Corticotrophin Releasing Hormone Modulates Morphine Effect on the Met-Enkephalin Activity in the Hypothalamic-Pituitary-Adrenal Axis in Lambs

The present study evaluated the effects of morphine or morphine together with corticotrophin releasing hormone (CRH) on Met-enkephalin synthesis, secretion, concentration and opioid receptors binding in the hypothalamus, anterior pituitary and adrenal cortex (HPA) in lambs. Lambs received a single i.v. injection of 0.9% NaCl (control) or morphine (MOR) or morphine in combination with CRH (MOR+CRH). Animals were decapitated after 60 min under anaesthesia and fragments of HPA tissues were dissected. Proenkephalin mRNA expression was measured by in situ hybridization, Met-enkephalin concentrations by RIA method, opioid secretion by in vitro incubation. Specific radioligands were used for each type of receptors H-DAGO for mu, H-DPDPE for delta and H-EKC for kappa binding sites. Acute injection of morphine affected the proenkephalin mRNA expression, native and cryptic Met-enkephalin concentrations as well as mu, delta and kappa receptors binding. There were multiple cases of the effects of morphine being reversed with CRH effects on the HPA axis level, however the most pronounced changes were observed in the hypothalamus. Interestingly, CRH reversed the effect of the morphine on the proenkephalin mRNA expression in all tested tissues. These results indicated an important role of CRH in the endogenous opioid peptides synthesis and opioid receptors binding.

