Release Of Methionine-enkephalin Research Articles

Study on the mechanism of the Pu-erh tea (Camellia sinensis var. assamica) extract inhibiting contraction of isolated mouse duodenum

The aims of this study were to investigate the effects of three different concentrations of the Pu-erh tea extract (PTE) on the contractile activity of the isolated mouse duodenum and explore their mechanism. The contraction amplitude and frequency of the isolated mouse duodenum were inhibited by all three concentrations of PTE. The high-concentration PTE significantly (P < 0.01) inhibited the promotion effects of acetylcholine chloride or BayK8644 on the amplitude and frequency of intestinal contraction, which were comparable to those of atropine sulphate and verapamil hydrochloride, respectively. The results of UV-Vis and ELISA showed that the content of methionine-enkephalin (Met-ENK) in the PTE-treated groups was decreased to varying degrees; contrarily, the activities of tyrosine hydroxylase (TH), total nitric oxide synthase, and the content of nitric oxide were increased to different degrees. The results suggest that PTE can inhibit the contraction of the isolated mouse duodenum, and the mechanism of action is that PTE can not only inhibit the signal transduction pathways of AC-cAMP-PKA and PLC-IP3-Ca2+, but also the Ca2+ signal systems mediated by G protein-coupled M receptors through the myenteric plexus. By reducing the release of Met-ENK from the motor neurons of the myenteric plexus, the GTP-cAMP-PKK signalling pathway and the Ca2+ signalling system mediated by G protein-coupled delta receptors were inhibited. By increasing the TH activity of the motor neurons in the myenteric plexus, the norepinephrine content was increased, thereby the AC-cAMP-PKA signal transduction pathway mediated by G protein-coupled β receptors was activated. This study increases knowledge regarding the medicinal value of the Pu-erh tea.

Nicotine Effects and the Endogenous Opioid System

Nicotine (NIC) is an exogenous ligand of the nicotinic acetylcholine receptor (nAChR), and it influences various functions in the central nervous system. Systemic administration of NIC elicits the release of endogenous opioids (endorphins, enkephalins, and dynorphins) in the supraspinal cord. Additionally, systemic NIC administration induces the release of methionine-enkephalin in the spinal dorsal horn. NIC has acute neurophysiological actions, including antinociceptive effects, and the ability to activate the hypothalamic-pituitary-adrenal (HPA) axis. The endogenous opioid system participates in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception is mediated by α4β2 and α7 nAChRs, while NIC-induced HPA axis activation is mediated by α4β2, not α7, suggesting that the effects of NIC on the endogenous opioid system are mediated by α7, not α4β2. NIC has substantial physical dependence liability. The opioid-receptor antagonist naloxone (NLX) elicits NIC withdrawal after repeated NIC administration, and NLX-induced NIC withdrawal is inhibited by concomitant administration of an opioid-receptor antagonist. NLX-induced NIC withdrawal is also inhibited by concomitant administration of an α7 antagonist, but not an α4β2 antagonist. Taken together, these findings suggest that NIC-induced antinociception and the development of physical dependence are mediated by the endogenous opioid system, via the α7 nAChR.

Modulation of enkephalin release by nociceptin (orphanin FQ)

