ACTH-Like Peptides, Pituitary-Adrenocortical Function and Avoidance Behavior

ACTH-Like Peptides, Pituitary-Adrenocortical Function and Avoidance Behavior E. ENDROCZI

Central Research Division, Postgraduate Medical School, Budapest, Hungary

INTERACTIONS OF ALP, CHOLINOMIMETICS AND OPIATES ON CORTICOTROPINRELEASING HORMONE (CRH) SECRETORY ELEMENTS Terenius (1975) showed that ACTH and its analogues have appreciable affinity for stereospecific opiate receptors in synaptosomal membranes of brain tissue. Interestingly, a-melanocyte stimulating hormone, vasopressin, substance P and thyroid releasing hormone proved t o be inactive. These observations supported the view of Zimmerman and Krivoy (1973) that ACTH-like peptides (ALP)interfere with morphine in the central nervous system. In addition to this, the findings are in accordance with observations that ALP can antagonize the analgesic effects of opiates (see the review of Gispen et al., 1976). Selye (1936) was the first t o demonstrate the morphine-induced hyperactivity of the adrenal cortex. This finding was recently confirmed, among others, by Munson (1973) and by deWied and deJong (1 974). Electrolytic lesions of the tubero-infundibular area prevented the morphine-induced rise of plasma corticosterone level (George and Leong Way, 1959), whereas local application of morphme into the basal and mid-hypothalamic nuclei increased the corticosterone production of the adrenal glands and elevated the plasma corticosterone level (van Ree et al., 1976). Local application of morphine into the dorsal and the caudal hypothalamic areas had no effect (Lotti et al., 1969). Cox et al. (1976) reported that hypothalamic sites where morphme elicits plasma corticosterone response are different from sites which are positive for temperature changes. Recently, we studied the effects of intrahypothalamic administration of morphine into different parts of the hypothalamus in adult male rats. The drug or its vehcle was given bilaterally in 11.11 physiological saline through a chronically implanted cannula; the rats were killed 45 min following injection of 2 yg morphine, and the plasma corticosterone concentration was determined by the protein-binding technique. The cannulae were implanted 10 days prior to the experiments into different parts of the hypothalamus. Bilateral injections of 2 yg morphme into the arcuate nucleus and the suprachiasmatic area caused an increase in the plasma corticosterone level (Fig. 1). Other sites, such as the posterior hypothalamus and rostra1 preoptic area, proved to be ineffective. Simultaneous administration of 2 pg morphine together with 10 yg naloxone (a morphine antagonist) into the caudal suprachiasmatic region produced a significant decrease of the morphine-induced plasma corticosterone response level (Fig. 2). It is worth mentioning that naloxone alone did not modify the plasma corticosterone level and did not inhibit the daily rhythm of pituitary-adrenal axis when the drug was injected bilaterally into the preoptic or suprachiasmatic areas (unpublished observations).

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F i g . 1 . Effect of bilateral microinjections of 2 gg morphine (MO) on the plasma corticosterone level in male rats. ARC, arcuate nucleus; SC, suprachiasmatic nucleus; HP, posterior hypothalamus; POR, preoptic region. Horizontal lines above bars give standard errors in this and the following figures. Fig. 2. Effect of naloxone given simultaneously with morphine into the suprachiasmatic nuclei on plasma corticosterone level in rats. Control rats received physiological saline as vehicle. MO, morphine, NAL, naloxone.

Intrahypothalamic administration of drugs which inhibit the cholinesterase activity is followed by a significant rise of the plasma corticosterone level in rats. Thus, the microinjections of 20 yg physostigmine and 5 yg di-isopropyl-fluorophosphate (DFP) via chronically implanted cannulae into different parts of the hypothalamus revealed that the preoptic area is highly effective in inducing the activation of pituitary-adrenal axis. The hypothalamic sites where cholinomimetics cause the plasma corticosterone level to increase, do not overlap the sites where morphine activates the pituitary ACTH release (Fig. 3). There is no causal relation between the inhibition of hypothalamic cholinesterase activity and the activation of pituitary ACTH release. Acetylcholinesterase activity was measured in

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Fig. 3. Localization of effective and ineffective sites for DFP and morphine (MO) to induce elevation of plasma corticosterone level. CA, commissure anterior; OC, optic chiasma.

