The effects of naloxone and picrotoxin on the sedative and anticonflict effects of benzodiazepines

Interactions between naloxone and the benzodiazepine, chlordiazepoxide (CDP), were investigated in rats in a Conditioned Suppression of Drinking Test (CSD), which is a model of experimental conflict behavior. Naloxone reversed the anticonflict activity of CDP in this test. Naloxone and picrotoxin were then tested against CDP in rats using the Geller Conflict Test, which is an operant model of conflict behavior. Both naloxone and picrotoxin antagonized the anticonflict effects of CDP. Naloxone and picrotoxin were also tested for their abilities to reverse CDP-induced loss of righting reflexes in mice. Both naloxone and picrotoxin antagonized the loss of righting reflexes induced by CDP. Naloxone had no effect on the loss of righting reflexes induced by barbiturates or meprobamate. These results suggest that naloxone may be useful in the management of benzodiazepine overdoses.     

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.     

Perinatal ACTH-naloxone treatment: Effects on physical and behavioral development

Pregnant mice were treated with either ACTH or naloxone, or both, from 2 to 5 days prior to parturition, until 5 days postpartum. Perinatal ACTH caused a retardation of growth, eye opening, and vaginal opening that was blocked by naloxone; perinatal naloxone produced a decrease in open field activity at 32 days of age that was blocked by ACTH; naloxone reduced the threshold for heat at 50 days of age with no interaction with the ACTH treatment. It is proposed that part of the role of ACTH in physical systems may be mediated via naloxone-sensitive receptors. The direct effect of naloxone on another class of receptors may mediate the changes in threshold to heat flinch. Effects of ACTH and naloxone on the mother may have indirectly produced alterations in physical and motor development of the offspring.     

Calcium content and binding in synaptosomal subfractions during chronic morphine treatment

Chronic exposure to morphine in mice produced an increase in Ca²⁺ content of synaptosomes, synaptic plasma membranes (SPM), and synaptic vesicles. Ca²⁺ binding capacity was significantly reduced in tolerant SPM fractions. Naloxone significantly reversed the increased calcium content and reduced binding capacity of SPM when administered to 72-h-treated mice. Scatchard analysis of binding curves reveals three distinct classes of Ca²⁺ binding sites. During tolerance, the high- and low-affinity sites exhibit a reduced capacity to bind calcium, which may be reversed by in vivo and in vitro administration of naloxone. The increase in SPM and synaptic vesicle calcium content may reflect adaptive changes in the cell membrane during tolerance development, which may contribute to changes in neurotransmitter and second messenger function.     

The effect of naloxone administration on pregnancy-associated seizures

Pregnant mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The possibility that the well-known increase in beta-endorphin concentration which accompanies pregnancy was involved in this effect was examined by testing whether naloxone administration could block the increased seizure susceptibility. Pregnant female, control female and male C3H mice were treated with 5-50 mg/kg naloxone 5 min before flurothyl seizure testing. Naloxone markedly increased clonic seizure susceptibility in all three groups at a dose of 50 mg/kg, but had little effect at lower doses. In contrast, naloxone had differential effects on myoclonic seizures in pregnant and control female mice, being anticonvulsant in the controls, but proconvulsant in the pregnant mice. A role for endogenous opiates is unlikely in mediating clonic seizures in pregnant mice, but may be involved in myoclonic seizures.     

The effect of MSH/ACTH 4-10 on delayed response performance and post-test locomotor activity in rats

Delayed response performance was measured in male, Long-Evans rats 1 hr after IP administration of various doses of MSH/ACTH 4-10 or control in a Hunter delayed reaction apparatus. Additional treatments consisting of naloxone 500 micrograms/kg (IP) and naloxone 500 micrograms/kg in conjunction with MSH/ACTH 4-10 95 micrograms/kg were also administered. Directly after delayed response performance was assessed, gross locomotor activity was determined. MSH/ACTH 4-10, at a dose of 95 micrograms/kg, significantly enhanced retention of a visual stimulus, while MSH/ACTH 4-10, at doses of 195 and 285 micrograms/kg, significantly impaired delayed response performance. Naloxone treatment resulted in significantly impaired delayed response performance when compared to control. However, naloxone plus MSH/ACTH 4-10 treatment failed to produce a significant difference from control in the delayed response performance paradigm. In post-test locomotor activity determination, an apparent dose-response existed for MSH/ACTH 4-10 with the two highest doses (190 and 285 micrograms/kg) resulting in significantly increased locomotor activity. The observed delayed response performance data support theories implicating MSH/ACTH peptides in attentional processes involving visual stimuli. The fact that large doses of MSH/ACTH 4-10 disrupt delayed response performance while increasing post-test activity suggest that an optimum level of effect caused by the MSH/ACTH peptide exists in this paradigm.     