Endogenous opioid peptides and their receptors play important roles in the regulation of many physiological processes via nervous, endocrine and immune systems interactions. Opioid peptides, belonging to families of enkephalins, dynorphins and endorphins are synthesized as large peptides (precursors) namely preproenkephalin (PENK), preprodynorphin (PDYN) and proopiomelanocortin (POMC), respectively. Among opioid peptides, enkephalins have the widest tissue distribution. They are released as native small molecules from multiple central nervous system structures and peripheral organs such as adrenals, gastrointestinal system, pancreas and cardiovascular systems (PIERZCHALA & . Met-enkephalin exists in the blood and tissues in two forms -1. native with five amino acid residues fragments and a very short half-life and 2. cryptic form in/or associated with large proteins and processed by proteases to active Met-enkephalin and Leu-enkephalin (PIERZCHALA & . Under in vitro conditions, native Metenkephalin might be released from cryptic (bound) precursor by enzymatic hydrolysis with trypsin and carboxypeptidase B.
Endogenous opioids act through specific receptors localized in both the brain and peripheral tissues. Classical pharmacological studies identified three classes of opioid receptors; mu (ì), delta (ä) and kappa (ê) which have been cloned. These classical opioid receptors belong to the family of the guanine regulatory binding (G) protein coupled receptor and are acting through the second messenger systems mainly by inhibition of Gi/Go proteins (BODNAR 2016). Pharmacological and molecular studies suggested that there is more than one type of mu opioid receptor. However, as only a single mu receptor gene has been reported, so the receptor may undergo extensive alternative splicing to generate a group of variants (PASTERNAK & PAN 2011).
Endogenous opioid peptides have been broadly studied as important factors modulating activity of hypothalamic-pituitary-adrenal axis (HPA) (DROLET et al. 2001;RUSSELL et al. 2008). Opioid-containing neurons have been shown to innervate the median eminence and paraventricular nucleus of the hypothalamus, thereby regulating inputs to ACTHcontrolling neurons in the anterior pituitary (STEIN & Z_LLNER 2009;DI MARZO et al. 2007). Thus, the hypothalamic-pituitary-adrenal axis represents a modulatory target for the action of exogenous and endogenous opioid ligands. Indeed, a growing body of evidence suggests that opioids regulate mechanisms activated during the stress response. Conversely, the endogenous opioid system is activated by stressful situations, raising the possibility that activation of the endogenous opioid system may play a role in stress-mediated events. Although stress is often linked to unpleasant events, the stress response can be beneficial. For example, exposure to mild stressors has been shown to activate the HPA axis, which is thought to play an important role in mediating cognitive adaptive changes that promote survival. Previous studies have shown that different stressors -emotional (isolation) and physical (exercise) affect tissue and plasma opioid concentrations and opioids receptors activity in the hypothalamus, pituitary and adrenal (PIERZCHA£A-KOZIEC et al. 2006) indicating a close relation between the opioid systems and corticotrophin releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and glucocorticoids. It was found that restraint stress increased plasma concentrations of Met-enkephalin and corticosterone in rats ( VAN LOON et al. 1990). Similarly in sheep, the stress of insulin-induced hypoglycemia increased plasma concentrations of both cortisol and Met-enkephalin (OWENS et al. 1988). Enkephalins are present in the hypothalamus and interact with the CRH neurones (SMITH & VALE 2006; VAN'T VEER et al. 2012;FUNK et al. 2014) and probably are secreted in order to modulate the activity of hypothalamic-pituitary-adrenal axis (BRUCHAS et al. 2009;LEMERRER et al. 2009;LEMOS et al. 2012). In addition, the opioid kappa receptor agonist, dynorphin, is co-localized with corticotrophin-releasing hormone (CRH) in hypothalamic neurons (CALOGERO et al. 1996).
Intravenous administration of either ì or ê selective opioid receptor agonists increases the circulating concentrations of adrenocorticotropin (ACTH) in fetal lambs (TAYLOR et al. 1997). Based on studies with the intracerebroventricular administration of Met-enkephalin and the consequent decrease in plasma concentration of ACTH, it can be assumed that Met-enkephalin can depress ACTH release acting at a central locus (WANG et al. 1988).
Morphine, an opium alkaloid acting through µ opioid receptors, has been widely used as an analgesic for decades, however, there are side effects such as respiratory depression, constipation and nausea at the central and peripheral levels (MATTHES et al. 1996;GENDRON et al. 2006). There is contradictory information on the effects of chronic morphine administration on hypothalamic CRH. Long term treatment with morphine was reported to decrease the CRH synthesis and concentrations in the hypothalamus (LAORDEN et al. 2002). In contrast, chronic morphine administration was found to increase hypothalamic CRH mRNA (MCNALLY & AKIL 2002;HOUSHYAR et al. 2004). Moreover, morphine administration to rats increased plasma concentrations of ACTH (BUCK-INGHAM 1982) and stimulated the release of CRH (CALOGERO et al. 1996). In spite of fact that morphine mainly acts through µ receptors it has been demonstrated that acute or prolonged morphine treatment upregulates delta and kappa receptors (PIERZCHALA-KOZIEC et al. 2000;GENDRON et al. 2006). Naloxone-induced morphine withdrawal is accompanied by stress-related parvocellular CRH transcription (LIGHTMAN & YOUNG 1988; PASTER-NAK & PAN 2011) again supporting cross-talk between opioid receptors and CRH (BRUNTON et al. 2005;CHONG et al. 2006;LIKAR et al. 2007;CHARRON et al. 2008;RUSSELL et al. 2008).
These results suggest that morphine and CRH are involved into regulation of HPA axis in varying situations but whether their collective effects represent a global mechanism acting to restraint endogenous enkephalin peptides activity remains to be elucidated.
Thus, the goals of the present study were twofold: 1. to determine whether single injection of morphine affected the synthesis, secretion, concentration of native and cryptic Met-enkephalin and mu, delta and kappa opioid receptor agonist binding in the hypothalamus, anterior pituitary and adrenal cortex of lambs, and 2. to explore the effect(s) of CRH on the morphine-induced changes in these processes.

