D-Ala2-Met5-enkephalinamide Research Articles

Opioid peptide regulation of neurons in the bed nucleus of the stria terminalis: a microiontophoretic study

Single unit activity was recorded from neurons in the bed nucleus of the stria terminalis (BNST) in response to single or combined microiontophoretic ejection of d-Ala 2-Met 5-enkephalinamide (DAME) and naloxone. BNST neurons showed predominantly excitatory responses following small iontophoretic applications of DAME, and these responses were antagonized by naloxone. In contrast, inhibitory responses that were elicited by DAME required larger ejection currents and usually failed to show naloxone antagonism. The results indicate that activation of enkephalin-sensitive BNST neurons by DAME is primarily, but not exclusively, excitatory, which suggests that these responses may reflect differences in receptor sensitivity to the applied agonist.

D-Ala 2-Met 5-enkephalinamide impairs the acquisition of ethanol preference without influencing sucrose preference

The effects of subcutaneous (SC) administration of D-Ala 2-Met 5-enkephalinamide (DAME) (1, 10 and 100 μg/kg), synthetic analog of Met-enkephalin, on the acquisition of ethanol preference were studied in male Wistar rats. Under our procedural conditions, rats develop ethanol preference by a forced ethanol drinking session (conditioning session). Preconditioning administration of DAME (100 μg/kg) induced a reduction in ethanol consumption on the day of treatment and on subsequent testing days, but did not reliably modify later ethanol preference. Postconditioning administration of DAME (1, 10 and 100 μg/kg) markedly impaired the acquisition of ethanol preference. However, under the same schedule of treatment, DAME failed to affect subsequent rats' sucrose preference. These results suggest that, when administered after rats' first exposure to ethanol, DAME could interfere either with the reinforcement mechanisms of ethanol consumption induced by its intake, or with the storage of the information related to the ethanol incentive value.

Intra-accumbens implants of embryonic dopaminergic neurons reverse the behavioral supersensitivity to opiates evoked by lesion of the mesolimbic dopaminergic pathway

The ascending mesotelencephalic dopaminergic systems of rat pups of 3 days of age were bilaterally lesioned using 6-hydroxydopamine injected at the level of the lateral hypothalamus. A sub-group of lesioned pups received, 5 days after the lesion, a dopamine neuron-rich cell suspension graft implanted bilaterally into the striatum and nucleus accumbens. Behavioral tests were conducted 6 months later. The lesion induced an increase in the locomotor activation induced by d-Ala 2-Met 5-enkephalinamide injected into the nucleus accumbens (2.5 μg/side) as compared to the activation observed in control animals. Locomotor activation by systemic apomorphine (0.1 mg/kg s.c.) was also increased while that induced by amphetamine (1.5 mg/kg i.p.) was abolished. The presence of DA neuron implants reversed each of these post-lesion modifications.

Enkephalins interfere with early phases of voluntary ethanol drinking

The relationship between the opiate peptides Leu-enkephalin and [D-Ala 2-Met 5]enkephalinamide (DAME) and the initial expression and maintenance of ethanol preference was studied in male Wistar rats. Subcutaneous administration of both peptides prior to the first choice test between water and ethanol induced reductions on ethanol intake and subsequently on total fluid intake. Leu-enkephalin treatment also diminished ethanol preference in the day of treatment and in consecutive days. Neither Leu-enkephalin nor DAME treatments modified rats sucrose preference or intake. The results suggest that the enkephalins studied, when administered in the early phases of ethanol preference, interfere with the mechanisms involved in the propensity to drink ethanol.