Nociceptin (orphanin FQ) is an endogenous peptide agonist for the newly discovered receptor (opioid receptor-like 1 receptor, ORL 1) that bears striking homology to opioid receptors. Initial reports claimed that this peptide had hypoalgesic effects following i.c.v. or i.t. administration. The present study demonstrates that, in the presence of opioid receptor blockade, nociceptin can substantially alter the magnitude of the stimulated release of methionine-enkephalin from the guinea pig myenteric plexus. This effect is concentration dependent. Low doses (1 or 10 nM) inhibit whereas higher concentrations (100 or 1000 nM) enhance evoked enkephalin release. In contrast, in the absence of opioid receptor blockade, a statistically significant inhibition of stimulated enkephalin release is observed in response to 1, 100 or 1000 nM nociceptin. However, the magnitude of this effect did not differ among these concentrations. Furthermore, at 10 nM nociceptin, either an inhibition or enhancement of stimulated enkephalin release is manifest. The ability of naloxone to alter the nociceptin modulation of enkephalin release suggests that a component of the nociceptin modulation of enkephalin release is mediated via opioid receptors. This is consistent with the observation that this peptide has modest affinity for opioid receptors (μ>κ>δ) which, under appropriate conditions, should be sufficient to permit interactions with multiple opioid receptor types. This complicates dose responsiveness for nociceptin since both the naloxone-resistant (ORL 1-mediated) and naloxone-sensitive (opioid receptor-mediated) component exhibit a concentration-dependent bimodality (albeit in opposite directions). Determination of i.c.v. or i.t. nociceptin dose responsiveness over several orders of magnitude is suggested before concluding the physiological effects of this peptide.

Involvement of nitric oxide in intracerebroventricular β-endorphin-induced neuronal release of methionine-enkephalin

Previous work has suggested that the antinociceptive effect of nitrous oxide (N 2O) in rats is mediated, at least in part, by β-endorphin (β-EP) and that centrally administered β-EP stimulates release of methionine-enkephalin (ME) in the rat spinal cord. Since inhibition of central nitric oxide (NO) production has been found to suppress N 2O antinociception, we examined the possible involvement of NO in the release of spinal cord ME by i.c.v. β-EP. Urethane-anesthetized, male Sprague-Dawley rats were intrathecally (i.t.) perfused with artificial cerebrospinal fluid (aCSF) and fractions of perfusate were assayed for immunoreactive (i.r.) ME. The β-EP-induced increase in ME concentration in the i.t. perfusate was significantly suppressed by perfusing the animal with aCSF containing 100 μM L-N G-nitro arginine (L-NOARG), an inhibitor of NO synthase (NOS). The further addition of 50 μM L-arginine (L-ARG), but not D-arginine (D-ARG), to the aCSF reversed the suppression of the ME change by L-NOARG. However, the potency of L-ARG decreased with increasing concentrations of L-ARG. On the other hand, increasing the concentration of L-NOARG in the aCSF to 250 μM failed to produce a greater suppression of the β-EP-induced increase in ME. These findings suggest that NO may mediate the β-Ep-induced release of ME in the spinal cord and that interference with this mechanism might be an explanation for the antagonism of N 2O antinociception in rats by NOS inhibitors.

Characterization of methionine-enkephalin release in the rat striatum by in vivo dialysis: Effects of gamma-hydroxybutyrate on cellular and extracellular methionine-enkephalin levels

The opioïd system is implicated in mediating the effects produced upon administration of gamma-hydroxybutyrate. Gamma-hydroxybutyrate occurs endogenously in the mammalian brain, and is most probably involved in the regulation of some basic brain functions, particularly those concerning the dopaminergic nigrostriatal pathway, which is closely linked to the expression of enkephalins in the striatum. In the present study, in vivo microdialysis was used to examine the basic characteristics of methionine-enkephalin (met-enkephalin) release in the striatum of Wistar rats, using a high performance radioimmunoassay. Administration of gamma-hydroxybutyrate to the rats induced a dose-dependent decrease in the extracellular release of met-enkephalin. In parallel, a dose- and time-dependent gamma-hydroxybutyrate-induced accumulation of met-enkephalin in striatum was observed. These two phenomena (tissue accumulation and inhibition of release) were blocked by NCS-382, a gamma-hydroxybutyrate receptor antagonist. The striatal met-enkephalin accumulation does not seem to be exclusively due to the inhibition of its release. Thus, a gamma-hydroxybutyrate mediating effect on met-enkephalin synthesis is suggested, most probably occurring via functional modulation of striatal dopamine synthesis and release. To understand the role of this dopaminergic mechanism, unilateral lesions of the nigrostriatal dopaminergic pathway were carried out. In gamma-hydroxybutyrate-treated rats, striata exhibited a similar increase in met-enkephalin content. In untreated rats, only the lesioned striatum showed an identical increase in met-enkephalin levels. Thus, striatal met-enkephalin accumulation could be attributed to the suppression of the dopaminergic impulse flow, due to gamma-hydroxybutyrate or to the action of 6-hydroxydopamine. In the extracellular spaces (microdialysis experiments), gamma-hydroxybutyrate administration induced identical modifications of met-enkephalin release in lesioned or non-lesioned striata. These modifications could be reproduced by peripheral or striatal administration of sulpiride, a D 2/D 3 antagonist. From a functional point of view, the dopaminergic D 2 receptor blockade or the gamma-hydroxybutyrate-induced inhibition of dopamine release could be considered to induce similar results, with identical consequences on striatal met-enkephalin accumulation and release. These results suggest that gamma-hydroxybutyrate-induced modifications in metenkephalin release, presumably potentiated by 6-hydroxydopamine treatment, act via a functional modification of the nigrostriatal dopaminergic pathway.