Materials and Methods
The study employed 3 months old lambs of the Polish Mountain Sheep breed. These were maintained in a controlled environment (photoperiod 14L: 14D with lights on from 7a.m to 7 p.m) and at a room temperature 20°C. The lambs had free access to food and water. The protocol was approved by the First Local Ethical Committee on Animal Testing in Kraków (64/OP/2005/I LKE).
Eighteen female lambs were assigned to 3 groups (n=6): control (C), morphine (MOR) and morphine plus corticotrophin releasing hormone (MOR+CRH). Each lamb received one intravenous injection of saline (0.9% of NaCl in the control group), morphine (MOR) (1 mg/kg b. w.) or morphine and CRH (1 µg/kg b.w. Sigma, St. Louis, MO, USA). Blood samples were taken to the heparinized tubes just before (0 time) and 10, 20, 30 and 60 minutes after the injection. The samples were immediately centrifuged and the plasma was stored at -80°C. The site of injection was left jugular vein, the blood was taken from the right jugular vein. Animals were decapitated after anaesthesia. Hypothalami, anterior pituitary glands and adrenal cortex were dissected out one hour after treatment injections. Tissues were divided into four parts and directed to: 1. in situ hybridization to estimate proenkephalin gene expression, 2. estimation of native and cryptic Met-enkephalin by radioimmunoassay, 3. opioid receptors binding and 4. in vitro secretion of native Met-enkephalin.
Proenkephalin mRNA gene expression was estimated by modified method described by LIGHTMAN & YOUNG (1987). Briefly, the frozen fragments of hypothalamus, anterior pituitary and adrenal cortex were sliced (14 ìm sections) using a Leica cryostat microtome (-22°C). The sections were thaw-mounted on gelatin-covered microscopic slides, and stored for 3 days at -20°C before the assay. Then, tissue sections were thawed and fixed in 4% formaldehyde in phosphate buffered saline (PBS; pH 7.4) for 10 min. Then, sections were acylated for 10 min in triethanolamine/acetic anhydride (0.25%). Sections were dehydrated by immersion through graded ethanol (70%, 80%, 95%, 100%) and in air dried.
After pre-hybridization, a synthetic deoxyoligonucleotide, complementary to the fragment of rat proenkephalin (PENK), was labeled using 35 S-dATP (1200 Ci/nmol) to obtain a specific activity about 4×10 6 cpm/ìl. The probes were diluted in a hybridization buffer (formamide, dextran sulfate, Saline-Sodium Citrate buffer (SSC), Denhardt's solution, yeast tRNA, herring sperm DNA). Hybridization occurred during 20 h in humidified chamber at 37°C. Then, the sections were washed once in SSC for 10 min, then four times for 15 min, each in SSC/ 50% formamide at 40°C, rinsed in SSC and distilled water at room temperature and air-dried. The sections were exposed to Kodak film for four weeks (-80°C). The photo-stimulated luminescence (PSL) density of the irradiated plates was measured with BAS-1000 readout system. The PSL/mm 2 at the resultant film images was determined using computer image analysis system.

Native and cryptic Met-enkephalin concentrations
Native and cryptic Met-enkephalin concentrations in the tissues and plasma were estimated by the radioimmunoassay method of PIERZCHA£A & . Briefly, fragments of tissues were homogenized in phosphate buffer, pH 6.5, centrifuged (4000×g, 4°C, 20 min) and supernatants were stored at -80°C until further processing. Enkephalin containing peptides (cryptic enkephalin) were hydrolyzed with trypsin (1 mg/ml, 37°C, for 30 min) followed by carboxypeptidase B (5 mg/ml) plus trypsin inhibitor (2.5 mg/ml) for 15 minutes.

Receptor binding assays
Receptors binding assays were performed according to procedures reported by BELCHEVA et al. (1994) andHYTREK et al. (1996) with some modification (PIERZCHA£A-KOZIEC et al. 2015). Briefly, the dissected tissues were homogenized in icecold buffer 50 mM Tris-HCl, pH 7.4 and the homogenate was centrifuged at 20,000 xg for 15 min. Cells membrane preparations (1 ml, 1 mg of protein) were incubated at 30ºC for 30 min with tritiated agonists for each type of opioid receptors: for mu receptors -26.

In vitro Met-enkephalin secretion
Met-enkephalin secretion from fragments of tissues was estimated according to the method of KOWALSKI & GIRAUD (1993) with some modifications. Briefly, fragments of tissues (20-30 mg) sliced by microtome were placed into 24-well plates with 1 ml of Krebs-Ringer bicarbonate buffer (medium). After a 20 min preincubation period, tissues were incubated at 37°C for five successive 20 min periods in 500 :l medium according to the sequence: 1. basal medium; 2. stimulating medium with 100 nM of naltrexone; 3. basal medium; 4. basal medium; 5. stimulating medium with 56 mM KCl. Stimulation with KCl served to validate survival of the tissue through the experiment. The concentration of Met-enkephalin in the basal media were not significantly different so the results were pooled and presented as Met-enkephalin release under basal conditions.