Spinal paralysis and catalepsy induced by intrathecal injection of opioid agonists

The intrathecal administration of high (1.05 mumol) doses of D-Ala2-Met5-enkephalinamide (DAMA), D-Ala2-Leu5-enkephalinamide (DADLE), Try-D-Thr-Gly-Phe-Leu-Thr, MR2034-TA, dextrorphan tartrate, U50,488H, levorphanol tartrate, methadone hydrochloride, and 1-methyl-4-phenyl-4-propionoxypiperidine induced spinal hypokinesia. The first 5 of these compounds caused spinal paralysis, whereas the other compounds and lower doses of the first 4 induced waxy catalepsy that was restricted to the hindquarters of rats. The paralysis induced by DAMA was not reversible by IT injections of 50 micrograms naltrexone, indicating, together with the paralytic effects of dextrorphan, that traditional opiate receptors are not involved in this behavioral effect. The spinal catalepsy induced by 0.26 mumol of DAMA was prevented by IT pretreatment with 10 micrograms of naltrexone. In view of this finding and the observation that spinal catalepsy can be induced by agonists of all opiate receptor classes, it seems likely that spinal catalepsy is produced by activation of specific opiate receptors, although the subtype remains to be established.

Effect of D ala2 metenkephalinamide on feline jejunal and ileal water and electrolyte transport

The aim of this investigation was to compare the effect of an opioid, D ala2 metenkephalinamide (DAMA), on net jejunal and ileal water and electrolyte fluxes using the gut perfusion technique in the anesthetized cat. Intestinal transport was measured during intravenous infusion of serial doses of 2, 6, and 18 micrograms.kg-1.h-1 of DAMA in 6 cats. Each cat was its own control during an intravenous infusion of 150 mmol/l NaCl preceding the first dose of peptide and following the last dose of DAMA. Both jejunal and ileal segments were isolated by inflated balloons and were studied at the same time. Fifteen ml of an iso-osmolar test solution with hypo-osmolar ion contents and complementary mannitol were administered in the upstream tube and collected 1 h later in the downstream tube. In the jejunum, water secretion was dose-dependently reversed to an absorption from a control value of +0.5 +/- 0.4 to -0.83 +/- 0.5 ml.h-1.10 cm-1; in the ileum, water absorption was increased from -0.5 +/- 0.3 to -1.5 +/- 0.2 ml.h-1.10 cm-1. The net absorption of all electrolytes, ie sodium, chloride, bicarbonate, potassium and calcium also increased during peptide administration. However, a qualitative difference in the ion transport was observed between the jejunum and the ileum.

Study of the relationship between δ-receptor agonist D-Ala 2-Ser(OBZ) 5 and pain threshold

In the rat, morphine and the opioid peptide [D-Ala2]-metenkephalinamide (D-ALA) increased the reaction time in the tail-flick test as well as the threshold for vocalization and vocalization afterdischarge after electrical stimulation of the tail. The analgesic effect of either opiate was potentiated by selective inhibition of brain type B MAO with deprenyl or pargyline and antagonized by selective inhibition of the A form of the enzyme with chlorgyline. β-Phenylethylamine (PEA), a specific substrate of brain type B MAO, also potentiated the opiate analgesia, and the enhancement was especially marked after combined treatments with deprenyl and PEA. The effect of PEA appeared to be preferentially related to the affective component of the pain response (threshold for vocalization afterdischarge). A more general role of PEA in brain function as a neuromodulator of the opiate receptor is proposed.

INHIBITION OF SYMPATHETIC NEUROTRANSMISSION BY THE OPIOID δ‐RECEPTOR AGONIST DAMA IN THE PITHED RAT

1. The effects of [D-Ala2,Met5]enkephalinamide (DAMA), an analogue of [Met5]-enkephalin that acts selectively on opioid receptors of the delta-subtype, were studied on pressor responses elicited by sympathetic stimulation in pithed rats. 2. Intravenous injections of bolus doses of 0.1 mg/kg and 0.3 mg/kg of DAMA did not affect either the basal blood pressure or pressor responses to noradrenaline. 3. Pressor responses elicited either by electrical stimulation of the spinal sympathetic outflow or by stimulation of sympathetic ganglion cells with the muscarinic agonist McN-A-343 were reduced by DAMA. 4. Naloxone (1 mg/kg + 0.5 mg/kg per h) had no significant effect on the basal blood pressure or on pressor responses to spinal sympathetic stimulation, but antagonised the inhibitory effect of DAMA. 5. These results indicate that activation of opioid delta-receptors on sympathetic vasomotor nerve terminals can inhibit noradrenergic neurotransmission.

