The effects of opiate agonists on growth hormone and prolactin release in rats

Various opioid receptor agonists, including Met5-enkephalin amide, Leu5-enkephalin amide, [D-Ala]2-Met5-enkephalin amide, [D-Ala]2-Leu5-enkephalin amide, morphine sulfate, d-methadone hydrochloride, and l-methadone hydrochloride were administered to adult male rats by subcutaneous injection. All opioid receptor agonists except Leu5-enkephalin amide significantly stimulated growth hormone and prolactin release. Naloxone and naltrexone blocked the hormone stimulatory effects of the opioids and both naloxone and naltrexone, when administered alone, significantly reduced serum growth hormone and prolactin concentrations. The dopaminergic agonist apomorphine, but not the alpha-adrenergic agonist clonidine, blocked opiate stimulation of prolactin. Morphine sulfate caused growth hormone release in rats pretreated with alpha-methyl-p-tryosine, a catecholamine synthesis inhibitor. Cholinergic agonists, physostigmine and pilocarpine, antagonized the growth hormone and prolactin release induced by morphine sulfate. The data suggest that the opiates stimulate prolactin via an interaction with catecholaminergic neurons controlling prolactin release and stimulate growth hormone via a mechanism independent of alpha-adrenergic or general catecholaminergic influence. The mechanism through which cholinergic agonists act to inhibit opiate agonist stimulation of growth hormone is presently unknown.     

Effects of antipsychotic drugs on action potential production in skeletal muscle. II. Haloperidol: nonspecific and opiate drug receptor mediated effects.

The effects of haloperidol, an antipsychotic butyrophenone, on excitability and action potential production in frog's sartorius muscle fibers were studied. This drug produced a local-anestheticlike effect which developed slowly over 1 to 5 h with lower concentrations (2.7 to 5.3 X 10(-6 M) but was completely reversed by exposing the muscles to a drug-free solution. In studies with intracellular microelectrodes, evidence was obtained showing that haloperidol decreased excitability and depressed action potential production by inhibiting the specific increase in sodium conductance (gNa) which normally follows an adequate stimulus. Evidence also was obtained showing an inhibition of the secondary increase in potassium conductance (gK). Haloperidol is structurally related to meperidine and it was found that the inhibition of gNa produced by haloperidol is partially antagonized by low concentrations of naloxone (2.8 X 10(-8) and 2.8 X 10(-7) M); as was previously shown for meperidine. Thus haloperidol, like meperidine, suppresses action potential production by two mechanisms of action: one, a nonspecific local-anaestheticlike effect; and the other, a specific inhibition of gNa mediated by means of an opiate drug receptor associated with the muscle fiber membrane. Naloxone did not antagonize the effects of chlorpromazine on gNa.     

Stereospecific opioid drug receptors on excitable cell membranes

In the past decade evidence has accumulated showing that there are stereospecific opioid drug receptors located on the surface membranes of neurones and skeletal muscle fibres which can block action potentials when opioid drugs are applied to the cells. The nature of this evidence and the difficulties involved in this area of investigation are discussed in detail. Two different mechanisms have been described for this depression of excitability; one, a direct blocking action on sodium channels (or the calcium channels in the case of cells with calcium action potentials) and the other, a stimulation of membrane potassium conductance which results in hyperpolarization and increased membrane conductance both of which decrease excitability. For the most part these different mechanisms seem to prevail in different cell types as will be discussed. Some other points discussed are as follows: first, in higher doses (or concentrations) opioids produce a local anestheticlike effect which cannot be antagonized by opioid antagonists; next, the opioid antagonists which are easily easily available and commonly employed change from antagonists to agonists as their concentration is increased; and finally, the evidence required for the demonstration of stereospecific opioid receptors is considered in some detail.     

Nalorphine: actions at an opiate receptor on frog skeletal muscle fibers

Intracellular microelectrode studies were conducted to investigate the actions of the partial agonist-antagonist nalorphine at an opiate receptor on functional frog skeletal muscle fiber membranes. In high bath concentrations (greater than or equal to 10(-4) M), nalorphine alone produces agonist actions similar to the "full" opiate agonists. These actions were (i) to depress both the sodium and potassium (gNa and gK) conductance increases due to electrical stimulation by a nonspecific local anestheticlike mechanism and (ii) to depress gNa by a specific opiate receptor mediated mechanism. In a much lower bath concentration (1 X 10(-8) M) nalorphine acts to antagonize the specific opiate receptor mediated depression of gNa produced by the "full" agonist meperidine. Thus in this preparation nalorphine, "the partial antagonist," has the same actions as naloxone, which is often considered to be a full antagonist. The quantitative differences observed in the effects of these two opiate antagonists are discussed.     