Influence of labor and neonatal hypoxia on sympathoadrenal activation and methionine enkephalin release in calves

Summary Labor and delivery stimulate increased release of catecholamines and endogenous opioid peptides in neonates. Catecholamines promote adaptation to the extrauterine environment after birth. Enkephalins are stored together with catecholamines in the adrenal medulla and have an inhibitory effect on catecholamine release. We investigated the influence of labor and neonatal hypoxia on epinephrine, norepinephrine, and met-enkephalin release in calves. Blood samples were taken from the umbilical artery before rupture of the umbilical cord and from the jugular vein repeatedly after birth. Highest plasma norepinephrine concentration was found in calves delivered at the end of gestation (term calves) before umbilical cord rupture. In calves delivered before the physiologic end of gestation (preterm calves), norepinephrine values increased after cord rupture, but remained lower than values in term calves. Epinephrine release followed a similar pattern, but norepinephrine was clearly predominant. In term calves, met-enkephalin values were significantly higher than values in preterm calves. In calves of both groups, met-enkephalin release increased after cord rupture. During birth, the increase in catecholamine release seems to take place earlier than that of enkephalins. Norepinephrine-dominated stimulation during expulsion of the calf might be followed by increasing enkephalinergic inhibition after cord rupture and onset of respiration. Reduced release of catecholamines and enkephalins in preterm calves may be connected with delayed adaptation to the extrauterine environment.

Interleukin-2 regulates monoamine and opioid peptide release from the hypothalamus.

The present study determined whether interleukin-2 (IL-2) and IL-2 receptors are present in the adult rat hypothalamus. In addition, we determined whether IL-2 is involved in the regulation of neurotransmitter release from hypothalamic slices. In the hypothalamus, the highest levels of endogenous IL-2 and IL-2 receptors were localized to the median eminence and arcuate nucleus. Application of exogenous IL-2 to hypothalamic slices produced significant decreases of potassium (25 mM)-evoked [3H]noradrenaline release (24%) without significantly affecting the evoked release of [14C]glutamate, [14C]5-hydroxytryptamine, [3H]dopamine or [3H] gamma-amino butyric acid. In addition, IL-2 increased the potassium-evoked release of methionine-enkephalin (by 78%) and beta-endorphin (by 38%) from hypothalamic slices without affecting the release of leucine-enkephalin. In contrast, the spontaneous release of neurotransmitters and neuropeptides was not affected by exogenous IL-2. Overall, the findings of this study suggest that hypothalamic IL-2 is an endogenous neurokine that may be of consequence to the regulation of hypothalamic function.