Statistical analysis
Results are presented as means ± SEM. The analysis was performed using the O Dell Statistica, ver.13, (1984-2016. Repeated measures ANOVA or paired t-test followed by Fisher test were used to determine the effects of morphine and CRH on the synthesis, secretion, plasma levels of Met-enkephalin as well as mu, delta and kappa receptor binding in the HPA axis.
There was no effect of morphine on hypothalamic PENK mRNA (Fig. 1). In contrast, an injection of morphine was followed by decreased (P<0.001) PENK mRNA expression by 47% for the anterior pituitary and by 55% in the adrenal cortex ( Fig. 1). Administration of morphine together with CRH influenced PENK expression. PENK mRNA was increased (P<0.001) in the hypothalamus, anterior pituitary gland and adrenal cortex in lambs receiving CRH and morphine compared to either control lambs or animals receiving morphine alone (Fig. 1).

Met-enkephalins concentrations
Native Met-enkephalin There were much higher concentrations (P<0.001) of native Met-enkephalin in the anterior pituitary gland (138.3±8.92 pmol/g wt.) than either hypothalamus (29.1±1.9 pmol/g w.t.) or adrenal cortex (33.5 ± 1.9 pmol/g wt.). The effects of morphine or morphine in combination with CRH on concentrations of native Met-enkephalin in the Injection of morphine alone or in combination with CRH was not accompanied by changes in the concentrations of native Met-enkephalin in the hypothalamus (Fig. 2). Morphine administration was accompanied by a 23% decline (P<0.05) in the concentration of native Met-enkephalin in the anterior pituitary gland (Fig. 2). Moreover, injection of both morphine and CRH was followed by a larger (80%) decrease (P<0.001) in the concentration of native Met-enkephalin in the anterior pituitary gland (Fig. 2). Morphine administration also depressed (P<0.01) the concentration of native Met-enkephalin in the adrenal cortex by 27% from 33.5 ± 1.9 to 24.3 ± 1.6 pmol/g wt. (Fig. 2). Native Met-enkephalin concentrations after treatment with morphine and CRH were intermediate of the concentrations seen in control and morphine but did not differ significantly from either (Fig. 2).

Cryptic Met-enkephalin
There were much higher concentrations (P<0.001) of cryptic Met-enkephalin in the anterior pituitary gland (216.9 ± 19.7 pmol/g wt.) than the hypothalamus (43.1 ± 2.7 pmol/g wt.) with adrenal cortex intermediate (160.9 ± 18.3 pmol/g wt.). Figure 3 summarizes the effects of morphine or morphine together with CRH on the concentrations of cryptic Met-enkephalin in the hypothalamus, anterior pituitary gland and adrenal cortex of lambs.
Acute administration of morphine was accompanied by increased (P<0.01) concentrations of cryptic Met-enkephalin in the hypothalamus and anterior pituitary by, respectively, 35% and 33% (Fig. 3). In contrast, the concentration of cryptic Met-enkephalin in the adrenal cortex was reduced (P<0.001) by 78% in lambs receiving injections of morphine (Fig. 3). Administration of CRH in combination with morphine reversed the effect of morphine on hypothalamic concentrations of cryptic Met-enkephalin (Fig. 3). Anterior pituitary concentrations of cryptic Met-enkephalin did not differ between lambs receiving morphine alone or morphine together with CRH (Fig. 3). Adrenal cortical concentrations of cryptic Met-enkephalin were reduced in lambs receiving morphine plus CRH with the decrease less (P<0.05) compared to morphine injected group (Fig. 3).
Delta ()) receptor binding (Fig. 5) There were similar DOR concentrations, as indicated by binding of 3 H-DPDPE, in adrenal cortical, hypothalamic and anterior pituitary tissue -39.12±4.11, 53.01±4.78 and 44.40±3.87 fmol/mg protein, respectively. Lambs receiving a single injection of morphine exhibited decreased  (P<0.001) DOR binding in the hypothalamus by 87%, anterior pituitary by 86% and adrenal cortex by 59% (Fig. 5). CRH given together with morphine caused small but statistically significant increases in DOR binding in the hypothalamus and anterior pituitary. In contrast, CRH potentiated the inhibitory effect of morphine on the delta receptor binding in the adrenal cortex.
Kappa (ê) receptor binding (Fig. 6) There were marked tissue differences (P<0.001) in kappa receptor binding as indicated by 3 H-EKC binding. Kappa receptor binding ranged from lowest in the hypothalamus (19.2 ± 1.44 fmol/mg protein), intermediate in the anterior pituitary gland (79.0 ± 6.39 fmol/mg protein) and highest in the adrenal cortex (355.1 ± 37.2 fmol/mg protein in of control lambs). There was no effect of morphine alone or in combination with CRH on the KOR agonist binding in the hypothalamus (Fig. 6). In contrast, ê receptor agonist binding was increased (P<0.001) in both anterior pituitary and adrenal cortical tissue from lambs treated with morphine (Fig. 6); the increases being, respectively, 53 and 122%. Administration of CRH with morphine further increased (P<0.001) ê receptor agonist binding in anterior pituitary tissue. However, injection of CRH with morphine attenuated (P<0.01) KOR binding in the adrenal cortex.