Receptor-related interactions of opiates with PGE-induced adenylate cyclase in brain

The inhibition by opiates of the PGE2-induced formation of cAMP in slices from rat brain striatum was investigated. A maximal, 3.5-fold increase over the basal level of cAMP was obtained with an EC50 for PGE2 of 3 microM. Opiate agonists of both mu and kappa type were inhibitory. The IC50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. These values were similar to the potencies of the compounds in displacing stereospecifically bound 3H-etorphine in rat brain membranes. As evidenced by the inactivity of dextrorphan, the inhibition of PGE2-dependent cAMP formation was stereospecific. Also ineffective were the opiate antagonists naloxone, naltrexone and MR 2266. These compounds did, however, reverse the inhibition by agonists, displaying thereby selectivity toward the putative mu and kappa opiates. Thus, the inhibition by morphine was antagonized to a greater degree by naloxone than by MR 2266, and the action of EKC was blocked more effectively by MR 2266 relative to naloxone.     

Calcium currents modulated by adrenergic receptors in sympathetic neurons

The superior cervical sympathetic ganglion is currently being used as a model neuronal system for the study of Ca2+-dependent processes in the mammalian nervous system. We have characterized a regenerative calcium conductance in postganglionic neurons. This Ca2+ current contributes to the shoulder of the action potential. In addition, Ca2+ influx during the spike activates a K+ conductance, which generates a hyperpolarizing afterpotential. These Ca2+-dependent potentials are antagonized by catecholamines. Pharmacologic studies suggest that alpha 2-adrenergic receptors inhibit the regenerative voltage-dependent Ca2+ influx that occurs during the action potential. Alpha-adrenergic agonists were also found to reduce the depression of the compound action potential following a train of preganglionic stimuli. We hypothesize that alpha 2-receptors function primarily to antagonize Ca2+ influx and thereby exert significant control over neuronal excitability and release of neurotransmitters.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Effect of the anticonvulsant U-54494A on cortical neuron excitability: comparison to the kappa agonist U-50488H.

U-54494A, a 1,2-diamine anticonvulsant, and U-50488H, a structurally related agonist for opiate kappa receptors, were tested for effects on spontaneous and glutamate-evoked firing rates in cerebral cortex of urethane-anesthetized male Sprague-Dawley rats. Iontophoretic application of 1,2-diamines, glutamate diethyl ether (GDEE), or procaine depressed spontaneous and amino acid-induced firing of cortical neurones. With continued ejection of 1,2-diamines or procaine, firing was silenced completely, but GDEE could maintain a partial suppression. A rapid rebound of excitation followed cessation of procaine ejections, but not of other agents. Procaine, but not U-54494A, blocked axonal conduction of rabbit sciatic nerve. Intravenous U-54494A and U-50488H significantly depressed spontaneous firing rates of cortical neurones, but only the U-50488H effects were antagonized by naloxone. It is concluded that U-54494A inhibits neuronal excitability by a mechanism independent of the analgesic kappa receptor. Biochemical and physiological studies have demonstrated that U-54494A and the kappa opioid agonist U-50488H (a structurally related diamine) (1) have anticonvulsant activity (2, 3). U-54494A lacks kappa analgesic and sedative properties, and it has been suggested that the mechanism of action of this compound may be mediated by a subtype of kappa opioid receptor (3). The effects of kappa analgesics on neuronal firing in nociceptive pathways have been described (4, 5). However, no previous electrophysiological studies on U-54494A have been done. Since U-54494A antagonizes amino acid-induced seizures (3), the interactions of this compound with glutamate are of interest. In the present study, the antagonist efficacies of U-54494A and U-50488H for inhibiting spontaneous and 1-glutamate stimulated neurons of the rat prefrontal cerebral cortex were assessed after i.v. and microiontophoretic administration of the compounds. Effects observed with these routes of administration allow the observation of neuronal changes occurring immediately after administration and take advantage of the high temporal resolution provided by the electrophysiological recording techniques of single cells. A preliminary account of portions of this work have been previously disclosed (6).     