Stimulus frequency and intensity: critical determinants of opioid enhancement or inhibition of evoked methionine-enkephalin release

Responses to opioids can be bimodal depending on the concentration of opioid used. For example, low concentrations (nM) enhance whereas higher concentrations (10–100 nM) inhibit the electrically evoked release of enkaphalin from the myeteric plexus. The nature of the responsiveness of the enkephalin release process to opioids is also dependent on the intracellular and/or extracellular milieu of enkephalin-containing neurons or other neurons of this plexus. Intracellular levels of cAMP, availability of pertussis toxin- and cholera toxin-sensitive guanine nucleotide binding proteins and intracellular calcium concentration have all been shown to be important determinants of opioid excitatory versus inhibitory actions. The present data indicate that the inhibition of enkephalin release by U50,488H or sufentanil is no longer observed when the applied voltage is increased 3- or 2-fold, respectively. Under this condition, a previously inhibitory concentration of opioid produces an enhancement of stimulated enkephalin release. Increasing the frequency of the applied stimulation from 5 to 60 Hz (at a constant voltage) also reverses the sufentanil-induced inhibition to a facilitation of enkephalin release. These data indicate that the intensity (voltage) or frequency of the stimulation that is used to release enkephalin is a critical determinant of the nature of its modulation by opioids. The possible relevance of these findings to known differences in opioid sensitivity between different types of pain is discussed.

Diethylstilbesterol- and Pregnancy-Induced Changes in Rat Neurointermediate Lobe Oxytocin, Arginine Vasopressin, Methionine Enkephalin and Dynorphin

The neurointermediate lobe of the pituitary (NIL) contains the opioid peptides methionine enkephalin (MENK) and dynorphin 1-8 (DYN) in addition to oxytocin (OT) and vasopressin (AVP). If the opioids have a functional role, such as feedback control on OT or AVP release, the content or release of the opioids might be expected to change under conditions in which OT or AVP change. This expectation was examined by studying the synthesis, storage and release of the 4 peptides under conditions in which OT and AVP dynamics are known to be altered. Diestrus, diethylstilbesterol(DES)-treated, and day 22 pregnant rats were decapitated, the hypothalamo-neurohypophysial system (HNS) excised and either superfused in oxygenated Krebs buffer at 37 degrees C or stored at -80 degrees C for measurement of paraventricular and supraoptic nuclei mRNA content by in situ hybridization analysis. Peptide content of superfusates and NIL homogenates were determined by specific RIAs. Compared with diestrus animals, DES treatment increased NIL OT but decreased MENK. In term pregnant rats, NIL OT, AVP, and DYN were increased over diestrus values, while MENK was again decreased. Release of the peptides from the isolated HNS paralleled changes in NIL content. The hypothalamic mRNA for OT was increased in DES-treated and pregnant rats while MENK mRNA was decreased. AVP and DYN mRNA was increased in pregnant animals. Although the NIL was found to contain much more immunoreactive OT and AVP than MENK or DYN, under basal conditions the release of MENK was equal to or greater than the release of OT, while the release of DYN approached that of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalin Expression in Spinal Cord Neurons is Modulated by Drugs Related to Classical and Peptidergic Transmitters.

The effects of various neurotransmitter agonists and antagonists on the synthesis and release of methionine enkephalin (mENK) in neuronal cultures of mouse spinal cord and dorsal root ganglia have been measured. Blockade of electrical activity with tetrodotoxin between days 9 and 13 in culture caused a > 95% decrease in the number of mENK-immunoreactive neurons. This effect was also seen upon the blockade of glycine and beta-adrenergic receptors with strychnine and propranolol, respectively, and stimulation of GABA receptors with muscimol. Stimulation of beta-adrenergic receptors with isoproterenol, or blockade of glutamate and GABA receptors with 2-aminophosphonovalerate and strychnine, respectively, had a qualitatively opposite action on both the number of mENK-immunoreactive neurons and enkephalin peptide levels measured by radioimmunoassay. Application of substance P also enhanced the mENK cell number. These data suggest that, at least in the spinal cord, characteristics other than the average level of impulse activity in the afferent input may be critical to the regulation of expression of mENK.