Opioid modulation of LH secretion in the ewe

Administration of opioid agonists and antagonists and measurement of resulting hormone changes were used to study the possible effects of opioids on reproductive function in the ewe. Intravenous administration of the long-acting methionine-enkephalin analogue FK33-824 (250 micrograms/h for 12 h) to 3 ewes during the follicular phase of the oestrous cycle depressed episodic LH secretion. This effect was reversed by administration of the opiate antagonist naloxone (25 mg/h) in combination with the FK33-824 treatment; in fact LH secretion was enhanced by the combined regimen. Naloxone (25 mg/h for 12 h) administered alone to 3 ewes in the follicular phase also enhanced LH secretion. In 3 animals treated with FK33-824 during the follicular phase, progesterone remained basal for 14 days after treatment, suggesting that ovulation was blocked. Jugular venous infusion of naloxone (25, 50 or 100 mg/h for 8h) into 5 ewes during the early and mid-luteal phase of the cycle resulted overall in a significant increase in mean plasma LH concentrations and LH episode frequency. To investigate whether endogenous opioids suppress LH release in seasonally anoestrous sheep, naloxone was infused intravenously into mature (25, 50 or 100 mg/h for 8 h) and yearling ewes (12 . 5, 25 or 50 mg/h for 8 h) during early, mid- and late anoestrus and plasma LH concentrations were measured. In the mature ewes, there was a trend for naloxone to increase LH values during the early anoestrous period but naloxone was without effect during mid- and late anoestrus. In the yearlings, naloxone infusion consistently increased plasma LH concentrations as a result of a significant increase in LH episode frequency. These experiments indicate that endogenous opioid peptides probably modulate gonadotrophin secretion during both the follicular and luteal phases of the oestrous cycle. However, the follicular phase of the sheep cycle is of short duration, and there may be residual effects of luteal-phase progesterone during this period. Secondly, there may be an age-dependent effect of naloxone on LH secretion during seasonal anoestrus in the ewe, with opioids playing a part in the suppression of LH in young but not in mature animals.     

Interactions between different antidepressants and morphine alter gastrointestinal transit in mice

To determine different serotoninergic antidepressants' effects on the gastrointestinal (GI) inhibiting effect induced by morphine, mice were pretreated with mianserin (a tetracyclic antidepressant with multiple 5-HT receptor subtypes interactions) and with fluoxetine (a selective 5-HT reuptake inhibitor). Mianserin alone, produced gastrointestinal inhibition in a dosedependent manner. Naloxone did not reverse this inhibiting effect, indicating that different mechanism of action are involved in morphine- and mianserin-induced inhibition of the gastrointestinal transit. Fluoxetine injected alone produced an increased propulsive motility of the GI transit. This effect was not reversed by naloxone. Fluoxetine did not reduce significantly mianserin-induced inhibition of GI transit. Fluoxetine also mildly reversed morphine-induced gastrointestinal inhibition, suggesting some degree of involvement of the opiates through the serotoninergic system.     

Serum luteinizing hormone, 13,14-dihydro-15-keto-prostaglandin F2alpha and cortisol profiles during postpartum anestrus in Brahman and Angus cows