Opiate receptor antagonism by l-prolyl-l-leucyl-glycinamide, MIF-I

Chronic administration of l-prolyl-l-leucyl-glycinamide (MIF-I) to mice slightly reduced morphine's antinociceptive activity in the hot-plate test and modified the biphasic motor activity response to morphine. MIF-I antagonized the initial depression of activity and potentiated the increased motor activity phase. Chronic treatment of rats with MIF-I prevented morphine's antinociceptive activity in the tail flick test. MIF-I partly antagonized the inhibition by morphine of the coaxially stimulated guinea-pig ileum preparation. The inhibition of the ileum produced by ethylketocyclazocine was weakly antagonized by MIF-I. In contrast, MIF-I had no effect on the inhibition of the stimulated mouse vas deferens produced by Leu-enkephalin. The findings show that MIF-I weakly and selectively inhibits μ-type opiate receptors which suggests that MIF-I could be an endogenous inhibitor of opiate receptors.     

Endogenous opioids and opiate antagonists modulate the blood pressure of the spontaneously hypertensive rat

Endogenous opioids have been implicated as modulators of the central nervous system regulation of blood pressure and heart rate. Whether these neuropeptides participate in blood pressure regulation in hypertension is unknown. To begin to study this question, we examined the response to opiate antagonists and agonists in the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY) rat. The long-acting opiate antagonist naltrexone, 2.5 micrograms/kg, was injected into the lateral ventricle of the brain in awake, freely-moving SHR and produced a significant 19 mmHg decrease in mean arterial blood pressure compared to basal blood pressure (p less than 0.01); a decrease was not observed at a two logarithm lower dose. In contrast, naltrexone had no effect on the blood pressure of normotensive Wistar-Kyoto (WKY) rats. To evaluate a possible regulatory role for the predominantly kappa receptor active opioids, alpha- and beta-neo-endorphin, 10 micrograms each, was administered to SHR on separate days by intracerebroventricular injection. alpha- and beta-neo-endorphin caused significant decreases in mean arterial blood pressure of 11 and 9 mmHg respectively, effects reversed by pre-treatment with the opiate antagonist, naloxone. Heart rate was unaffected by any of the injected opioids or antagonists. Our naltrexone results support the hypothesis that an endogenous opioid(s) contributes to the hypertensive state of the SHR. Additionally, alpha- and beta-neo-endorphin can lower blood pressure in this model.     

Opiate receptor antagonism by l-prolyl-l-leucyl-glycinamide, MIF-I

Chronic administration of l-prolyl-l-leucyl-glycinamide (MIF-I) to mice slightly reduced morphine's antinociceptive activity in the hot-plate test and modified the biphasic motor activity response to morphine. MIF-I antagonized the initial depression of activity and potentiated the increased motor activity phase. Chronic treatment of rats with MIF-I prevented morphine's antinociceptive activity in the tail flick test. MIF-I partly antagonized the inhibition by morphine of the coaxially stimulated guinea-pig ileum preparation. The inhibition of the ileum produced by ethylketocyclazocine was weakly antagonized by MIF-I. In contrast, MIF-I had no effect on the inhibition of the stimulated mouse vas deferens produced by Leu-enkephalin. The findings show that MIF-I weakly and selectively inhibits μ-type opiate receptors which suggests that MIF-I could be an endogenous inhibitor of opiate receptors.     

Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates

1. Nanomolar concentrations of opiates inhibit phagocytosis in the ciliated protozoan Tetrahymena thermophila. 2. Naloxone and naltrexone counteract the effect of the opiate agonists tested. 3. The dose-response curves are U-shaped, with no detectable effect at low or high concentrations. 4. An increase in extracellular calcium and dopamine counteract the inhibition caused by metenkephalin. 5. The recognition mechanism for opiates in Tetrahymena cannot be classified as belonging to any of the mammalian opiate receptor subtypes and is perhaps a primitive receptor.     

