Opiate antagonist counteracts reproductive inhibition by porcine ACTH extract.

The effects of naloxone, an opiate receptor antagonist, on the maturation of female mice chronically pretreated with ACTH from weaning, were examined. Naloxone treatment, begun at 40 days of age, blocked ACTH-induced delay of puberty. Naloxone also reversed decreases in weights of the reproductive organs produced by ACTH. Naloxone treatment also resulted in estrous cycling and increased the number of ACTH-treated mice with ovarian corpora lutea. These changes are reflected in increased LH levels in ACTH-naloxone treated vs. ACTH-saline treated animals. Naloxone alone may increase reproductive function. Effects of ACTH on spleen and thymus weights, presumably via the adrenals, were partially reversed by naloxone. ACTH may have a different mechanism in adrenal function as compared to reproductive function.     

Role of endogenous opiates and extracellular K+ accumulation in the inhibition of frog spinal reflexes by electrical skin stimulation

Electrical skin stimulation of the hind limb (10-100 Hz, 30 s-5 min) at the intensity which leads only to the excitation of low threshold afferents depressed (for 1-30 min) the flexor reflex evoked in spinal frogs by nociceptive stimuli. The inhibition, which lasted for longer than 5 min was blocked by naloxone. Short-term poststimulation effects were associated with an increase of extracellular K+ concentration (delta [K]e) and were not blocked by naloxone. Enkephalins or morphine applied to the spinal cord surface increased the threshold for flexor reflexes while naloxone decrease their threshold. The stimulation was followed by short-term hyperpolarization of primary afferents (PAH; 1-5 min) and by depression of dorsal root potentials (DPRs) which had a similar time course to the delta [K]e, and were not blocked by naloxone. This period was frequently followed by longlasting PAH and enhancement of DRPs (5-30 min), which were abolished by naloxone. Superfusion of the isolated spinal cord with opioids produced PAH and enhanced DRPs evoked by nociceptive stimuli, while naloxone or increase of [K] in Ringer solution depolarized primary afferents and depressed DRPs. It is suggested that the antinociceptive effects of electrical stimulation of low threshold cutaneous afferents in spinal frogs involves at least two mechanisms. The short-term effect may result from delta [K]e, especially at high stimulus strength and is equally effective against noxious and non-noxious stimuli. The longlasting effects selectively affecting nociceptive transmission appear to be produced by endogenous opioids.     

Naloxone does not antagonize PCP-induced stimulation of the pituitary-adrenal axis in the rat

Phencyclidine (PCP) has been shown to stimulate the pituitary-adrenal axis in the rat. The purpose of the present study was to determine whether opiate receptors are involved in this effect by testing whether pretreatment with the opiate antagonist naloxone can antagonize PCP-induced ACTH and corticosterone release. PCP (10.0 mg/kg) produced increases in plasma ACTH and corticosterone 60 min after s.c. administration. Pretreatment with naloxone (2.0 mg/kg s.c.) did not reduce the rise in plasma levels of ACTH or corticosterone produced by PCP. These results indicate that naloxone-sensitive opiate receptors are not involved in the PCP-induced stimulation of the pituitary-adrenal axis in rats.     

Emetic and antiemetic properties of regulatory peptides

Emetic and antiemetic properties of opioid peptides, substance "P", beta-lipotropin, and ACTH1-39 have been investigated in experiments on cats. It was shown that morphine, enkephalin, beta-endorphin and DADLE caused vomiting in animals, which was blocked by naloxone. Substance "P", gamma- and des-tyr-gamma-endorphin manifested antiemetic properties similar to those of naloxone. Selective antagonists of delta-opioid receptors ICI 154, 129 blocked emetic action of delta-agonist DADLE but did not prevent vomiting caused by mu-agonist morphine. It is suggested that the vomiting mechanisms of endogenous opioid peptides involve stimulation of mu- and delta-opioid receptors in the chemoreceptor trigger zone of the vomiting centre.     

Local loperamide injection reduces mechanosensitivity of rat cutaneous, nociceptive C-fibers

Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.     