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P’ig. 4. Effect of 5 pg DFP injections into the preoptic area on the plasma corticosterone level and the acetylcholinesterase (AChE) activity of the hypothalamic tissue in rats. The enzyme activity was expressed in IU/g wet weight. The rats were killed at different time intervals following intrahypothalamic injections. Fig. 5. Effect of atropine treatment (4 mg/kg body weight, subcutaneously) on the physostigmine(PHYS) and DFP-induced increase of plasma corticosterone level in rats. Five pg DFP and 10 pg physostigmine were injected into the preoptic area and the rats were killed 45 min later.

those tissue pieces where the DFP was injected. Following administration of 5 pg DFP into the rostra1 preoptic area the elevation of plasma corticosterone level normalized within 24 h, although the enzyme activity was still suppressed by at least SO%, and returned to control values only 5-7 days later (Fig. 4). Simultaneous administration of the anticholinergic drug, atropine, with physostigmine and/or DFP into the preoptic area or the suprachiasmatic nuclei, revealed that atropine can inhibit the physostigmine-induced increase of plasma corticosterone level, but it failed t o suppress the effect of DFP (Fig. 5). These observations led us to assume that factors other than an excess of free acetylcholine at the synapses may be involved in DFP-induced activation of pituitary ACTH release. In higher doses, atropine blocks both muscarinic and nicotinergic receptors, and the local application of 10 pg atropine in our studies may be considered a large dosage.

OPIATE RECEPTORS AND CHOLINOMIMETICS Combined injections of DFP with different doses of naloxone into the suprachiasmatic nuclei led t o a dose-dependent decrease of the DFP-induced rise in plasma corticosterone level. Thus, 8-10 pg naloxone and 2 pg DFP resulted in at least 50% suppression of the presumed activation of the pituitary ACTH release (Fig. 6 ) . The experimental findings that naloxone can affect the DFP-induced acitvation of corticotrophin releasing hormone ( C M ) neurosecretory elements supports the assumption that tlus organophosphorous substance acts at the opiate receptors, or else produces conformational changes of receptive sites. Irreversible inactivation of acetylcholinesterase by

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Fig. 6. Dosedependent action of naloxone (NAL) o n the DFP-induced increase of plasma corticosterone level in rats. The drugs were given simultaneously into the suprachiasmatic area (SC). Horizontal lines indicate the mean and standard deviations of resting plasma corticosterone concentration. Vertical lines indicate mean i standard deviation of results. Fig. 7 . Inhibitory action of 4 fig ACTH 4-10 on the morphine- (MO) induced increase of plasma corticosterone level in male rats. The drugs were injected into the suprachiasmatic nuclei in single or combined microinjections.

DFP as a result of the phosphorylation of serine residues of the enzyme molecule is a known mechanism for inhibition of esterase activity. However, phosphorylation of membrane macromolecules possessing other receptive sites cannot be excluded. Separate administration of naloxone into the arcuate nucleus and DFP into the preoptic area did not modify the DFP-induced activation of CRH neurosecretory elements. This observation led t o the assumption that both drugs act upon the same pool of cells. However, it remains unclear whether these cells possess both cholinoreceptive and opiate receptor sites, or, whether they are separate elements. Activation of pituitary ACTH release by microinjections of morphine into the suprachasmatic nuclei was decreased by local application of ACTH 4-10. The ACTH analogue itself did not change the resting plasma corticosterone level. Intrahypothalamic injection of 4 pg ACTH 4-10 was performed simultaneously with 2 pg morphine through chronically implanted cannulae, the animals being killed 45 min later. ACTH 4-10 produced a significant decrease of the morphine-induced elevation of plasma corticosterone level (Fig. 7). It is worth mentioning that combined injections of morphine plus atropine into the suprachiasmatic nuclei in different doses did not influence the morphine-induced increase of plasma corticosterone level. This finding indicates that opiate receptors are involved in the activation of CRH neurosecretory elements but the way in which neurotransmitters are involved is still unclear. ACTH-INDUCED BEHAVIORAL REACTIONS AND BRAIN NOREPINEPHRINE NEURONAL SYSTEM Considerable evidence suggests that brain catecholamines are involved in the organization of behavioral reactions, although the mechanisms behind these processes are still the subject

