Glen R. Van Loon

Nicotine protects against μ-opioid receptor antagonism by β-funaltrexamine: Evidence for nicotine-induced release of endogenous opioids in brain

We have hypothesized that some effects of nicotine are mediated through endogenous opioids. This study was designed to demonstrate in rats that nicotine releases endogenous opioids in brain. In the control group, subcutaneous morphine (8 mg/kg) produced analgesia or antinociception as measured by prolongation of tail flick latency. Intracerebroventricular administration 24 h earlier of beta-funaltrexamine (beta-FNA, 2.5 micrograms), an antagonist which irreversibly alkylates opioid receptors, markedly reduced (66%) morphine analgesia. Subcutaneous administration of nicotine (0.1 mg/kg) prior to beta-FNA attenuated (31%) the inhibitory effect of beta-FNA on morphine analgesia. These data support our hypothesis that endogenous opioids released by nicotine bind to mu-opioid receptors in brain and protect them against inactivation by beta-FNA.

Opioid mediation of cocaine-induced hyperactivity and reinforcement

The mechanisms by which cocaine produces hyperactivity and reinforcement remain poorly understood. Since reinforcement is also a property of other drugs of abuse including opiates, we examined the possible mediation of these cocaine-induced behaviors by endogenous opioid peptides. In this study, we have confirmed reports that cocaine increases locomotor activity and conditioned place preference in rats. We have also demonstrated that opioid receptor blockade with naloxone antagonizes completely the locomotor-activating effect of cocaine and attenuates the strength of the place preference conditioning produced by cocaine. These data support the thesis that endogenous opioids are involved in mediation of cocaine-induced behavior.

NICOTINE-INDUCED ALTERATION IN TYR-GLY-GLY AND MET-ENKEPHALIN IN DISCRETE BRAIN NUCLEI REFLECTS ALTERED ENKEPHALIN NEURON ACTIVITY

Nicotine acts in CNS, but the pathways and mechanisms of its actions are poorly understood. Recent studies suggest an interaction between brain nicotinic receptors and endogenous opioid peptides. Acute administration of nicotine may alter enkephalin release without affecting brain enkephalin level. Tyr-Gly-Gly has been shown previously to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A. Concentrations of Tyr-Gly-Gly in brain were used to provide an index of enkephalin release in vivo. Thus we examined the thesis that nicotine alters brain neuronal enkephalin release, by measuring Tyr-Gly-Gly levels in specific brain nuclei from rats treated with nicotine 0.3 mg/kg SC 10 min before decapitation. Of 30 brain regions investigated, acute nicotine increased Tyr-Gly-Gly immunoreactivity in nucleus accumbens and in lower brain stem areas including dorsal raphe, pontine reticular formation, gigantocellular reticular formation, locus coeruleus, sensory trigeminal nucleus and the caudal part of ventrolateral medulla oblongata. Concomitantly, nicotine produced a significant decrease in native Met-enkephalin in central amygdala, flocculo-nodular lobe of cerebellum, caudal part of the ventrolateral medulla and intermediolateral cell column of the spinal cord. It is probable that the effects of nicotine to increase Tyr-Gly-Gly and alter Met-enkephalin concentration are mediated by nicotine-induced release of enkephalin at these brain sites. Furthermore, some of the physiologic and pharmacologic effects of nicotine may be mediated by such enkephalin release.

Nicotine-induced alterations in brain regional concentrations of native and cryptic Met- and Leu-enkephalin.

The distribution of cryptic forms (larger enkephalin-containing peptides) in neostriatum, hypothalamus, spinal cord T3-L1 and neurointermediate lobe of pituitary were determined by radioimmunoassay. Optimal conditions for enzymic hydrolysis of the cryptic enkephalins by trypsin and carboxypeptidase B were established. The proportion of total Met- and Leu-enkephalin represented by native pentapeptide varied markedly among these central nervous system regions. Also, the distributions of native and cryptic Met-enkephalin were distinct from that of Leu-enkephalin. Chromatographic separation by HPLC of immunoreactive Met-enkephalin peptides revealed only two peaks corresponding to Met-enkephalin and Met-enkephalin sulfoxide in rather equal amounts. Hydrolysis of cryptic Met-enkephalin also produced only two HPLC-separable peaks of immunoreactive Met-enkephalin, again corresponding to Met-enkephalin and Met-enkephalin sulfoxide. Bioactivity of cryptic striatal Met-enkephalin after hydrolysis was demonstrated by antinociception and catalepsy in rats following its intracerebroventricular injection. Repeated short-term administration of nicotine, 0.1 mg/kg IP six times at 30 min intervals, produced significant increases in native and cryptic Met-enkephalin in striatum, consistent with an increase in neuronal release of Met-enkephalin together with increases in synthesis and processing of proenkephalin A in this brain region. This regimen of nicotine also decreased levels of native Met-enkephalin and of both native and cryptic Leu-enkephalin in neurointermediate lobe, consistent with nicotine-induced release of both proenkephalin A- and prodynorphin-derived peptides from neurointermediate lobe.