The anticonvulsant effects of DADLE are primarily mediated by activation of delta opioid receptors: interactions between delta and mu receptor antagonists

Dose-response comparisons of the ability of the selective delta antagonist ICI 154,129 (12.5-50 nmol), the nonselective antagonist naloxone (29-290 nmol), and the irreversible selective mu antagonist beta-fNA (1.3-21 nmol) to alter the threshold response to DADLE or etorphine was studied in the rat flurothyl seizure test. DADLE (35 nmol, i.c.v.) and etorphine (122 nmol/kg, s.c.) both caused increases in seizure threshold which were differentially antagonized by pretreatment (i.c.v.) with the respective antagonists. For DADLE, only ICI 154,129 and naloxone produced a dose-related blockade of the increase in seizure threshold, with ICI 154,129 being more potent than naloxone. In contrast, the anticonvulsant action of etorphine was not antagonized by ICI 154,129 (50 nmol), but was blocked by a low dose of naloxone (29 nmol) or beta-fNA (21 nmol). In addition, prior occupancy of mu-sites with beta-fNA (21 nmol) significantly diminished the abilities of either ICI 154, 129 (50 nmol) or naloxone (290 nmol) to antagonize the anticonvulsant action of DADLE. The results of this study demonstrated that the effects of DADLE to increase seizure threshold in the rat were primarily mediated by activation of a delta-opioid receptor system. Furthermore, evidence has been provided for a functional interaction between delta and mu receptors in the opioid regulation of seizure threshold.     

Pressor, tachycardic and behavioral excitatory responses in conscious rats following ICV administration of ACTH and CRF are blocked by naloxone pretreatment

Experiments were conducted to compare the blood pressure and heart rate responses of conscious rats given intracerebroventricular (ICV) injections of adrenocorticotropin (ACTH 1-24) and corticotropin releasing factor (CRF). Under sodium pentobarbital anaesthesia, rats were implanted with a stainless-steel cannula into the lateral cerebral ventricle and had their right femoral artery and vein cannulated. Upon recovery (24-48 hr later) conscious, unrestrained rats were given ICV injections (total volume 5 microliter by gravity flow) of sterile saline, ACTH (1-24) (0.85 and 1.7 nmoles) or CRF (0.55 and 1.1 nmoles) and blood pressure and heart rate were monitored over the next 2 hr (from the abdominal aorta via the femoral arterial catheter). Both ACTH and CRF caused mean arterial pressure (MAP) to increase, which was paralleled with increases in mean heart rate (MHR). Moreover, these elevations in MAP and MHR were temporally associated with excessive grooming (for ACTH) and locomotor activity (for CRF), which occurred before and lasted as long as MAP and MHR were enhanced. Intravenous (IV) pretreatment whereby naloxone was given 10 min before ICV administration of ACTH (1.7 nmoles) or CRF (1.1 nmoles), showed that naloxone blocked the behavioral, pressor and tachycardic effects of both ACTH and CRF. The results demonstrate that the pressor, tachycardic and locomotor effects evoked in conscious rats by ICV administration of ACTH or CRF are antagonized by naloxone and that their hemodynamic changes may, in part, be mediated by prior behavioral activation.     

ACTH and corticosterone response to naloxone and morphine in normal, hypophysectomized and dexamethasone-treated rats

The effect of opiate receptors blocker naloxone on ACTH and corticosterone secretion in normal, dexamethasone-treated and hypophysectomized rats was studied. A dose-related increase in plasma corticosterone level was found at 45 min after s.c. injection of naloxone in a dose range of 0.25-2.0 mg kg-1. The rise in plasma corticosterone was preceded by a slight increase in plasma ACTH. Acute morphine administration in a relatively low dose (6 mg kg-1 s.c.) induced a significant rise in both plasma ACTH and corticosterone levels. Dexamethasone treatment was followed by low basal corticosterone level, by total inhibition of the stress response and response to morphine injection, while the response to ACTH administration was normal. Under these circumstances as well as in rats 6 days after hypophysectomy, naloxone failed to increase plasma corticosterone levels. It is concluded that a direct stimulation of corticosteroid biosynthesis in adrenal cortex is not involved in the mechanism of naloxone-induced activation of pituitary-adrenocortical function.     

Ontogenetic changes in porcine adrenocortical adrenocorticotropic hormone receptors.

1. Binding of [125I]ACTH(1-38) analog to adrenal receptors was measured in fetal pigs (Sus domesticus) at 15-day intervals from midpregnancy (60 days) to near term (105 days; pregnancy length 114 days). 2. Binding was greatest at day 60 (0.42 +/- 0.03 fmol/200 micrograms protein or 0.50 +/- 0.08 fmol/50 micrograms DNA), and least at day 105 (0.13 +/- 0.03 fmol/200 micrograms protein or 0.16 +/- 0.04 fmol/50 micrograms DNA). Total adrenal binding was constant (0.61 +/- 0.02 fmol/paired adrenals). 3. Scatchard analyses at day 60 and day 105 showed comparable apparent affinities of ACTH receptors (Ka day 60 = 1.51 +/- 0.72 x 10(9) M-1 vs Ka day 105 = 1.94 +/- 0.78 x 10(9) M-1). 4. DNA per paired adrenals and membrane-associated protein increased 1.6-fold, providing a constant protein: DNA ratio. Concentrations of adrenal cortisol were constant from 60 to 90 days of gestation age but increased dramatically by day 105. 5. These data suggest that during 60-105 days of gestation age the number of ACTH receptors per cell is reduced.     