Pluriparous suckled Brahman and Angus cows were utilized to evaluate the effect of breed, day after calving and endogenous opioid peptides (EOP) on hormonal profiles during postpartum anestrus. On Days 17 and 34 after calving, blood samples with and without heparin were collected at 15- and 30-min intervals, respectively, for a 7-h period via jugular cannula. Two hours after the start of blood sampling, cows of each breed were administered either 1 mg/kg iv naloxone or saline. Three hours later, all animals received 10 ng/kg iv GnRH. On Day 34 after calving cows received 0.2 IU/kg iv ACTH. Mean LH, basal LH and area under the LH curve increased (P < 0.01) from Day 17 to Day 34 after calving. Height of LH pulses increased (P < 0.05) by Day 34 after calving. Brahman cows had higher (P < 0.05) mean LH, basal LH, LH pulse frequency and area under the LH curve than Angus cows. Naloxone increased postchallenge area under the LH curve in treated cows above that of control cows (P < 0.06). Naloxone also increased the postchallenge area under the LH curve above that of the prechallenge level (P < 0.01). No breed differences in the response to the naloxone challenge were observed. The LH response to naloxone challenge occurred earlier on Day 34 than on Day 17 after calving but the amount of LH released was similar between days. The GnRH-induced LH release was greater in Brahman than in Angus cows (P < 0.04). Mean cortisol concentrations and area under the cortisol curve decreased (P < 0.05) between Day 17 and Day 34 after calving. Mean cortisol concentrations and area under the cortisol curve were lower (P < 0.01) in Brahman than in Angus cows. Cortisol secretion after ACTH treatment was similar between Brahman and Angus cows. The cortisol response after ACTH challenge was positively correlated (r=0.68; P < 0.001) to the prechallenge area under the cortisol curve. Under optimal environmental conditions Brahman cows have a greater LH release and their anterior hypophysis is more sensitive to GnRH challenge than the Angus cows.     

Naloxone and methylprednisolone sodium succinate enhance sympathomedullary discharge in patients with septic shock

Naloxone and methylprednisolone sodium succinate (MPSS) may act in synergy to improve hemodynamics in patients with septic shock by enhancement of sympathomedullary discharge. This randomized double-blind study describes the effect of various dosing regimens of naloxone and MPSS upon hemodynamics and plasma catecholamines in patients with septic shock (n = 57). Consecutive bolus doses of naloxone were given 30 minutes apart (10 μg/kg;–100 μg/kg) and a single dose of MPSS (30 mg/kg); bolus doses of 5% dextrose in water solution plus single dose of MPSS as above; bolus dose of naloxone (30 μg/kg) followed by continuous infusion (30 μg/kg/hr for 1 hour) with single dose of MPSS as above; a bolus and continuous infusion of naloxone as above without MPSS; MPSS alone and standard therapy alone. In patients treated with bolus doses of naloxone in combination with MPSS, plasma levels of epinephrine and norepinephrine were increased approximately five-to tenfold. In patients treated with bolus plus continuous infusion of naloxone given with or without MPSS, only plasma epinephrine levels were increased. Systolic blood pressure and left ventricular stroke work index were improved within 15 minutes in groups which received naloxone and corticosteroids regardless of dose. In those groups, there were no changes in heart rate or filling pressure. Systematic vascular resistance improved significantly only in the group which received low dose bolus and continuous infusion of naloxone and MPSS. Naloxone and MPSS quickly improved cardiac function in patients with septic shock by enhanced sympathomedullary discharge and may be useful as an adjunct in the therapy of this disorder.     

In vivo bidirectional regulation of mouse natural killer (NK) cell cytotoxic activities by Leu-enkephalin: reversibility by naloxone.

Intraperitoneal injection of Leu-enkephalin (LENK, 10 or 7.5 mg/kg) induced bidirectional modulation of natural cytotoxic activities in spleens of CBA mice (suppression followed by enhancement). NK-cytotoxic activity was more affected than the ADCC. Early suppression of NK activity could be reversed by 4 x M excess of naloxone injected 20 min before LENK, suggesting that the suppression was mediated by opioid receptors. Subsequent increase of NK activity could not be abrogated by naloxone, at least not completely. Naloxone itself decreased NK activity 12 hours after treatment, but enhanced ADCC at 24 and 48 hours. This increase was abrogated by LENK. In addition to functional alterations, LENK also induced phenotypic changes of spleen cells, i.e. a decrease in the percentage of asialo-GM-1+ cells 24 hours posttreatment. There was no correlation between LENK-induced alterations of cytotoxic function and the percentage of cells with NK phenotype (GM-1+). Thus, LENK modulates cytolytic functions and the phenotype of NK cells in vivo in a complex way, which besides opioid mechanisms may also include non-opioid ones.     