Modulation of μ-mediated antinociception by δ agonists: characterization with antagonists

The functional interactions between supraspinal μ and δ receptors were characterized in the mouse using μ receptor-selective antagonists. The effects of pretreatment with the μ opioid antagonists, β-funaltrexamine (β-FNA) and naloxonazine on the modulation of morphine antinociception by the δ agonists [D-Pen 2,D-Pen 5]enkephalin (DPDPE) and [D-Ala 2, Met 5]enkephalinamide (DAMA) were studied. When co-administered in the same i.c.v. injection, a sub-antinociceptive dose of DPDPE consistently and significantly increased the antinociceptive potency of morphine in control animals, while a sub-effective dose of DAMA decreased morphine antinociception; both the respective increase and the decrease of morphine potency by DPDPE and DAMA had been previously shown to be blocked by ICI 174,864, a δ antagonist. Pretreatment of mice with the non-equilibrium μ antagonist β-FNA 4 h prior to testing, a pretreatment which had no effect on i.c.v. DPDPE or DAMA antinociception, prevented the modulation of morphine antinociception by both DPDPE and DAMA. Pretreatment with the long acting μ 1 antagonist naloxonazine, 24 h prior to testing, failed to affect the modulation of morphine antinociception by either DPDPE or DAMA; such a pretreatment had no effect on the antinociceptive effects of DPDPE or DAMA when given alone. These results provide further support for the concept of a functionally coupled μ-δ receptor complex which is sensitive to antagonism by β-FNA, but not naloxonazine, and support the notion that subtypes of opioid μ and δ (i.e. complexed and non-complexed) receptors may exist.

Inhibition of stimulated lacrimal secretion by [D-Ala2]Met-enkephalinamide

The synthetic enkephalin analogue, [D-Ala2]Met-enkephalinamide (DALA) was used to investigate opioid peptide modulation of lacrimal protein secretion. By use of an in vitro perifusion system, the secretion of peroxidase by rat lacrimal gland fragments was measured during continuous stimulation for up to 60 min. DALA had no effect on unstimulated secretion of peroxidase. However, the addition of DALA to the perifusion medium resulted in a dose-dependent (10 nM-10 microM) inhibition of carbachol-stimulated peroxidase release. The maximum effect of DALA was achieved at a dose of 10 microM, which resulted in a 54% inhibition of carbachol-induced secretion. The opiate antagonist naloxone (10 nM-10 microM) did not alter basal or carbachol-stimulated peroxidase release. The effect on 10 microM carbachol-stimulated secretion by the addition of 3 microM DALA, however, was reversed in a dose-dependent manner by naloxone. The extent of inhibition of vasoactive intestinal peptidergic (VIPergic) stimulation (50 nM VIP) by DALA was similar to the inhibition of cholinergic stimulation of peroxidase release by gland fragments. Neither alpha 1-adrenergic stimulation of secretion nor a synergistic stimulation by VIP and carbachol was inhibited by the enkephalin analogue. We conclude that DALA exerts an inhibitory modulation of cholinergic and VIPergic stimulation of in vitro lacrimal protein secretion and suggest a physiological role for methionine enkephalin as an inhibitory peptide involved in the regulation of lacrimal gland function.