Naloxone-induced enhancement of carbon dioxide stimulated respiration

In unanesthetized rats, naloxone (5 mg/kg, s.c.) produced an increase in both respiratory frequency and tidal volume as compared to saline administered animals. Maximal respiratory stimulation was observed within 5 minutes after naloxone injection and duration of the response was greater than 30 minutes. Exposure to different atmospheres of carbon dioxide potentiated the increase in ventilation in a step-wise manner as the carbon dioxide concentration was increased. Pretreatment with low doses of morphine sulfate (2 mg/kg daily for 2 days) or naloxone HCl (5 mg/kg daily for 5 days) enhanced respiratory stimulation induced by naloxone. It was concluded that naloxone increases the sensitivity of central ventilatory response to carbon dioxide as a result of displacement of endogenous endorphins from central opioid receptors.     

Effects of the muscarinic agonist, 5-methylfurmethiodide, on contraction and electrophysiology of Ascaris suum muscle

Contraction and electrophysiological effects of 5-methylfurmethiodide (MFI), a selective muscarinic agonist in mammals, were tested on Ascaris suum muscle strips. In a contraction assay, MFI produced weak contraction and was less potent than levamisole and acetylcholine. Atropine (3microM) a non-selective muscarinic antagonist in mammalian preparations, did not affect contractions produced by MFI. Mecamylamine (3microM) a nicotinic antagonist in A. suum preparations, blocked the MFI contractions indicating that MFI had weak nicotinic agonist actions. In two-micropipette current-clamp experiments MFI, at concentrations greater than 10microM, produced concentration-dependent depolarizations and small increases in membrane conductance. The depolarizing effects were not abolished by perfusing the preparation in a calcium-free Ascaris Ringer solution to block synaptic transmission, suggesting that MFI effects were mediated by receptors on the muscle and were calcium-independent. A high concentration of mecamylamine, 30microM, only reduced the depolarizing responses by 42%, indicating that MFI also had effects on non-nicotinic receptors. Three non-nicotinic effects in the presence of 30microM mecamylamine were identified using voltage-clamp techniques: (i) MFI produced opening of mecamylamine-resistant non-selective-cation channel currents; (ii) MFI inhibited opening of voltage-activated potassium currents; and (iii) MFI increased the threshold of voltage-activated calcium currents. We suggest that a drug that is more selective for voltage-activated potassium currents, without effects on other channels like MFI, may be exploited pharmacologically as a novel anthelmintic or as an agent to potentiate the action of levamisole. In a larval migration assay we demonstrated that 4-aminopyridine (4-AP: a potassium channel blocker) potentiated the effects of levamisole but MFI did not.     

The effect of low-dose naloxone infusion on plasma ACTH and LH in patients with Cushing's and Addison's diseases

The ACTH, cortisol and LH responses to low dose (0.8 mg/h) naloxone 90 min infusion were investigated in seven patients with untreated Cushing's disease, six patients with Addison's disease and four control subjects. Naloxone had no effects on ACTH hypersecretion or normal ACTH levels. These data confirm that naloxone cannot provide additional diagnostic or therapeutic approaches in ACTH hypersecretion syndromes, mainly in Cushing's disease. The mean percentage LH levels did not significantly change during low dose naloxone in controls or patients with Cushing's and Addison's diseases. This suggests that increased endogenous opioid peptides in these diseases may not modify the LH responses to low dose of naloxone. However, since three of five adults with Cushing's disease had increased LH levels during naloxone, further studies may be indicated.     