Nigrostriatal dopamine mediates the stimulatory effects of corticotropin releasing factor on methionine-enkephalin and dynorphin release from the rat neostriatum

This study examined the effects of corticotropin-releasing factor (CRF) on the in vitro release of methionine-enkephalin (Met-enkephalin) and dynorphin in neostriatal slices taken from rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the nigrostriatal DA pathway. In neostriatal slices from control saline-infused animals and in those from the contralateral hemisphere of 6-OHDA-lesioned animals, CRF (10 −10 M) administered as a 90-min pulse exerted potent stimulatory effects on both Met-enkephalin and dynorphin release. In the neostriatal slices of 6-OHDA-lesioned striata, both the basal release and tissue content of Met-enkephalin were significantly ( P < 0.01) higher (2-fold) than those of control animals and the contralateral hemisphere of 6-OHDA-lesioned animals; however, neither the basal release nor the tissue content of dynorphin in 6-OHDA-lesioned striata was significantly different from control striata. In response to CRF (10 −10 M) the release of both Met-enkephalin and dynorphin were significantly diminished in slices of 6-OHDA-lesioned striata. These data support previous studies suggesting that nigrostriatal DA itself may exert a tonic inhibitory action on the activity of striatal Met-enkephalin neurones; however, DA may not have the same influence on striatal dynorphin neurons. However, the results of this study demonstrate that the action of CRF on Met-enkephalin as well as dynorphin release from the rat neostriatum is DA dependent. The data suggest that CRF receptors in the rat neostriatum may be localized on nigrostriatal/nigropallidal DA terminals/collaterals.

Opioids can enhance and inhibit the electrically evoked release of methionine-enkephalin

The stimulated (40 Hz) release of enkephalin from the myenteric plexus can be modulated by multiple types of opiate receptor. The direction of the modulation is not fixed but is bimodal. Both an inhibition and an enhancement of evoked release is observed depending on the concentration of opioid agonist that is used. Each of these effects can be antagonized by naloxone. Following pretreatment of guinea pig myenteric plexus in vitro with forskolin (0.5 μM) or the lipid soluble cAMP analog 8-(4-chlophenyltion)_cAM (8-CPT-cAMP; 100 μM) the inhibition of stimulated Met-enkephalin release that is produced by sufentanil (10 −8 M), [ d-Pen 2- d-Pen 5]enkephalin (DPDPE, 10 −8 M) or dynorphin (10 −7 M) is no longer observed. On the contrary, in forskolin- or 8-CPT-cAMP treated plexus a previoulsy inhibitory concentration of the above opioids now produces an enhancement of the magnitude of the stimulated Met-enkephalin release. Excitatory responses (enhanced release) to lower concentrations of sufentanil (1 nM) or DPDPE (5 nM) are not affected by pretreatment with the same concentration of forskolin or 8-CPT-cAMP. These data suggest that the ability of opioids to enhance or inhibit evoked enkephalin release is mediated via different biochemical processes (separate second messenger systems). This could imply that the opioid enhancement of enkephalin release is due to a direct facilitation of release and not to disinhibition. The ability of opioids to enhance the release of at least some neurotransmitters should be taken into account when attempting to explain the physiological sequelae of the acute and chronic effects of narcotics.

Corticotropin-releasing factor stimulates the release of methionine-enkephalin and dynorphin from the neostriatum and globus pallidus of the rat: in vitro and in vivo studies