Intracerebroventricular N-ethylmaleimide differentially reduces supraspinal opioid analgesia in mice

I.c.v. injection of 1 nmol N-ethylmaleimide (NEM) into mice interfered with opioid-induced supraspinal analgesia, as assessed in the warm water tail-flick test. This effect of NEM was long-lasting (more than 3 days), non-competitive and differentially inhibited by the opioids studied. The analgesia induced by [D-Ala 2,D-Leu 5]enkephalin (DADLE), [D-Ala 2,Met 5]enkephalinamide (DAME) and [D-Pen 2,D-Pen 5]enkephalin (DPDPE) was greatly reduced in NEM-treated mice. The antinociception elicited by [D-Ala 2,N-MePhe 4,Gly-ol 5]enkephalin (DAGO) was also impaired although to a lesser extent. In contrast, the activity of morphine and etorphine remained unchanged. NEM-sensitive ipioids coadministered with morphine antagonized the analgesia elicited by the alkaloid in NEM-treated mice. The administration of naltrexone or DADLE, DAGO, [D-Ala 2,N-MePhe 4,Met-(O) 5-ol]enkephalin (FK-33824) and morphine in doses equivalent to the ED 90 doses for inducing analgesia, a few minutes before NEM prevented it from interfering with DADLE-elicited supraspinal analgesia when evaluated 24 h later. In contrast, the selective δ antagonist, ICI 174864, did not protect the DADLE-induced analgesia against the effect of NEM. We suggest that NEM produced its effect by acting upon a site that appears to be distal to the receptor binding site, presumably located on the guanine nucleotide binding regulatory proteins, G i/G o. Therefore, these transducer proteins might play a key role in the effects displayed by opioids when acting via the μ receptor-G i/G o complexes.

Sufentanil, Morphine, Met-enkephalin, and k-Agonist (U-50,488H) Inhibit Substance P Release from Primary Sensory Neurons

An in vitro model system for analysis of presynaptic inhibitory actions of spinal opioids has been applied. Embryonic sensory neurons derived from chick dorsal root ganglia were grown in primary cell culture, and the release of substance P was evoked by electrical field stimulation during exposure to drugs with well-demonstrated affinity for opioid receptors. This allowed a pharmacologic characterization of the inhibitory actions of specific opioid agonists on the release of substance P as measured by radioimmunoassay (RIA). Sufentanil (0.5 microM), a high affinity mu receptor agonist, U-50,488H (25 microM), a selective kappa receptor agonist, and morphine (10 microM), an agonist with high affinity for mu and delta receptors, inhibited the evoked release of substance P by approximately 60%, 40%, and 50%, respectively. For sufentanil the response was demonstrated to be dose-dependent. As is the case for its analgesic action in vivo, morphine was approximately 50-fold less potent than sufentanil on a molar basis in this assay. The actions of sufentanil, U-50-488H and morphine were mimicked by the endogenous opioid peptide met-enkephalin, and its stable synthetic analog D-ala2-met5-enkephalinamide (DAME). Naloxone (25 microM), an opioid receptor antagonist, blocked the inhibitory action of sufentanil (0.5 microM), morphine (5 microM), and DAME (5 microM), but not U-50,488H (10 microM). The action of U-50,488H was partially blocked by the antagonist naltrexone (25 microM). Stereo-selectivity of agonist action was confirmed by the failure of dextrorphan (50 microM), an inactive opioid isomer, to inhibit the release of substance P.(ABSTRACT TRUNCATED AT 250 WORDS)

Microinjections of d-Ala 2-Met 5-enkephalinamide placed into nucleus accumbens suppress hypothalamically elicited hissing in the cat

The effects of d-Ala 2-Met 5-enkephalinamide (DAME) upon the hissing component of hypothalamically elicited affective defense behavior in the cat were examined in this study. Microinjections of DAME placed into the nucleus accumbens significantly suppressed this response in a dose and time dependent manner. This dose dependent suppression of affective defense decreased toward baseline levels at 60 and 90 min following delivery of 1 and 10 μ g 0.5 μ l of DAME, respectively. Similar injections placed into the caudate nucleus had no effects upon this response. Neither vehicle control nor naloxone placed into nucleus accumbens was found to significantly alter latencies for hissing. Naloxone injected into nucleus accumbens prior to administration of either a 1-μg or a 10-μg dose of DAME blocked the suppressive effects of DAME that were observed when this drug was administered alone. These findings suggest that opioid receptors in the nucleus accumbens play an important role in the regulation of the hissing component of hypothalamically elicited affective defense behavior in the cat.

