II - Prostaglandin hyperalgesia: The peripheral analgesic activity of morphine, enkephalins and opioid antagonists

Morphine, enkephalins, nalorphine, naloxone and pentazocine are shown to have a peripheral analgesic effect. In our modification of the Randall-Selitto test these substances were 50–100 times more potent than a standard local anaesthetic, lidocaine. At this peripheral site, naloxone did not antagonize the effect of morphine. Morphine had a marked analgesic effect on the hyperalgesia induced by PGE₂ and PGI₂, BaCl₂, Ca²⁺ ionophore A23187, isoprenaline but not on that induced by dibutyryl cyclic AMP. It was suggested that the peripheral analgesic effect of morphine is due to an inhibition of adenylate-cyclase activity.     

III - Prostaglandin hyperalgesia: Relevance of the peripheral effect for the analgesic action of opioid-antagonists

Morphine injected into the rat cerebral ventricles had a marked analgesic effect, while no effect was observed with pentazocine and naloxone or nalorphine caused a strong hyperalgesia. Administered systemically (IP) naloxone and nalorphine caused a transitory analgesia followed by a long lasting hyperalgesic effect; morphine and pentazocine showed only an analgesic effect. It was concluded that the site of analgesic action of opioid-antagonists is peripheral rather than central. The peptidase-resistant enkephalin-analog, BW 180c, which does not cross the blood brain barrier, caused a marked analgesia by IP administration to paws made hyperalgesic by PGE₂ or carrageenin. It is suggested that agents derived from morphine, morphine-antagonists, enkephalins or cGMP devoid of central effect but having a strong peripheral effect may constitute a new class of safer analgesics.     

Changes in morphine self-administration in rats induced by prostaglandin E1 and naloxone

Interactions of prostaglandin E₁ (PGE₁) with morphine have been reported in several test systems and an hypothesis has been advanced for a role of prostaglandins in morphine analgesia and physical dependence. In rats self-administering morphine intravenously, a simultaneous and continuous infusion of naloxone hydrochloride at 56 to 560 μg/kg/day caused the expected increase in injection rate for morphine. Infusion of PGE₁ by itself at 56 or 180 μg/kg/day had no effect on the rate of morphine intake. Likewise the addition of PGE₁ at 180 μg/kg/day did not potentiate the increase caused by naloxone (56 or 180 μg/kg/day) when it was added to the naloxone infusion. These results do not support a role for prostaglandins in the behavioral aspects of morphine addiction. However, larger doses of PGE₁ (1 and 1.8 mg/kg/day), which were without overt effects in normal rats, caused severe and incapacitating prostration in morphinized rats.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Effects of morphine, enkephalins and naloxone on postprandial gastric acid secretion, gastric emptying and gastrin release in dogs

The effects of intravenous infusions of morphine, met-enkephalin and leu-enkephalin on gastric acid secretion, gastrin release and gastric emptying were investigated in four dogs with gastric cannulas stimulated by a liquid peptone meal. The actions of a potent opiate antagonist, naloxone, used alone or combined with opiates were also studied. Morphine, met-and leu-enkephalin decreased the fractional gastric emptying rate. Acid secretion was decreased by enkephalins and increased by high doses of morphine. Enkephalins and to a lesser degree morphine inhibited gastrin release during the first hour following the administration of the meal. Only leu-enkephalin decreases significantly the integrated gastrin response. Naloxone at the doses used antagonized partly or totally the effects of opiates on gastric emptying but not those on gastric secretion or gastrin release. Naloxone infused alone had no significant effect on the gastric functions tested. These studies indicate that in dogs stimulated by a liquid test meal, enkephalins inhibit gastric emptying, acid secretion and gastrin release. Morphine inhibits gastric emptying and gastrin release and enhances acid secretion.     

Morphine diminishes the constancy of spontaneous uterine contractions, antagonizes the positive inotropic effects of prostaglandin E2, but not of prostaglandin F2α and inhibits prostaglandin E and F outputs from the uterus of ovariectomized rats

The effects of morphine on the constancy of spontaneous contractions (isometric developed TENSION = IDT and contractile FREQUENCY = CF), in uterine strips isolated from ovariectomized rats and the influence of naloxone, were explored. The inotropic responses to added prostaglandins (PGs) E₂ and F_2α and the influences of morphine and of morphine in the presence of naloxone on PG actions, were also determined. Moreover, the synthesis and outputs of PGs E and F from uteri and the effects of morphine alone and of morphine plus naloxone, were studied. Morphine (10⁻⁶ M) significantly depressed uterine constancy of IDT during the first hours following delivery, but its action on CF did not differ from controls. Naloxone, neither at 10⁻⁸ M nor at 10⁻⁶ M, altered the negative inotropoic influence of morphine on IDT. Exogenous PGs E₂ and F_2α, stimulated uterine inotropism in a concentration-dependent fashion. Morphine altered dose-response curves for exogenous PGE₂, evoking a parallel surmountable shift to the right, but did not affect the inotropic action of added PGF_2α. This antagonistic effect of the opioid was not altered by preincubation with naloxone. Basal synthesis and outputs of PGs E and F in uteri from ovariectomized rats were significantly depressed by morphine (10⁻⁶ M) but not altered by incubating tissues with morphine in presence of naloxone. Results are discussed in terms of a presumptive dual action of morphine on uterine motility, i.e., antagonizing PGE₂ receptors and inhibiting the synthesis of some PGs by the uterus. These influences of morphine do not appear to be subserved by the activation of μ opioid receptors. Moreover, the possibility that endogenous opioids could play a relevant role modulating uterine PG influences, is also discussed.     