Mu receptors at discrete hypothalamic and brainstem sites mediate opioid peptide-induced increases in central sympathetic outflow.

Synthetic human beta-endorphin, 7.25 nmol intracisternally, in conscious, freely moving, cannulated adult male rats increased plasma concentrations of the 3 catecholamines, epinephrine, norepinephrine and dopamine. Similarly administered equimolar morphine increased only plasma epinephrine concentration significantly. A 10-fold greater intracisternal dose of morphine significantly increased plasma concentrations of all 3 catecholamines. This effect was inhibited by prior intra-arterial naloxone administration. Intracisternal administration of the selective mu receptor agonist [D-Ala2,NMe-Phe4,Gly-ol5]enkephalin (DAGO), 2.9 nmol, also increased plasma concentrations of the 3 catecholamines and, furthermore, these effects were significantly greater than those noted in response to equimolar beta-endorphin. The greater potency of DAGO than beta-endorphin to increase catecholamine secretion suggests that this opioid peptide-induced effect is mediated at mu receptors. Administration of DAGO, 0.1 nmol, directly into either the hypothalamic paraventricular nucleus (PVN) or brainstem nucleus of the solitary tract (NTS) significantly increased plasma concentrations of all 3 catecholamines when compared with either saline-infused controls or animals administered DAGO into other brain areas. These catecholamine-stimulating effects of DAGO administered into either PVN or NTS were prevented by prior intra-arterial naloxone administration. Heart rate, but not mean arterial blood pressure, increased in response to DAGO administration into the NTS while no significant cardiovascular changes were noted among the experimental groups in response to DAGO administered into the PVN. These data support a hypothesis that mu receptors at discrete and anatomically distant brain sites mediate opioid peptide-induced catecholamine secretion through activation of the central sympathetic outflow to the adrenal medulla and sympathetic nerve terminals.

Role of sympathoadrenomedullary system in cardiovascular response to stress in rats

Sympathetic nerve and/or adrenal medulla contributions to stress-induced cardiovascular responses were investigated by factoring out their influence using adrenal demedullation (DMED) and/or chemical sympathectomy with guanethidine (GUAN). Rats divided into 4 groups [sham-operated/saline (SHAM/SAL), SHAM/GUAN, DMED/SAL and DMED/GUAN] were injected i.p. over 4 weeks with either saline or GUAN (25 mg/kg/day). At the end of this treatment period, blood pressure (BP) and heart rate (HR) were monitored via carotid catheter prior to and during restraint in conscious rats. Treatments did not alter basal BP or HR when compared to controls. Restraint increased HR (delta 72 bpm) and systolic, diastolic and mean BP (delta approximately 20 mm Hg) in control animals. Restraint-induced HR change was significantly greater in DMED/SAL animals (delta 88 bpm), but less in SHAM/GUAN animals (delta 40 bpm) than in controls. DMED/GUAN was not different from SHAM/GUAN alone in altering HR response to stress, supporting the greater influence of sympathetic nerves over adrenal medulla in controlling HR. Chronic GUAN abolished normal pressor responses to restraint stress. DMED increased diastolic blood pressure response to stress. However, in DMED/GUAN rats, not only did stress fail to increase blood pressure but rather stress produced hypotension (delta - 34 mm Hg MAP), demonstrating the role of adrenal medulla in maintaining BP during stress. Differential effects of the various treatments on diastolic and systolic pressure suggest that the treatments had effects on peripheral vasculature. These results demonstrate that sympathetic nerves and adrenal medulla have important influences on cardiovascular function during stress and that in the absence of either, the other system may partially compensate.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin increases the activity of tuberoinfundibular and nigroneostriatal dopamine neurons: Prolactin antiserum inhibits the haloperidol-induced increases in dopamine synthesis rates in median eminence and striatum of rats

The role of PRL in mediating the haloperidol-induced increase in tuberoinfundibular dopamine synthesis rate was assessed by studying the effects of administration of PRL antiserum. Antiserum to PRL generated in rabbits and not cross-reacting with other anterior pituitary hormones was administered IV to adult, male rats which received haloperidol 2.5 mg/kg or tartaric acid vehicle SC 22 hr and 12 hr before measurement of dopamine turnover. Comparable groups of haloperidol or vehicle-treated animals received normal rabbit serum as control. Dopamine synthesis or turnover rate was estimated by measurement of accumulation of L-dihydroxyphenylalanine following inhibition of L-aromatic amino acid decarboxylase with m-hydroxybenzylhydrazine. Haloperidol increased median eminence dopamine synthesis rate, and PRL antiserum completely prevented this effect, supporting the thesis that the haloperidol-induced increase in tuberoinfundibular dopamine turnover is mediated by PRL. PRL antiserum did not alter basal median eminence dopamine synthesis rate in male rats. In addition to its effect in median eminence, PRL antiserum blunted the haloperidol-induced increase in striatal dopamine synthesis rate, suggesting that the haloperidol-induced increase in nigroneostriatal dopamine turnover is mediated in part by PRL. Neither haloperidol nor PRL antiserum altered serotonin synthesis rate in mediobasal hypothalamus or striatum. The data provide further support for a mechanism by which PRL can regulate its own secretion. They also suggest that prolactin alters the activity not only of tuberoinfundibular but also of nigroneostriatal neurons.