Some properties of adenylate cyclase in the segmental ganglia of the leech Hirudo medicinalis

1. The effects of serotonin, D-ala 2met-enkephalinamide, GTP and several non-physiological modulators of adenylate cyclase were examined on cAMP system of the segmental ganglia of the leech Hirudo medicinalis. 2. Serotonin stimulated both cAMP levels and adenlate cylase, while the opiate slightly, but significantly, inhibited cAMP synthesis. 3. Adenylate cyclase activity was dually regulated by GTP, stimulated by GTPyS and Gpp(NH)p and inhibited by GDPβS. 4. Fluoride ions greatly enhanced the enzyme activity, while forskolin was ineffective on basal and serotonin-stimulated cAMP synthesis.

Multiple brain sites sensitive to feeding stimulation by opioid agonists: A cannula-mapping study

Evidence suggests that brain opioid receptorrs of the mu, delta and kappa subtypes may be involved in the control of feeding behavior. However, limited information is available regarding the specific anatomical location of these feeding relevant opioid receptors. To address this problem, we microinjected three opioid agonists, morphine, (D-Ala 2)-Met-enkephalinamide (DALA) or MR 2034, into one of 15 different brain areas and measured the subsequent feeding responses of satiated rats. Morphine (25 nmol) and DALA (6.8 nmol) both elicited strong feeding responses from the same five brain areas, namely, the paraventricular, dorsomedial and lateral hypothalamus, as well as from sites within the septum and amygdala. No other brain sites yield significant responses to these opioid receptor agonists. In contrast to this anatomically specific pattern of effects, the opioid agonist MR 2034 (8.5 nmol) produced a feeding response which was generally smaller in magnitude and had little anatomical specificity. These findings suggest that opioid receptor systems for stimulating feeding exist in multiple discrete brain areas. Of the regions tested, specific sites within the hypothalamus, septum and amygdala are distinguished as being most sensitive to feeding stimulation by morphine and DALA.

Effects of d-Ala 2-Met 5-enkephalinamide microinjections placed into the bed nucleus of the stria terminalis upon affective defense behavior in the cat

This study examined the effects of intracerebral injections of d-Ala 2-Met 5-enkephalinamide (DAME) upon hypothalamically eliited hissing behavior in the cat. The bed nucleus of the stria terminalis (BNST) was selected for investigation because of its anatomical connections with the medial hypothalamus, its relatively high concentrations of enkephalins and opiate receptors and its demonstrated ability to modulate hypothalamically elicited aggressive reactions in the cat. DAME microinjected into the BNST in 1.0 or 10.0 μg/0.5 μl quantities resulted in significant dose dependent increases in mean latencies for elicitation of the hissing response. Suppression of hissing following the 1.0 μg dose of DAME was selectively diminished by prior administration of naloxone. These findings suggest that the opiate receptors within the BNST play a role in the regulation of the hissing component of hypothalamically elicited affective defense behavior.

Evidence that long-term administration of a methionine-enkephalin analogue stimulates the growth and steroidogenic capacity of rat inner adrenocortical cells

A prolonged infusion with D-ala 2-met-enkephalinamide (DALA) caused a significant increase in both basal and ACTH-stimulated corticosterone secretion by dispersed inner adrenocortical cells of rats whose hypothalamo-hypophyseal axis was pharmacologically interrupted. This effect of DALA was associated with a notable hypertrophy of isolated cells. These findings suggest that enkephalins are involved in the stimulation of the growth and steroidogenic capacity of the cells of the inner layers of rat adrenal cortex.