Effects of (D-ala2, met5)-enkephalinamide and naloxone on ACTH and corticosterone secretion

An intravenous administration of (D-ala2, met5)-enkephalinamide (DALA) caused a significant elevation of plasma ACTH and corticosterone at 10 to 20 min after injection in unanesthetized freely moving rats. An intraperitoneal administration of cyproheptadine tended to reduce plasma ACTH and corticosterone levels at 60 min after injection, but it did not attenuate the DALA-induced ACTH and corticosterone elevation. A large dose of naloxone (1-10 mg/kg body weight) caused a significant elevation in plasma corticosterone, but naloxone at 10 mg/kg body weight reduced the basal ACTH level and DALA-induced ACTH elevation. When both DALA and naloxone were injected, the steroidogenic effect was attenuated. Neither DALA nor naloxone affected the basal ACTH release and CRF-induced ACTH stimulation in rat anterior pituitary cell cultures. These results suggest that DALA acts at the extra-hypophyseal level to stimulate ACTH and corticosterone and that the naloxone stimulatory effect on steroidogenesis acts on the adrenal gland or is mediated by stimulating corticosterone stimulating factors other than ACTH.     

Opioids and hibernation. II. Effects of kappa opioid U69593 on induction of hibernation in summer-active ground squirrels by "hibernation induction trigger" (HIT)

A "Hibernation Induction Trigger" (HIT) isolated from plasma of winter-hibernating woodchucks induced hibernation in summer-active ground squirrels (Citellus tridecemlineatus). Effects of kappa opioid U69593 on the HIT-induced hibernation were examined. U69593 alone did not elicit marked behavioral alteration or hibernation in summer-active ground squirrels. U69593, however, antagonized hibernation induced by HIT in summer active ground squirrels. In the guinea pig ileum myenteric plexus-longitudinal muscle preparation, woodchuck HIT depressed the electrically-induced contraction. The depression was, however, neither reversed nor blocked by naloxone even when naloxone was used at high doses. This study demonstrates that kappa opioid, at least in the case of U69593, was unable to induce hibernation in the summer-active ground squirrels. The results also demonstrate that woodchuck HIT, like the bear HIT, did not act directly at opioid receptors. Together with our previous observation that naloxone blocked summer hibernation induced by HIT (Bruce et al., Life Sci.., this issue), it is tempting to suggest that HIT may not mediate its effects through kappa opioid receptors but may do so through other types of opioid receptors such as mu or delta. U69593 may antagonize HIT-induced hibernation as a mu or delta receptor antagonist.     

Altered excitability of rat cutaneous mechanoreceptors during transcranial electrical stimulation

Functional aspects of high, medium, and low threshold mechanoreceptor units were investigated in acute experiments under hexanol-induced anesthesia during transcranial electrical stimulation (TES). It was found that low and medium threshold receptor units ceased responding to mechanical stimulation 1 and 5 min respectively after the start of TES. Firing ended by 18–20 min of TES in high threshold units responding to mechanical stimuli produced by a needle. No significant alteration in parameters of response were produced by TES after prior i.p. or intracutaneous injection of naloxone. Potential mechanisms of TES action are discussed. It is suggested that changes produced in the function of cutaneous mechanoreceptors by TES may be put down to endogenous opioids acting on the receptors.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 495–500, July–August, 1990.     

Adrenocorticotrophin responses to hypoxaemia in fetal sheep are sustained in the presence of naloxone.

We have examined the effects of fetal hypoxaemia, produced by reducing the percent oxygen in maternal inspired air, on fetal plasma concentrations of corticotrophin releasing hormone (CRH), adrenocorticotrophin (ACTH) and cortisol and determined the effects of an opioid receptor antagonist, naloxone on these responses. Hypoxaemia (fetal PO2, 15-18 mmHg) for 60 min provoked a significant (P < 0.05) increase in fetal plasma ACTH and cortisol concentrations at days 125-130 of pregnancy, but did not affect circulating CRH. There was no effect of naloxone administered either intravenously (1.25 mg bolus followed by a 2.5 mg/h continuous infusion for one hour; fetal body weight approximately 2.5 Kg) or via the lateral cerebral ventricle (50 micrograms bolus followed by a 100 micrograms/h infusion for one hour) on this pattern of ACTH and cortisol change nor on the lack of CRH response to hypoxaemia. We conclude that the increase in fetal ACTH and cortisol in response to acute hypoxaemia is not accompanied by an increase in systemic CRH concentrations, nor is the response dependent on short-term opioid regulation.     