Catatonic or hypotonic immobility induced in mice by intracerebroventricular injection of mu or kappa opioid receptor agonists as well as enkephalins or inhibitors of their degradation

The intracerebroventricular injections in mice of the mu receptor agonists morphine and fentanyl induced an immobility state (the animals staying motionless with the head down on a 45° inclined plane) which was apparently hypertonic (catatonia ?) or at least enabled them to remain hanging on a horizontal wire with their forepaws. In similar conditions, injections of the kappa receptor agonists ketocyclazocine and bremazocine induced an immobility state which was hypotonic, in contrast with the preceding one. In a similar way to the mu agonists, Met-enkephalin or Leu-enkephalin injected i.c.v. in association with the inhibitor of enkephalinase thiorphan induced an apparently hypertonic immobility which was easily antagonized by naloxone. The association of thiorphan with bestatin ( an inhibitor of aminopeptidases involved in enkephalins inactivation ) produced similar results. In contrast, the hypotonic immobility induced by the kappa receptor agonists required relatively high doses of naloxone to be antagonized. The opiate antagonist MR 2266 antagonized equipotent doses of all the above mentioned agents with a similar efficacy. From these data it is suggested that enkephalins could induce an apparently tonic immobility by stimulating mu receptors and that endogenous enkephalins could be involved in a tonic mediation modulating the locomotor activity or regulating the muscular tone.     

Highly selective agonists for substance P receptor subtypes.

The existence of a third tachykinin receptor (SP-N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N-methylation of individual peptide bonds in the C-terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl-[Asp6,Me-Phe8]SP6-11, elicits half-maximal contraction of the guinea pig ileum through the neuronal SP-N receptor at a concentration of 0.5 nM. At least 60,000-fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP-P and SP-E). The action of selective SP-N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP-N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.     

Central, stereoselective receptors mediate the acute effects of opiate antagonists on luteinizing hormone secretion

Although a central site of acute opiate action in regulating luteinizing hormone (LH) secretion has been suggested by the ability of centrally implanted opiate antagonists to increase LH levels, opiate antagonists are lipophilic and could influence the pituitary in situ. Also, the physiological significance of opiate receptor blockade with antagonists rests on the assumed, but untested, stereoselectivity of these receptors. Therefore, a lipophobic quaternized derivative of naltrexone (MRZ 2663-Naltrexone methobromide) and dextro- (+) and levo- (-) stereoisomers of naloxone were used to study the site- and stereoselectivity of gonadotropin responses to opiate antagonists in vivo. Male rats were injected intracerebroventricularly (icv) or intravenously (iv) with the quaternary or tertiary congeners of naltrexone and subcutaneously (sc) with (-) or (+)-naloxone. Rats injected icv with 20 ug of quaternary naltrexone displayed significant increases in serum luteinizing hormone (LH). The onset of the response was rapid with serum LH levels being significantly elevated 15 minutes after the injection and returning to basal levels 30 minutes later. Rats injected iv with 10 mg/kg of quaternary naltrexone failed to show significant LH responses. Rats injected either centrally or periphally with equivalent doses of tertiary naltrexone showed LH responses that were similar to those found in animals injected icv with quaternary naltrexone. As little as 0.5 mg/kg of (-)-naloxone resulted in significant elevations in serum LH that were higher than those elicited by up to 10 mg/kg of (+)-naloxone, indicating that this effect of naloxone is stereoselective. These data support the argument that opioids can acutely modulate LH secretion through actions at stereoselective opioid receptors in the central nervous system.     

Counteraction by naltrexone of stress-induced inhibition of TSH release: role of noradrenergic system

The purpose of the present study was to examine the effect of administering an opiate receptor antagonist, naltrexone (NALT) on the decline in pituitary thyrotropin (TSH) release induced by both acute and chronic stress, and to determine whether norepinephrine (NE) is involved in the mechanism by which opiate receptor blockade counteracts inhibition of TSH release during stress. Administration of NALT, a specific opiate receptor antagonist, significantly attenuated the decrease in plasma TSH observed after acute exposure to ether or restraint stress. The ability of NALT to prevent suppression of circulating TSH in ether-stressed rats was blocked by pharmacological suppression of NE activity induced by pretreatment with diethyldithiocarbamate (DDC) or phenoxybenzamine (PB), both NE antagonists. In chronically stressed rats, thrice daily injections of NALT attenuated the sustained decline in circulating TSH, and resulted in a significant elevation in plasma TSH when compared with stressed, saline-treated animals. Pretreatment with DDC prior to NALT injection abolished this stimulatory effect of NALT. These observations indicate that opiate/receptor interaction is prerequisite for the decrease in circulating TSH release during both acute and chronic stress, and support the hypothesis that endogenous opioid peptides (EOPs) mediate the suppressive effect of stress on TSH release. The finding that uninterrupted NE function is necessary for NALT's action on TSH release during stress suggests that the suppressive effect of stress on TSH and its reversal by opiate antagonists involves alterations in hypothalamic NE activity.     