Opioids and sexual behavior in the male rat

Naloxone in the doses of 4 or 16 mg/kg failed to effect copulatory behavior of testosterone-treated castrated male rats. Morphine 10 mg/kg, administered 60 min before behavioral observation, reduced the proportion of animals displaying sexual behavior. Doses of 2.5 or 5 mg/kg reduced the latency to the second ejaculation, whereas the few animals still copulating after morphine 10 mg/kg showed a reduced latency to the first ejaculation. The same doses of morphine administered 5 min before behavioral observation produced a dose-dependent reduction of mount, intromission and ejaculation percentages. However, those animals that did copulate showed a normal copulatory behavior. D-Ala 2-Met 5 enkephalinamide (DALA) infused into the left cerebral ventricle in a dose of 5 μg 5 or 60 min before tests had no effect. When the peptide was infused 30 sec after the first intromission, the number of intromissions as well as the latency to ejaculation were reduced. Opioids may facilitate ejaculatory mechanisms, perhaps as a consequence of their rewarding properties. Moreover, in animals treated with DALA after the first intromission, the number of intromissions and the latency to ejaculation were similar for the first and second copulatory series, while these parameters were much reduced upon the second ejaculation for control animals. It is possible that liberation of endogenous opioids is the cause of ejaculation-induced facilitation of subsequent sexual behavior.

Effects of the desensitization by morphine of the opiate-dependent adenylate cyclase system in the rat striatum on the activity of the inhibitory regulatory G protein

Opiates act through a specific receptor to inhibit the striatal adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and stimulate a high-affinity GTPase (EC 3.6.1). The present study analyzes the functions of the striatal adenylate cyclase complex following chronic morphine treatment in the rat. The inhibitory effects of GTP on basal adenylate cyclase activity, between 10 −6 and 10 −4 M, were reduced. Moreover, the half-maximal inhibitory concentration of the opiate receptor agonist ( d-Ala 2Met 5-enkephalinamide (DAME) on striatal adenylate cyclase activity was increased by about four times, whereas the maximal effect was reduced in membranes from treated rats. In parallel, the half-maximal stimulatory concentration of DAME on GTPase was increased by two times, and the maximal stimulation was reduced from 60 to 25%. Binding studies performed with [3,5- 3] DAME (saturation curves) and with [ 3H]naloxone (competition curves) did not show any change in opiate receptor numbers and affinity. Moreover, the kinetics of the activation of the inhibitory GTP binding protein (G i) which transduces the opiate receptor effect on adenylate cyclase showed a small but significant delay. Therefore, hypofunction of G i can be, at least in part, responsible for the observed desensitization by morphine of the opiate-dependent GTPase and adenylate cyclase.

Hypothalamo-pituitary-interrenal responses to opioid substances in the trout: II. Effects of morphine and d-Ala 2, Met 5-enkephalinamide on plasma cortisol titres in vivo

The influence of morphine, d-Ala 2, Met 5-enkephalinamide (DALA), and naloxone on plasma cortisol titres has been studied in vivo in fingerling and adult trout. The responses were complex and variable. A single ip injection of morphine or DALA into fingerlings usually resulted in a rise in plasma cortisol after 0.5 hr followed by a fall below control values within 2 hr. In similar experiments with adult trout, only an inhibitory effect was observed. Naloxone reduced the rise in plasma cortisol following saline injection, but only when the hypothalamo-pituitary-interrenal response was intense. The antagonist also blocked the morphine-induced rise in cortisol secretion. Prolonged morphine treatment diminished both the postinjection and stress-induced secretion of cortisol in adult fish. Morphine had no effect on the spontaneous or ACTH-induced secretion of cortisol by interrenal tissue incubated in vitro. The results support the concept of inhibitory and stimulatory sites of action by opiates and opioid substances on the hypothalamo-pituitary-interrenal axis. These findings are discussed with reference to the action of morphine on hypothalamic and pituitary tissue of the trout in vitro and with the opioid control of hypothalamo-pituitary-adrenal function in mammals.