The met-enkephalin analog FK 33-824 directly inhibits ACTH release by the rat pituitary gland in vitro

Systematic administration of the enkephalin analog FK 33-824 was previously shown to stimulate PRL secretion and to inhibit ACTH secretion in man. Naloxone prevented the effect on PRL release, but not on ACTH release. In this study, the direct action of this analog on hormone release by rat anterior pituitary lobes $\text{in}$$\text{vitro}$ were investigated. 1 uM FK 33-824 inhibited basal ACTH secretion by anterior pituitary glands in vitro, while 0.1 uM and 1 uM attenuated the lysine vasopressin stimulated ACTH release. Naloxone did not reverse the inhibitory action of the analog on ACTH release. β-Endorphin (0.01 - 1 uM) did not directly affect ACTH release. Basal and dopamine-induced inhibition of PRL release by anterior pituitary glands was neither influenced by FK 33-824 (0.1 and 1 uM), nor by β-endorphin (0.1 and 1 uM) with or without bacitracin. This study shows that the long-acting met-enkephalin analog FK 33-824 differentially affects PRL and ACTH secretion by the pituitary gland. It seems to stimulate PRL release at a suprapituitary site and this action probably involves u opiate receptors, because naloxone prevents these stimulatory effects. The inhibitory effect of FK 33-824 on ACTH release, however, is mediated via a direct effect at the pituitary level, which does not involve u receptors, as naloxone did not prevent this effect. In this respect, its action differs from that of β-endorphin, which does not directly affect ACTH release by the anterior pituitary gland.     

"Paradoxical" analgesia and aggravated morphine dependence induced by opioid antagonists

Chronic treatment with naloxone (Nx) or naltrexone (Ntx) induces paradoxical analgesia. In the present study, the effects of chronic treatment with opioid receptor antagonists, such as nor-binaltorphimine (nor-BNI) for kappa and naltrindole (NTI) for delta receptors, on analgesic response using the hot plate test and on morphine physical dependence in rats were examined. The hot plate latency was significantly increased by pretreatment with Nx (5 mg/kg, s.c.), nor-BNI (20 mg/kg, i.p.) or NTI (20 mg/kg, i.p.) for 5 days. After chronic pretreatment with these antagonists, the rats were treated with morphine-admixed food (0.5 mg/g of food) for 3 days. Chronic pretreatment with Nx and NTI significantly increased Nx precipitated body weight loss in morphine dependent rats, while chronic pretreatment with nor-BNI produced small increase. These results indicate that chronic treatment with nor-BNI or NTI as well as with Nx induces obviously paradoxical analgesia, and that chronic blockade of mu or delta may enhance the development of physical dependence on morphine.     

Effects of a kappa-opioid agonist on adrenocorticotropic and diuretic function in man

Although kappa-opiate receptors represent an important fraction of the total opiate receptor capacity in human brain their endocrine function is unknown. We determined the effects of a kappa-opiate receptor agonist on the secretion of vasopressin, ACTH and cortisol and on diuresis. The racemic benzomorphan kappa agonist MR 2033 or its opiate active (-)-isomer, MR 2034, inhibited the release of cortisol and ACTH in 12 trials in a naloxone reversible manner; plasma levels of vasopressin were not altered. The (+)-isomer, MR 2035, did not affect the secretion of cortisol or ACTH. Surprisingly, in five other subjects large increases were observed in vasopressin, ACTH and cortisol following the kappa-agonist, which were probably elicited indirectly by aversive effects of the opioid. The subjects in whom vasopressin release was not altered by MR 2033 and MR 2034 displayed large decreases in urine osmolality which were not antagonized by naloxone. The opiate inactive (+)-isomer, MR 2035, caused no diuretic response. Subjects in whom vasopressin release was stimulated did not show decreases in urine osmolality indicating that vasopressin is capable of antagonizing the diuretic action of the kappa-agonist. Our data show that a kappa-agonist inhibits secretion of cortisol and ACTH by acting at stereospecific opiate receptors and elicits diuresis by acting at stereospecific, but naloxone-insensitive non-classical opioid receptors. These data support the concept that different types of kappa-receptors can be distinguished in man.     

Effects of morphine on cold-induced TRH release from the median eminence of unanesthetized rats

The effect of morphine perfusion into the median eminence on cold-induced TRH secretion was studied in unanesthetized rats by push-pull cannulation. Perfusion with 10(-6)M morphine blocked the cold-induced TRH peak occurring about 40 min after the transfer of rats from 24 degrees C to 4 degrees C. This inhibition by morphine was blunted by concomitant administration of naloxone (10(-6)M or 10(-5)M), but naloxone alone had no effect on either basal or cold-induced TRH release. We conclude that specific opiate receptors may be located on TRH nerve endings in the ME, and that endogenous opiates may not have any physiological role in the cold-induced TRH response, at least during the two hours that follow cold exposure.