This study examined the changes in the in vitro and in vivo release of methionine-enkephalin (Met-enkephalin) and dynorphin from the rat neostriatum in response to corticotropin-releasing factor (CRF). The levels of each opioid peptide were measured in the same sample collected at each time interval by specific radioimmunoassay methods. The in vitro release experiments were conducted using neostriatal slices (250 μm) obtained from adult male Wistar rats whereas in the in vivo studies, the release of both Met-enkephalin and dynorphin were assessed in push-pull perfusates of the caudate nucleus (containing both Met-enkephalin and dynorphin cell bodies/fibres) and the globus pallidus (containing Met-enkephalin and dynorphin terminals) of chloral hydrate-anaesthetised adult male Wistar rats. In the in vitro studies, CRF (10 −12, 10 −10 and 10 −8 M) (applied in pulses of 75 min) stimulated both Met-enkephalin and dynorphin release from the neostriatal slices in a dose-related manner; in the presence of the CRF receptor antagonist, α-helical CRF 9–14 (10 −16 M) release of both Met-enkephalin and dynorphin in response to CRF (10 −8 M) were completely blocked. Push-pull perfusion experiments conducted in both the caudate nucleus and the globus pallidus, also demonstrated a dose-related increase in the release of both Met-enkephalin and dynorphin in response to CRF (10 −12, 10 −10 and 10 −8 M) applied in 60-min pulses. In addition, in each of the two brain sites, the release of both Met-enkephalin and dynorphin in response to CRF (10 −8 M) was completely blocked by α-helical CRF 9–41 (10 −6 M). Both the in vitro and in vivo studies thus demonstrate that CRF can exert potent effects on Met-enkephalin and dynorphin release within the neostriatum-pallidum of the rat and that such effects are mediated via receptors specific for CRF, probably located on both the cell bodies and terminals of these opioid-containing neurones. The data obtained in this study thus substantiate the view that CRF, in addition to its regulation of pituitary opioid peptides, can communicate to opioid-containing neurones within the central nervous system and that many of the effects elicited by CRF may be ascribed to the opioid peptide released by CRF. The effects of CRF on the activity of Met-enkephalin and dynorphin neurones within the rat neostriatum, two distinct populations of chemically identified striatal projection neurones, suggest that CRF may play an integral role in the regulation of somatic motor processes with relevance to disorders of movement that originate from the basal ganglia.

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment.

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.

Meal-stimulated release of methionine-enkephalin into the canine jejunal lumen.

Application of enkephalins to the luminal surface of the bowel augments intestinal absorption. However, to date, endogenous enkephalins have not been demonstrated within intestinal luminal fluid. To determine whether enkephalins are present in the intestinal lumen, five adult dogs had 25-cm chronic jejunal Thiry-Vella loops constructed. Dogs were studied in the awake, fasted state. Jejunal loops were perfused with isoosmotic, neutral Krebs buffer containing protease inhibitors. After basal sampling, the dogs received a high fat meat meal. Collections were made during the meal and for 60 min postprandially. Luminal met-enkephalin levels were determined by radioimmunoassay and confirmed by HPLC. HPLC separation of luminal samples demonstrated two immunoreactive peaks which co-eluted with pure met-enkephalin and met-enkephalin-sulfoxide. Basal met-enkephalin outputs averaged 52 +/- 13 ng/min. The meal significantly increased mean luminal met-enkephalin output to 137 +/- 71 ng/min. During the initial 20-min postprandial period, output remained elevated (180 +/- 73 ng/min), after which it returned to basal levels. We conclude that met-enkephalin is present in the jejunal lumen, and that luminal release of this opioid is augmented by a meal.

Effects of hemorrhage and naloxone on adrenal release of methionine-enkephalin and catecholamines in halothane anesthetized dogs