Modulatory effects of Gs-coupled excitatory opioid receptor functions on opioid analgesia,tolerance, and dependence

Electrophysiologic studies of opioid effects on nociceptive types of dorsal root ganglion (DRG) neurons in organotypic cultures have shown that morphine and mostμ, δ, and κ opioid agonists can elicit bimodal excitatory as well as inhibitory modulation of the action potential duration (APD) of these cells. Excitatory opioid effects have been shown to be mediated by opioid receptors that are coupled via Gs to cyclic AMP-dependent ionic conductances that prolong the APD, whereas inhibitory opioid effects are mediated by opioid receptors coupled via Gi/Go to ionic conductuances that shorten the APD. Selective blockade of excitatory opioid receptor functions by low (ca. pM) concentrations of naloxone, naltrexone, etorphine and other specific agents markedly increases the inhibitory potency of morphine or other bimodally acting agonists and attenuates development of tolerance/dependence. These in vitro studies have been confirmed by tail-flick assays showin that acute co-treatment of mice with morphine plus ultra-low-dose naltrexone or etorphine remarkably enhances the antinociceptive potency of morphine whereas chronic co-treatment attenuates development of tolerance and naloxone-precipitated withdrawal-jumping symptoms. Special issue dedicated to Dr. Eric J. Simon.     

Phospholipid changes in synaptic membranes by lipolytic enzymes and subsequent restoration of opiate binding with phosphatidylserine.

A study has been made of the role of phosphatidylserine in stereospecific opiate binding to neural membranes, utilizing specific lipolytic enzymes to attack the lipid. At very low concentrations phospholipase A2 from bee venom will preferentially hydrolyze C22:6-fatty acid; and even after a few percent of the total phosphatidylserine is hydrolyzed, opiate binding is greatly inhibited. The addition of brain phosphatidylserine will restore opiate binding; however, when the inhibition approaches 50% restoration is only partial. Exposure of membranes to phosphatidylserine decarboxylase will partially inhibit opiate binding; and the binding returns to the control level after the addition of phosphatidylserine. The partial inhibition of opiate binding by low concentrations of Triton X-100, which presumably remove lipids, can be partially reversed by phosphatidylserine. The binding of 3H-naloxone, an opiate antagonist, is similar to agonists in its behavior towards phospholipases and phosphatidylserine; however, binding of naltrexone, also an antagonist, is far less responsive. It is concluded that the phosphatidylserine associated with the opiate receptor is the C18:0, 22:6-diacyl form, which is closely associated with protein.     

Naltrexone precipitated opiate withdrawal in methadone addicted human subjects: Evidence for noradrenergic hyperactivity

The noradrenergic alpha-2 receptor agonist, clonidine, suppresses many of the effects of opiate withdrawal in both humans and other animals and this is consistent with the data indicating important interactions of opiate and noradrenergic systems in brain. This evidence supports the hypothesis that central noradrenergic hyperactivity is involved in the expression of major signs and symptoms of the opiate withdrawal syndrome, but to date clinical studies have not provided biochemical data consistent with this idea. In order to assess whether naltrexone precipitated opiate withdrawal in methadone addicted human subjects is associated with changes in noradrenergic function, a double-blind study was completed in which 15 methadone-dependent subjects received naltrexone and 8 subjects received placebo. Signs and symptoms of the opiate abstinence syndrome increased significantly in the 15 subjects who received naltrexone compared with the 8 who received placebo. Plasma concentrations of free MHPG also increased significantly in those subjects who took naltrexone compared to the placebo treated subjects. In addition, withdrawal signs and symptoms were significantly correlated with plasma MHPG concentration. Since several compounds which suppress central noradrenergic activity also reduce the severity of the withdrawal syndrome, the noradrenergic hyperactivity suggested by the present clinical study may be a functional mechanism for at least part of the opiate withdrawal syndrome.     

Depression and facilitation of synaptic responses in cat dorsal horn by substance P administered into substantia gelatinosa

The effects of substance P and an enkephalin analogue administered by electrophoresis into the substantia gelatinosa have been examined on the synaptic responses of dorsal horn neurons evoked by peripheral stimulation. Extracellular neuronal firing was studied in cats under pentobarbitone anesthesia. The enkephalin produced naloxone-reversible depression of responses to noxious heat stimulation without affecting responses to non-noxious stimuli. Substance P caused a selective enhancement or depression of noxious responses. It was tentatively concluded that substance P may modify the release of a sensory transmitter and produce direct post synaptic changes in membrane excitability.