Neuropeptides in spinal cord injury: Comparative experimental models

The possible role of endogenous opioids in the pathophysiology of spinal cord injury was evaluated utilizing a variety of experimental models and species. In the cat, we have shown that β-endorphin-like immunoreactivity was increased in plasma following traumatic spinal injury; such injury was associated with a decrease in spinal cord blood flow (SCBF) which was reversed by the opiate receptor antagonist naloxone. Naloxone treatment also significantly improved functional neurological recovery after severe injury. Thyrotropin-releasing hormone (TRH), possibly through its “anti-endorphin” actions, was even more effective than naloxone in improving functional recovery in the cat. In a rat model, utilizing a similar trauma method, TRH proved superior to naloxone in improving SCBF after injury. In addition, naloxone at high doses attenuated the hindlimb paralysis produced by temporary aortic occlusion in the rabbit. The high doses of naloxone required to improve neurological function after spinal injury suggest that naloxone's actions, if opiate receptor mediated, may be mediated by non-μ receptors. Dynorphin, an endogenous opioid with a high affinity for the κ receptor, produced hindlimb paralysis following intrathecal administration in rats. Taken together, these findings suggest that endogenous opioids, possibly acting at κ receptors in the spinal cord, may serve as pathophysiological factors in spinal cord injury.     

Antiarrhythmic action of naloxone: direct, non-opiate effect on the rat heart

The opiate antagonist naloxone reduced the incidence and severity of cardiac arrhythmia induced in rats by intracarotid administration of adrenaline. Naloxone also reversed the adrenaline-induced arrhythmia in isolated heart preparations, suggesting a local antiarrhythmic action of the opiate antagonist. Similar effects were obtained with the (+) stereoisomer of naloxone which is inactive as an opiate antagonist. Thus, the direct action of naloxone at the rat heart is probably not mediated by opiate receptors.     

Thermoregulatory (core, surface and metabolic) responses of unrestrained rats to repeated POAH injections of beta-endorphin or adrenocorticotropin

Repeated preoptic-anterior hypothalamic (POAH) injections of saline and 10 or 25 micrograms/microliters of beta-endorphin or ACTH were given to groups of male Sprague-Dawley rats. One hr after the fifth injection of beta-endorphin or ACTH, each rat received a POAH injection of naloxone HCl (10 micrograms/microliters). Core (Tre-rectal) and surface (Tt-tail) temperatures, metabolic (VO2) and behavioral responses were recorded 30 min before and 60 min after each drug injection. The initial POAH injection of either dose of beta-endorphin produced a hyperthermia. Peak hyperthermia was reduced in the group given 10 micrograms/microliters of beta-endorphin repeatedly. TtS rose after each beta-endorphin injection but temporally lagged Tre increases. Metabolic rate (VO2) was increased with repeated POAH injections of beta-endorphin. Naloxone reduced the elevated Tre seen with beta-endorphin by increasing Tt's further and reducing VO2. POAH administration of ACTH evoked only a slight hyperthermic Tre response, but elevated TtS and VO2S, due to enhanced grooming and explorative behavior. With repeated ACTH injections, TreS did not change from those on the first day as TtS and VO2 remained enhanced. Naloxone reduced VO2 and TtS of the ACTH-treated rats but TreS still were unchanged. Results suggest that the hyperthermia of unrestrained rats given an acute as opposed to repeated POAH beta-endorphin injections is mediated by different effector mechanisms. With the doses used, the slight and unchanging TreS seen with ACTH occurred because this peptide increased heat production due to locomotor activation yet also exaggerated heat loss by vasodilating the peripheral vasculature.     

Suppression of narcotic withdrawals and plasma ACTH by auricular electroacupunture.

Auricular electroacupuncture (AES) has been found to be successful in the suppression of withdrawal symptoms of morphine-addicted mice. In abrupt withdrawals precipited by naloxone, the plasma adrenocorticotropin (ACTH) rises to a high level which can also be effectively suppressed by AES. This elevation of plasma ACTH is not due to naloxone, as naloxone has no effect on the ACTH level in non-addicted mice. The possible physiological effect produced by AES is discussed.     