Concurrent levels of methionine-enkephalin and catecholamines in adrenal vein, femoral vein and femoral artery were measured under baseline conditions and during graded hemorrhage in halothane anesthetized dogs and compared to a non-bled control group. Naloxone was administered in both groups at the end of the experiment. Normotensive hypovolemia with a remaining blood volume of 76% led to a moderate decrease in mean arterial blood pressure from baseline and a 15- to 20-fold increase in norepinephrine, epinephrine and dopamine, and a 5-fold increase in enkephalin in the adrenal vein. Subsequent induction of hypotensive hypovolemia with a remaining blood volume of 51% resulted in a profound drop in blood pressure and evoked a further increase in the level of catecholamines (40- to 50-fold from baseline) and enkephalin (8-fold from baseline) in the adrenal vein. In the control group only a 3- to 4-fold increase from baseline in adrenal vein hormone levels was observed over time. Naloxone administration at the end of the experiment, led to a 2- to 6-fold further increase in hormones at the 3 collection sites in both groups of dogs. Joint calculation of the partial correlation coefficients for the influence of preceding blood volume and blood pressure, and concurrent blood volume and blood pressure on hormone secretion in the adrenal vein revealed that these variables explained the variation in hormone levels between 56 and 92% during normotensive hypovolemia and 62–83% during hypotensive hypovolemia. In one dog with bilateral adrenalectomy, hemorrhage was poorly tolerated, and naloxone administration did not lead to increased systemic plasma levels of catecholamines and enkephalin or improved hemodynamics. In the hemorrhage group, molar ratios of norepinephrine/epinephrine in the adrenal vein showed a significant increasing trend during the experiment. Findings in these experiments support the idea of differential monoaminergic and enkephalinergic regulation in adrenal medullary cells.

Evidence for the coexistence and co-release of [met]enkephalin and noradrenaline from sympathetic nerves of the bovine vas deferens

The localization and neurosecretion of methionine-enkephalin was studied in sympathetic nerves of the bovine vas deferens. Immunostaining showed methionine-enkephalin-like immunoreactivity in a network of varicose nerve fibres in the smooth muscle layers of the vas deferens. When vas deferens homogenates were subjected to differential and sucrose density gradient centrifugation, methionineenkephalin was found to parallel the distribution of noradrenaline in the more dense region of the gradient, where “heavy” or large dense-cored vesicles are present. Electron microscopic immunochemistry confirmed this finding and showed methionine-enkephalin-like immunoreactivity in large dense-cored vesicles. The release of methionine-enkephalin upon electrical stimulation was studied in superfusion experiments. The methionine-enkephalin secretion was shown to be Ca 2+-dependent and was inhibited by adding the adrenergic neuron blocking drug guanethidine to the superfusion medium. We conclude that in the bovine vas deferens methionine-enkephalin is only present in large dense-cored vesicles of adrenergic neurons and that the peptide is released from these vesicles together with noradrenaline by a Ca 2+-dependent mechanism.

Does nitrous oxide induce release of brain opioid peptides?

Increased concentrations of immunoreactive methionine-enkephalin but not leucine-enkephalin were measured in fractions of cerebrospinal fluid perfusate collected from ventricular-cisternally perfused, urethane-anesthetized rats exposed to a mixture of 75% nitrous oxide/25% oxygen for 60 min. These findings suggest that nitrous oxide might be capable of inducing the release of methionine-enkephalin in the central nervous system.

GABA and baclofen potentiate the K +-evoked release of methionine-enkephalin from rat striatal slices

GABA potentiates the potassium-evoked release of methionine-enkephalin (ME) from slices of rat corpus striatum. This potentiation is observed only when a submaximal concentration (30 mM) of K + is used to evoke release. The effect of GABA is dose-dependent between 100 and 1000 μM. The basal release of ME is not altered by these concentrations of GABA. Baclofen, but not muscimol, mimics the effect of GABA on the evoked release of ME. This effect is not stereoselective as both the (+)- and (−)-isomers of baclofen enhance ME release. Picrotoxin (100 μM) blocks the enhancement of ME release produced by both GABA and baclofen. Bicuculline methiodide (100 μM) does not block the effect of GABA. The effect of GABA on ME release may be mediated by an atypical GABA receptor which is activated by baclofen.

Peristalsis abolishes the release of methionine-enkephalin from guinea-pig ileum in vitro

The release of methionine-enkephalin from superfused guinea-pig ileum has been demonstrated. Methionine-enkephalin, as measured by radioimmunassay, was shown to be released from tissues maintained at an intraluminal pressure of 0 cm water (resting conditions). Increased intraluminal pressure (5 cm water) induced peristalsis and abolished the release of methionine-enkephalin. These results provide further evidence that endogenous opioids may be involved in the modulation of contractile activity in the guinea-pig ileum.