Source of Stress-Induced Increase in Plasma Met-Enkephalin in Rats: Contribution of Adrenal Medulla and/or Sympathetic Nerves*.

The contribution of the adrenal medulla and/or the sympathetic nerves to the plasma levels of Met‐enkephalin was investigated. Rats were divided into four groups: sham‐operated/saline, sham‐operated/guanethidine, adrenal‐demedullated/saline, demedulla‐ted/guanethidine. After 4 weeks of injection with either saline or guanethidine (25 mg/kg/day), animals were cannulated in the left carotid artery for blood sampling. Three days later, blood samples were taken before and at 2 and 30 min of restraint stress. Adrenal demedullation lowered basal plasma epinephrine levels markedly and prevented entirely the increase induced by restraint stress. Chronic guanethidine treatment lowered basal plasma norepinephrine levels and decreased the response to stress. Guanethidine treatment increased the basal plasma epinephrine level without affecting the response to stress. The combination of guanethidine plus adrenal demedullation lowered basal plasma concentrations of all three catecholamines and further attenuated the norepinephrine response. Restraint stress increased plasma native and peptidase‐derivable Met‐enkephalin. Adrenal demedullation, resulting in greater than 95% depletion of adrenal catecholamines and significant depletion of adrenal Met‐enkephalin, did not inhibit the stress‐induced increase in plasma Met‐enkephalin, and in fact, was associated with a potentiated response to stress. Guanethidine treatment with or without demedullation increased baseline plasma native Met‐enkephalin and abolished the stress‐induced increase in plasma native and peptidase‐derivable Met‐enkephalin. Thus, the stress‐induced increase in plasma Metenkephalin results from release from sympathetic nerves, rather than adrenal medulla. However, the sympathetic nerves and adrenal medulla together do not appear to account entirely for basal concentrations of circulating Met‐enkephalin. Hepatic portal vein plasma concentration of native Met‐enkephalin was greater than that in the carotid artery, suggesting contribution from the gastrointestinal tract; however, evisceration did not decrease plasma native Met‐enkephalin. Some compensatory mechanism results in elevation of basal plasma native Met‐enkephalin in sympathectomized rats. Also, in the absence of the adrenal medulla there is a compensatory increase in the amount of Met‐enkephalin released into the circulation in response to stress.

Stabilization of methionine-enkephalin in human and rat blood

Methods of preventing the degradation of 3H-methionine-enkephalin (3H-ME) in human blood both at 37 degrees C and under conditions of immediate cooling were examined. We found that, contrary to previous suggestions, use of aprotinin (with or without immediate cooling) was ineffective in preventing the degradation of 3H-ME in blood. Thus, previous reports on the circulating levels of ME which relied on such procedures to stabilize the ME may have reported artifactually low values. However, we found that citric acid effectively prevents 3H-ME breakdown in both human and rat blood. Thus, we propose the use of citric acid, mixed with blood immediately upon collection, as an effective method for the stabilization of ME in blood.

Haloperidol-Induced Increase in Striatal Concentration of the Tripeptide, Tyr-Gly-Gly, Provides an Index of Increased Enkephalin Release In Vivo

A sensitive and specific radioimmunoassay has been developed for the tripeptide, Tyr‐Gly‐Gly, which has been shown previously to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A. Using this assay, we found a regional variation in Tyr‐Gly‐Gly immunoreactivity in rat brain, with highest levels in striatum and lowest in cerebral cortex. Intracerebroventricular administration of the aminopeptidase inhibitor, bestatin, produced a threefold increase in Tyr‐Gly‐Gly immunoreactivity in rat striatum, whereas thiorphan, an enkephalinase inhibitor, produced a 45% reduction in striatal Tyr‐Gly‐Gly immunoreactivity. These data suggest that the tripeptide, Tyr‐Gly‐Gly, is in a dynamic state in the brain, and provide further support for the hypothesis that its concentration in specific brain areas may reflect the release of endogenous enkephalins in these brain areas. Further confirmation of the validity of measurements of brain Tyr‐Gly‐Gly as indices of enkephalin release under conditions of altered neuronal activity was provided by our demonstration that chronic dopamine receptor blockade with haloperidol increased striatal concentrations of both Met‐enkephalin and Tyr‐Gly‐Gly.