Plasma concentrations of native Met-enkephalin
Plasma concentrations of native Met-enkephalin levels were unchanged in control lambs during the 60 min course of the experiment (Fig. 7). In contrast, injection of morphine was followed rapidly by decreased (P<0.01) plasma concentrations of native Met-enkephalin (Fig. 7) being decreased by 63% after 10 min (P<0.01) and by 79% to a nadir after 20 min (P<0.01) (Fig. 7). Plasma concentrations of native Met-enkephalin remained reduced (P<0.01) at 30 min and after 60 min (Fig. 7). CRH partially overcame the inhibitory effects of morphine on plasma concentrations of native Metenkephalin (Fig. 7). There was a transitory 44% decrease (P<0.01) in plasma concentrations of native Met-enkephalin 10 min after morphine plus CRH challenge (Fig. 7). After 20 min plasma level of native Met-enkephalin was not different from the value observed in control animals. At 30 minutes Met-enkephalin level was significantly higher compare to control and morphine treated animals (Fig. 7). Administration of morphine plus CRH was followed by what appeared to be a biphasic change in the plasma concentrations of native Met-enkephalin with an initial 10 min decrease (P<0.01), recovery at 20 min, overshoot at 30 min (P<0.05) and a second decrease (P<0.05).
Met-enkephalin secretion in vitro from the hypothalamus, anterior pituitary gland and adrenal cortex  termined in the presence or absence of the opioid antagonist, naltrexone (Fig. 8).

Hypothalamus
Basal release of Met-enkephalin from the hypothalamus in vitro was elevated in lambs receiving morphine; increasing from 8.2 ± 0.78 pmol/g/20 min in controls to 17.9 ± 2.1 pmol/g/20 min with morphine treatment (P<0.001) (Fig. 8). In contrast, Met-enkephalin secretion was reduced by 63% (P<0.01) in lambs receiving morphine and CRH (Fig. 8). In the presence of naltrexone, there was markedly increased release of Met-enkephalin from hypothalamus of control animals by 187% (P<0.01) (Fig. 8). However, there was reduced release of Met-enkephalin (by 72%, P<0.001) from tissue from lambs injected with morphine (Fig. 8). In contrast, there was increased secretion (by 250%, P<0.001) of Met-enkephalin from the hypothalamus in the presence of naltrexone in tissue from lambs injected with morphine and CRH (Fig. 8).

Anterior pituitary gland
Basal in vitro secretion of Met-enkephalin from sheep anterior pituitary tissue was 5.9 fold greater than from the hypothalamus (Fig. 8). Basal in vitro secretion of Met-enkephalin was lower (P<0.001) in lambs treated with morphine by 74% and with morphine together with CRH (Fig. 8). The effect of morphine was attenuated somewhat (P<0.05) in the presence of CRH with secretion depressed (P<0.001) by 62% in tissue from lambs receiving morphine + CRH compared to control lambs (Fig. 8).