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of intensive investigation. In our earlier work in rats we studied the effect of ALP on brain norepinephrine (NE) turnover, and certain correlations were found between ACTH-induced behavioral reactions and NE-neuronal activity (see the review of Endroczi, 1976). Intraventricular administration of ACTH 1-24 and ACTH 4-10 produced an increase of brain NE turnover which could not be simulated by ACTH 11-24. Moreover, it was found that ALP produced a marked disappearance rate of [3H]NE from the brain pool, which could not be prevented by blocking either the uptake or the biosynthesis (Endroczi et al., 1975). In studying the sites where ALP enhanced the activity of NE neuronal system we found that microinjections of ACTH 4-10 into the brain stem reticular formation at the level of the locus coeruleus were followed by a significant rise of NE turnover in the forebrain (Endroczi, 1976). Injections at other sites like septum, hippocampus, hypothalamus and neocortex were ineffective in this respect (Fig. 8). In earlier investigations we found that the local application of ALP into the brain stem increased the resistance against extinction of avoidance responses. Rats were trained in a shuttlebox until a near 100% criterion level was attained. Naloxone (8 pg) and ACTH 4-10 (2 pg) were then injected, in 1 physiological saline, into the brain stem reticular formation at the locus coeruleus level, and the extinction of avoidance response was studied by presentations of non-reinforced trials. Fifteen trials were presented in each daily session, with the intracerebral injections given 30 min prior to the session. Fig. 9 shows that ACTH 4-10 retarded the extinction of avoidance response, but the combined injection with naloxone blocked the effect. Naloxone, injected alone into the brain stem in doses ranging between 4 and 12 pg, failed t o influence the extinction rate. With regard to the inhibitory action of naloxone upon the ACTH-induced behavioral reaction, we have tested the effect of ACTH plus simultaneous administration of naloxone upon brain NE turnover. Injections of 2 pg ACTH 4-10 and 8 pg naloxone bilaterally into

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Fig. 8. Effect of 2 pg ACTH 4-10 on the forebrain NE turnover rates when given into the brain stem at the locus coeruleus (COER) level and into the septum. [3H]NE/[3H]tyrosine(T) ratio indicates the turnover rate. Time scale corresponds to 45 rnin. Vertical lines indicate mean f standard deviation. Fig. 9. Effect on the extinction of avoidance response in rats of 8 pg naloxone (NAL) given in combined injections with 2 fig ACTH 4-10. The drugs were administered into the brain stem reticular formation at the locus coeruleus level.

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Fig. 10. Effect of naloxone (NAL) o n the ACTH 4-10-induced increase of NE turnover of the forebrain in rats. The peptide (2 pg) and the drug (8 ug) were given into the brain stem reticular formation. Explanation of the symbols given in Fig. 8.

the brain stem reticular formation led to a marked increase in forebrain NE turnover. These observations indicate that ACTH-like peptides influence the NE neuronal system through a separate mechanism, and the blocking of opiate-receptors prevents the behavioral manifestations but not the activation of NE neurons ascending into the forebrain areas (Fig. 10). EFFECTS OF DIBUTYRYL CYCLIC GMP ON THE CRH RELEASE Recent findings suggest that analgesic effects of various drugs are mediated by more than one mechanism (Mayer and Price, 1976; Cohn et al., 1978). Enkephalins and morphine increase the cyclic GMP level in the brain (Minneman and Iversen, 1976; Racagni et al., 1976). Moreover, drugs affecting cholinergic transmission also altered the cyclic GMP content of the brain tissue (Ferrendelli et al., 1970). Centrally administered acetylcholine and cholinomimetics possess analgesic eftects that are blocked by atropine (Pedigo et al., 1975). Dibutyryl-cyclic guanosine 3’-monophosphate (dibutyryl-cGMP) administered directly into the central nervous system protects against noxious stimuli, without causing sedation or altering perception and locomotor activity. Unlike opiates, the analgesic properties of cyclic GMP (cGMP) are neither prevented nor reversed by naloxone (Cohn et al., 1978). It has not been established whether direct or indirect mechanisms are involved in morphme-induced elevation of cGMP content. For example, the cerebellar concentration of cGMP was altered by drugs affecting cholinergic transmission (Ferrendelli et al., 1970), and it is known that acetylcholine is the one of the transmitters implicated in the actions of both morphine and cGMP (Pert and Snyder, 1973; Ferrendelli et al., 1970). On the other hand, atropine does not antagonize the analgesic property of cGMP (Cohn et al., 1978), while iontophoreticdy applied atropine blocks the effect of acetylcholine but not of cGMP (Ferrendelli et al., 1970).

