHRH. Thus, opioids may have parallel actions on inhibitory (SRIF) and excitatory (GHRH) pathways to modulate the timing of secretory GH episodes. Reduction of both PRL and GH concentrations following Nal administration suggests that endogenous opioids were involved in the elevation of these hormones in heifers. Therefore, we conclude that endogenous opioid peptides regulate PRL and GH secretion in heifers. Implications

Third cerebral ventricle administration of the opioid agonist morphine into heifers resulted in a dose-related increase in serum prolactin concentrations, but growth hormone responses were variable. However, both pro-


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