Adrenal cortex
Met-enkephalin release in vitro from the adrenal cortex tissue is markedly lower than from hypothalamic and anterior pituitary tissue. In vivo injection of morphine increased secretion of Met-enkephalin (P<0.05) by 22%. In the presence of naltrexone, Met-enkephalin release was increased (P<0.001) in adrenal cortical tissue from control lambs (by 121%), from morphine treated animals (by 74%) and from morphine with CRH injected lambs (by 77%).

Discussion
There were novel and marked effects in vivo treatment with morphine or morphine combined with CRH on enkephalin related parameters and on opioid receptors (see Figs 1-8). The direct and magnitude of effects results are summarized in Table 1.

The effects of morphine and morphine with CRH on proenkephalin mRNA expression
Acute morphine injection did not change proenkephalin mRNA expression in hypothalamus but significantly decreased it in anterior pituitary and adrenal cortex (Fig. 1). It has been previously reported that morphine administration can acutely change opioid peptide gene transcription in rat brain (BASHEER & TEMPEL 1993) and in specific brain regions (YuKHANANOV & HANDA 1997). Treatment with CRH completely reversed the effect of morphine and increased the proenkephalin mRNA expression in all HPA axis levels.
There is evidence for cross-talk between CRH and the opioid systems. IREDALE et al. (2000) found extrahypothalamic CRH to be implicated in anxiety and aversion associated with opiate withdrawal. Type 1 CRH receptor (CRH-R1) antagonists attenuated behavioral signs of opiate withdrawal as well as footshock stress-induced reinstatement of heroin seeking and morphineconditioned place preference (WANG et al. 2006). CRH plays a role in the elevation of withdrawalinduced noradrenergic transmission (FUNADA et al. 1994). Moreover, chronic morphine selectively sensitizes locus coeruleus norepinephrine neurons to CRH (XU et al. 2004). These mechanisms contribute to the facilitated neuroendocrine stress response seen in morphine dependent rats after withdrawal (HOUSHYAR et al. 2004).

The concentrations of cryptic and native Met-enkephalin in the hypothalamic-pituitary-adrenal axis
Data in the literature regarding opiate-induced regulation of the brain enkephalin system are controversial. Thus, increases or decreases in enkephalin immunoreactivity were reported to occur in brain tissues after chronic treatment with morphine, whereas in other studies, no change was observed ( VAN BOCKSTAELE et al. 2000). These differences are probably due to the various experimental protocols used in chronic morphine treatment, the fragments of brain, different methods for enkephalin estimation, autoradiography or in situ hybridization and small opioid peptide extraction from tissues (NIETO et al. 2002). In the present experiments acute morphine caused increase of cryptic enkephalin in the hypothalamus without any serious changes in the native form of opioid. In spite of lower synthesis of proenkephalin as an effect of decreased gene expression after acute morphine, the concentration of cryptic Met-enkephalin in the anterior pituitary was increased. This higher level of cryptic enkephalin probably was an effect of lower activity of enzymes responsible for processing large precursor to native form what resulted in significantly lower concentration of native Met-enkephalin. Met-enkephalins, cryptic and native, were decreased after morphine injection in the adrenal cortex, probably due to inhibition of synthesis and enzymatic hydrolysis. Corticotrophin releasing hormone potentiated stimulating effect of morphine on the cryptic Met-enkephalin concentration and inhibiting effect of opiate on the native opioid in the anterior pituitary. CRH reversed completely (hypothalamus) and partially (adrenal cortex) morphine effects. It is interesting that administration of CRH with morphine did not influence the concentrations of either native or cryptic Met-enkephalin in the hypothalamus despite the elevated PENK mRNA expression (Table 1, Figs 1-3). It seems probable that mRNA expression did not mirror the proteins concentrations particularly these characterized by short half-life.