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Fig. 11. Effect of bilateral dibutyryl-cGMP injections (250 nmol) on the plasma corticosterone level in male rats. The nucleotide was given into the arcuate nucleus (ARC), suprachiasmatic nucleus (SC), preoptic region (POR) and the posterior hypothalamus (HP). Fig. 12. Effects o n plasma corticosterone level of injections of cCMP, either in combination with atropine (A), naloxone (NAL), or following dexamethasone (Dexam) pretreatment. The nucleotide and the drugs were injected into the arcuate nucleus 30 min prior to sacrificing the rat.

Microinjections in conscious rats of 150-250 nmol dibutyryl-cGMP, in 1 physiologica saline, produced a marked rise in plasma corticosterone level if the administration was performed into the arcuate nucleus. The plasma corticosterone concentration began to rise within 5-10 min, and had its peak about 25-40 min following the injections. Fig. 11 shows that injections of cGMP into the suprachiasmatic and preoptic nuclei or into the dorsal hippocampus did not increase the plasma corticosterone level which was measured 30 min later. Simultaneous administration of atropine or naloxone with dibutyryl-cCMP into the arcuate nucleus did not modify the nucleotide-induced activation of the pituitary-adrenal axis (Fig. 12). Administration of 0.5 mg/kg dexamethasone 12 h prior t o the intracerebral administration of nucleotide led t o a complete inhibition of plasma corticosterone response. EFFECTS OF cGMP ON BEHAVIORAL REACTIONS Bilateral intraventricular administration of 250 nmol cGMP produced n o remarkable changes in spontaneous behavior, with the rats showing normal orienting reactions and exploratory activity in a novel environment. The analgesic property of the nucleotide was not tested in these experiments although it was apparent, because the rats showed an increased resistance t o the pain caused by penetration of the sharp points of the clamp t o the tail. The behavioral effect of cGMP was tested on the extinction of avoidance response in adult male rats. The animals were trained in a shuttle-box by presentations of buzzer sound with painful electric shocks (0.5 mA). Twenty associations were presented in each daily session until a near 100%criterion level was reached. The cGMP was then administered

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Fig. 13. Effect of cGMP and cAMP o n the extinction of avoidance response in rats. The nucleotides were given bilaterally into the lateral ventricles (250 nmol in 5 g1 physiological saline) and the extinction was performed b y presentations of 15 trials per daily session. The administration of nucleotides was repeated each day 15 min prior to behavioral testing.