Prevention of damage in acute spinal cord injury by peptides and pharmacologic agents

Cats were used as models of traumatic spinal cord injury. Each experimental animal received a 500 g-cm force to the exposed dura at the level of thoracic fourth vertebra. Somatosensory evoked potentials (SEPs), carotid arterial blood pressure (BP), and abdominal aorta blood flow in the treated groups were compared with those of the control group. The three treated groups received naloxone (5 mg/kg), TRH (5 mg/kg), and a combination of methyl-prednisolone sodium succinate (MP, 35 mg/kg) and epsilon-aminocaproic acid (EACA, 350 mg/kg). The SEPs which were done only in the naloxone treated group approached "normalcy" 24-26 hours after trauma as compared with the absence of SEPs in traumatized untreated group. In all three groups, the treatment increased the blood flow in abdominal aorta significantly. Morphine sulfate increased substance P (SP) immunoreactivity in the dorsal and ventral gray matter. Naloxone not only reversed this effect, it depleted SP below the saline control level. In order to establish that lipid free radicals are responsible for damage to biological membranes, their effects were also investigated in vitro: 14C-GABA uptake by mouse cortical slices which had decreased by 33% in the presence of superoxide (. O-2) generating system, horseradish peroxidase (HRP), was reduced only by 9% when superoxide dismutase was added to the medium. The latter also protected the nerve endings from damage by (. O-2) as examined by electron microscopy. It is concluded that the agents used in this study produce their ameliorating effects by virtue of their anti-inflammatory, anti-oxidant, and membrane stabilizing properties in addition to their effect on enhancing the regional microcirculation. The release of SP by naloxone may be responsible for the increase in blood flow. The consequences of traumatic injury as depicted in Fig. 1 are discussed at length.     

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.     

Naloxone reversal of the pentobarbital induced blockade of ovulation in the rat

The effect of naloxone, an opiate antagonist, on the pentobarbital induced suppression of gonadotrophin secretion and subsequent ovulation was studied. Two injections of naloxone, the first at 13.00 h and the second, given simultaneously with an ovulatory blocking dose of pentobarbital at 13.30 h, partially reversed the inhibitory effect of the barbiturate. However, if the first injection of naloxone was given with pentobarbital at 13.30 h and a second at 14.30 h, then the opioid antagonist had no such effect. Naloxone administered alone, enhanced gonadotrophin secretion and increased the number of ova shed. These findings support the view, that endogenous opioids may exert an inhibitory influence on central neural processes involved with ovulation.     

Corticosteroidogenesis modulation by beta-endorphin and dynorphin1-17 in isolated rat adrenocortical cells

Two opioid peptides, beta-endorphin and dynorphin1-17 were bioassayed with isolated rat adrenocortical cells. beta-Endorphin increases basal production of corticosterone as well as the adrenal responsiveness to low doses of ACTH, these effects being partially reversed by naloxone. Dynorphin1-17, without affecting basal corticosterone synthesis, increases adrenocortical responsiveness to ACTH; naloxone does not influence this effect. It is suggested that peripheral opioid peptides may participate in the maintenance of the homeostatic balance by modulating adrenal corticosteroidogenesis.     

Naloxone potentiates ACTH and angiotension II but not potassium stimulated aldosterone secretion, in vitro

The effects of naloxone on basal and ACTH, Angiotensin II (AII) and [K+] o stimulated aldosterone secretion from superfused rat adrenocortical tissue were investigated. A high dose (10(-6) M) of naloxone inhibited while a smaller dose (10(-10) M) potentiated and doses of 10(-8) or 10(-12) M naloxone were without an effect on ACTH stimulated aldosterone secretion. A potentiation of AII stimulated aldosterone secretion was observed beginning 2 hrs after 10(-6) or 10(-10) M naloxone was administered while no effect was observed with 10(-4) M naloxone. No effects of 10(-6), 10(-8), 10(-12) M naloxone were detected on aldosterone secretion stimulated by transiently elevating extracellular potassium. Naloxone from 10(-4) to 10(-12) M did not appear to significantly influence basal steroidogenic activity under these conditions. These findings demonstrate that the "opioid antagonist" naloxone has prominent actions on adrenocortical tissue. Both the specificity and lack of specificity of the action of this agent to influence the activity of the 3 secretagogues suggest that naloxone and possibly a naturally occurring endogenous ligand interacts with one or more membrane receptor distinct from the ACTH receptor. A naturally occurring ligand for this receptor could play a prominent role in the physiological regulation of adrenal steroid secretion.