Opioid receptors binding
Mu receptor binding in the anterior pituitary gland was decreased in lambs receiving injections of morphine (Fig. 4). However, there were no effects in the hypothalamus and adrenal cortex suggesting an absence of down regulation and probably lack of receptor internalization in the hypothalamus and adrenal. Treatment with CRH along with morphine was accompanied with marked shifts in mu receptors with the suppressive effects of morphine on the anterior pituitary gland augmented, mu receptors binding in the hypothalamus increased and mu receptors binding in the hypothalamus decreased (Fig. 4). Administration of injection alone CRH was observed to increase the mu receptor binding in the hypothalamus (PIERZCHA£A-KOZIEC et al. 2015). It is possible that 3 H-DAGO could not bind in vitro to receptors that were persistently occupied by morphine or other opioid agonists. KISSIN et al. (1991) postulated that the absence of correlation between anal- Injection of morphine greatly reduced in vitro binding of 3 H-DPDPE to delta receptors in hypothalamic, anterior pituitary and adrenal cortical tissues (Fig. 5). In contrast, prolonged or acute morphine treatment has been reported to upregulate delta and kappa receptors in the HPA axis (PIERZCHALA-KOZIEC et al. 1990;GENDRON et al. 2006). Co-treatment of CRH with morphine partially reversed the effects of morphine on the hypothalamus and adrenal cortex but increased the inhibiting effect of morphine on the 3 H-DPDPE binding in the adrenal cortex. Similarly, administration of injection CRH reduced delta receptor binding in each level of the HPA axis (PIERZCHA£A -KOZIEC et al. 2015). These data agree with the report of agonist induced activation of the delta opioid receptor leads to receptor desensitization and finally to internalization (PRADHAN et al. 2009;. The level of kappa receptor binding varied markedly from the lowest in the hypothalamus, intermediate in the anterior pituitary gland and highest in adrenal cortex (Fig. 6). The presence of kappa opioid receptor in the lamb hypothalamus is consistent with the role for kappa receptors in modulating the release of luteinizing hormone (GOODMAN et al. 2004;LOPEZ et al. 2016), thyrotrophin releasing hormone and growth hormone releasing hormone (DEPAOLI et al. 1994;FUNK et al. 2014) and the presence of ê-opioid receptors located in GnRH neurones .
Dynorphin and alfa-neo-endorphin are the main agonists for kappa receptors. Dynorphin has been found to regulate neuronal excitability broadly in brain and can affect learning, cognition, seizures, nociception, and endocrine function. Recently, activation of the dynorphin/kappa receptor system has also been shown to be necessary and sufficient for stress-induced behavioral responses in animal models of anxiety, depression, and drug seeking behaviors (VANDERAH 2010).
In the present study, acute treatment with morphine alone or in combination with CRH did not influence kappa receptor binding in the hypothalamus of lambs (Fig. 6). This may be due to antagonistic properties of mu receptor agonists at the central level. PFEIFFER et al. (1986) reported that kappa receptor agonists are responsible for induction dysphoric and psychomimetic effects in contrast to mu receptors agonists. In contrast, there were increases following morphine injection in 3 H-EKC binding to kappa receptor in the anterior pituitary gland and adrenal cortex (Fig. 6). The effect was potentiated by CRH on the anterior pituitary gland but attenuated by CRH with adrenal cortical tissue (Fig. 6). These tissue differences may reflect different class of kappa receptors and this could be investigated using novel and new kappa agonists that never enter the CNS ( VAN-DERAH 2010). The role of CRH-induced activation of the kappa receptors and dynorphin opioid peptides has been implicated as a mediator of adverse responses to stress. Stress-induced release of dynorphins has been reported to potentiate the "reward" effects of drugs e.g. cocaine, ethanol and nicotine ( VAN'T VEER et al. 2012, 2013. Kappa receptors activity has been linked to circulating concentrations of corticosterone levels as there is a faster rate of increase of corticosterone following stress in mice lacking dynorphin (BILKEI-GORZO et al. 2008).