bilaterally via chronically implanted cannulae into the lateral ventricle 15 min prior to each behavioral test. Fifteen non-reinforced trials were presented in order to follow the extinction of avoidance response. The control rats received physiological saline, while another group of animals was treated with 250 nmol cyclic adenosine 5’-monophosphate (CAMP). Fig. 13 shows that intraventricular administration of 250 nmol cGMP resulted in a marked resistance t o extinction of avoidance response. The CAMPtreatment proved to be ineffective. It is worthwhile mentioning that retardation of extinction by cGMP administration was more powerful than the effect observed after ALP treatment. Moreover, the systemic administration of cGMP in doses ranging between 500 and 1200 nmol/l00 g body weight did not influence the extinction rate. GENERAL CONCLUSIONS More than 25 years ago Torda and Wolf (1952) reported that ACTH administration is followed by an increase of acetylcholine synthesis at the neuromuscular junction. It was demonstrated in other studies that ALP increase the NE turnover of the brain in both intact and adrenalectomized rats (Hokfeh and Fuxe, 1972; Versteeg, 1973; Endroczi et al., 1975; Endroczi, 1976). Correlation studies between the NE turnover rate and the extinction process of avoidance response postulated a direct role of the NE neuronal system in ACTHinduced behavioral changes (Endroczi, 1976). In the present investigations we have found that naloxone inhibits the behavioral action of ALP, although the effect of the peptide upon NE turnover remained unchanged. These observations indicate that ALP exert a multiple action on cells with different receptive sites, and that the activation of brain NE neuronal systems is not necessarily coupled t o extinction of conditioned responses. Terenius and Wahlstrom (1975) demonstrated the existence of an endogenous ligand of morphine receptors which was isolated from the cerebrospinal fluid. Terenius (1 975) showed

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that ALP have an appreciable affinity for opiate binding sites, observations that are in accordance with the view of Zimmernian and Krivoy (1974) that ALP interfere with morphine in the central nervous system. Many studies indicate that brain tissue contains endogenous compounds which resemble opiates in their action. The most intensively investigated of these substances are peptides such as enkephalins and endorphins. However, there is another type of endogenous morphine-like compound which is not a peptide, but w h c h binds to opiate receptors (Gintzler and Gershon, 1978). Immunocytochemical localization of such substances in various brain stem nuclei would imply their involvement with certain neuronal pathways associated with stimulus-produced analgesia. In connection with these findings, we must take into account that cGMP injections into the brain stem (e.g. periaqueductal gray matter and reticular formation) do not mimic the analgesic action of morphine, which suggests that opiates and nucleotides do not share a common mechanism of action. Involvement of the brain NE neuronal system in controlling the behavioral reactions has been supported by a number of observations, although the recent investigations presented here, plus the fact that (D-Phe7)-ACTH 4-10 is inactive in this respect are not in accordance with this view. Nevertheless, direct or indirect involvement of NE neurons in learning and memory processes is well-founded, although the mechanisms behind these events require further studies. Participation of cholinergic neurons in the central control of pituitary ACTH secretion has been supported by many observations. The present data are in accordance with the view that cholinergic transmission is involved in activation of CRH neurosecretory elements and the cholinoreceptive field located rostrally to the arcuate nucleus and the median eminence. These findings may help t o interpret the observations of Makara and Stark (1976), who reported that deafferentation of the basal and medial hypothalamus prevents the activation of pituitary ACTH release after local application of cholinomimetics. The DFP-induced activation of pituitary-adrenocortical function was partially inhibited by naloxone, which supports the assumption that in addition to cholinoreceptive field the opiate receptors are involved in controlling the CRH-producing elements. SUMMARY The discovery of behavioral effects of adrenocorticotropin-like peptides (ALP) had a profound influence upon the study of endocrine control over adaptive behavior, and greatly contributed t o the development of research into neuropeptides and peptidergic transmission in the brain (Mirsky et al., 1953; Murphy and Miller, 1955; Krivoy and Guillemin, 1961; deWied, 1965; Levine and Jones, 1965; Endroczi, 1972; Kastin et al., 1975). Considerable evidence suggests that ALP may act upon brain protein synthesis (Gispen et al., 1976) and catecholamine turnover (Endroczi et al., 1975), and that they bind t o opiate receptors (Terenius, 1975). The experimental observations that ALP of extrapituitary origin are present in the brain led t o the assumption that such neuropeptides are involved in neural transmission (Krieger et al., 1977; Pacold et al., 1978). The biologically active and immunoreactive ACTH content of the brain remained unchanged after either hypophysectomy or dexamethasone administration. These last observations indicate that other factors are involved in regulation of pituitary ACTH synthesis than that of the brain ALP. Whether the neuropeptides are directly involved in neural transmission or rather are playing a modulatory role, is still the subject of investigations.

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