Plasma native Met-enkephalin concentrations
Plasma concentrations of Met-enkephalin were markedly reduced following administration of morphine with the nadir 20 minutes after injection (Fig 7). Interestingly, to our best knowledge it is first report about morphine decreasing plasma concentrations of Met-enkephalin level. Plasma concentrations of Met-enkephalin showed tendency to increase by 60 min following morphine challenge (Fig. 7). The magnitude of the decline in plasma concentrations of Met-enkephalin (Fig. 7) coupled with the short half-life of Met-enkephalin suggests an even greater decrease in the release and/or production of Met-enkephalin. The present results with morphine administration are consistent with the anterior pituitary and/or the adrenal cortex being significant sources of circulating Met-enkephalin.
Combined injection of morphine and CRH evoked a biphasic effect on plasma concentrations of Met-enkephalin with an initial decrease, recovery and then increase up to 30 min. This is arguably analogous to the response of rats to stressors with an immediate increase of native Met-enkephalin (around 1 min), then return to basal levels and second peak of opioid after 30 minutes (PIERZCHALA et al. 1987). The response to morphine was completely abolished in the presence of CRH at 20 and 30 minutes following challenge and attenuated at 60 minutes (Fig. 7). It is suggested that CRH is effectively acting as an antagonist. It is further suggested that the loss of effectiveness of CRH in depressing the effects of morphine on plasma concentrations of Met-enkephalin 60 minutes after challenge was due to prolonged clearance of CRH and appears to be responsible for the sustained release of ACTH that occurs after injection of this hormone.
In vitro Met-enkephalin release from HPA Short-term in vitro incubations allowed direct examination of Met-enkephalin secretion (Fig. 8). There were marked tissue differences with hypothalamic and adrenal cortical release of Metenkephalin (increase) in morphine treated lambs and anterior pituitary decreased release of Metenkephalin in morphine treated lambs (Fig. 8). Morphine treatment in vivo influenced the release of Met-enkephalin in vitro; stimulatory for hypothalamus and inhibitory for the anterior pituitary gland. There is evidence that the effects of morphine are direct as they are reversed in the presence of the opioid receptors antagonist, naltrexone, in the incubation media (Fig. 8). It is also likely that basal release of Met-enkephalin from each tissue is influenced by an endogenous opioid receptor antagonist because there are shifts in the release of Met-enkephalin in the presence of naltrexone. Administration of CRH along with the morphine was accompanied by reduced release of Met-enkephalin from the hypothalamus compared to either control or morphine treated lambs (Fig. 8). Moreover, in vivo treatment with CRH along with morphine overcame the effects of morphine on release of Met-enkephalin from anterior pituitary tissue (Fig. 8).
Basal Met-enkephalin in vitro release from the adrenal cortex was lower than from hypothalamus and anterior pituitary. Interestingly, morphine injection increased the enkephalin basal release but CRH again reversed this effect. There were consistent in vitro stimulatory effects of naltrexone on release of Met-enkephalin from adrenal cortical tissue. These results provide clear evidences for a physiological role for Met-enkephalin in the adrenal cortex. Met-enkephalin and other opioid receptor agonists have been reported to increase production of both aldosterone and corticosterone by rat adrenocortical cells with the effects mediated by, respectively, mu and both mu and kappa receptors (KAPAS et al. 1995). Moreover, opioid agonists were reported to increase cortisol production by porcine adrenal cortical cells (KRAZINSKI et al. 2011).

Summary
The anterior pituitary gland had consistently higher concentrations of PENK mRNA, native and cryptic Met-enkephalin and basal release of Metenkephalin in vitro than either the hypothalamus or adrenal cortex. Acute morphine injection affected synthesis, concentrations of both forms of Metenkephalin, agonists receptor binding as well as opioid release in the hypothalamic-anterior pituitary-adrenal cortex axis. However, it must be pointed that the morphine effect on the hypothalamic Met-enkephalin changes were much smaller than in other tested tissues. This contention is supported by morphine decreasing the following: anterior pituitary gland PENK expression in both the anterior pituitary gland and adrenal cortex (Fig. 1), native Met-enkephalin in both the anterior pituitary gland and adrenal cortex (Fig. 2) and cryptic Met-enkephalin in the adrenal cortex (Fig. 3). Moreover, in the case of the anterior pituitary gland, tissue for morphine treated lambs exhibited much reduced in vitro release of Met-enkephalin (Fig. 8). Treatment with CRH either attenuated or augmented effects of morphine (Table 1) depending on the tissue and enkephalin activity.
In conclusion, the present data demonstrated acute effects of morphine on the synthesis, secretion and concentrations of native and cryptic Metenkephalin. The present results provide further support for the cross-talk between mu, delta and kappa receptors and the HPA axis. Furthermore, evidence is advanced supporting the ability of CRH to attenuate some of the effects of morphine and to augment other effects of morphine.