Release of endogenous prostaglandins by mild hyperosmotic saline inhibits tetragastrin-stimulated gastric acid secretion in rats

Filling of the gastric lumen of rats with 1.0 M NaCl solution (5 ml) for 10 min under urethane anesthesia caused an increase in the gastric fluid concentrations of prostaglandin (PG) E₂, 13, 14-dihydro-15-keto-PGE₂ and 6-keto-PGF_1α as determined by radioimmunoassay. PGE₂ was the major PG generated. The levels of PGE₂ in the gastric fluid were increased dose-dependently after filling the lumen with 0.3, 0.5, 0.7 or 1.0 M NaCl solutions. The pH of the gastric fluid increased similarly after 0.5 to 1.0 M NaCl solutions. Indomethacin (10 mg/kg, i.p.) suppressed the PGE₂ increase caused by 1.0 M NaCl solution, but did not prevent the increase of the pH of the gastric fluid induced by intragastric 1.0 M NaCl. Infusion of tetragastrin (62.5 μg/kg/hr, i.v., for 10 min) caused a marked increase of acid secretion without modifying intragastic concentration of PGE₂. The acid secretion due to tetragastrin was completely inhibited after intragastric administration of 1.0 M NaCl solution, while indomethacin restored the tetragastrin-induced acid secretion, with prevention of a rise of intragastric PGE₂ levels. These observations suggest that 1.0 M NaCl solutions suppress basal intragastric acid through a mechanism which is independent of prostaglandins. In contrast, the suppression of tetragastrin-induced acid secretion by intragastric 1.0 M NaCl solution appears to be mediated through a release of prostaglandins     

Chronic Activation of Inhibitory δ-Opioid Receptors Cross-Regulates the Stimulatory Adenylate Cyclase-Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108-15) Hybrid Cells

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E₁ (PGE₁) receptor system in neuroblastoma × glioma (NG108-15) hybrid cells. Persistent activation of δ-opioid receptors by morphine (10 µmol/L; 3 days) substantially down-regulates the number of PGE₁ binding sites by ～30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE₁ binding sites are still capable of interacting functionally with their associated stimulatory G proteins, G_s. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of G_s (G_sα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc^? reconstitution experiments, respectively. Evaluation of the functional interaction between PGE₁ receptors and G_s by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of G_sα revealed a significant increase in the ability of PGE₁ receptors to activate G_sα (3.3-fold increase in EC₅₀; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE₁ receptor/G_s coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE₁ receptor system represents a potential target of chronic δ-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.     

Studies on growth hormone secretion IX. Prostaglandins do not act like ionophores

To gain further insight on the mechanism of GH secretion in general and on the stimulation of this process by prostaglandins in particular, we compared the effects of PGE₁ and PGE₂ on hormone release and cyclic nucleotide levels with those of the ionophores A23187 and X537A under a variety of experimental conditions. All these substances (in the presence but not in the absence of calcium) enhanced GH release in incubated rat anterior pituitaries , prostaglandins being considerably more potent than ionophores. However, while PGE₂ caused a dose-dependent rise in pituitary cyclic AMP levels (from doubling at 10⁻⁷ M to a two-hundred fold increase at 10⁻⁵ M), the ionophores had no effect on the concentrations of this nucleotide. Neither PGE₂ nor the ionophores had any measurable effect on cyclic GMP levels. Exposure of tissues to ionophores for 60 minutes rendered them refractory to subsequent stimulation by PGE₁ or to ionophores themselves, whereas preincubation with PGE₁ did not diminish GH responses during a second incubation period. On the other hand, 60-minute preincubation of hemipituitaries in the presence of ionophores (10⁻⁵ M) did not suppress subsequent PGE₁-promoted cyclic AMP accumulation. Metabolic blockers inhibited PGE₂ and A23187-promoted GH-release but failed to suppress GH-response to X537A. Verapamil partially inhibited PGE₂ but not ionophore induced GH secretion. Ionophores particularly X537A, accelerated 45Ca efflux while PGE₁ did not influence this. Electronmicroscopy revealed extensive vacuolization localized chiefly at the Golgi apparatus when tissues were incubated with X537A. PGE₁ and A23187 had no such morphological effect. It is concluded that prostaglandins E and ionophores promote GH secretion by different mechanisms.     

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect,similar to the agonist,on the release of GnRHIn Vitro

Summary 1. Inin vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissiset al., 1993).2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37°C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH.3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release.4. Diallyl-G also inhibits the release of prostaglandin E₂ (PGE₂). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE₂ but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release.5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.     

Naloxone blocks the release of opioid peptides in periaqueductal gray and N. accumbens induced by intra-amygdaloid injection of morphine

The working hypothesis that the periaqueductal gray (PAG), N. accumbens and amygdala were connected serially in a unidirectional loop for antinociception, in which Met-enkephalin and β-endorphin were considered to be two important analgesic neurotransmitters, was examined by simultaneously perfusing the PAG and N. accumbens after microinjection of morphine into the amygdala. Intra-amygdaloid injection of morphine increased the release of enkephalins and β-endorphin in the PAG and N. accumbens. When the perfusion fluid contained 3 μM of naloxone, the release of enkephalins and β-endorphin was reduced in both the PAG and the N. accumbens. These results do not support the hypothesis of a unidirectional loop and its putative sequence.     

PGE2 secretion from organ cultured gastric mucosa: Correlation with cyclooxygenase activity and endogenous substrate release

In gastrointestinal research the in vitro release of prostaglandins from incubated or cultured biopsies is a widely used method to estimate prostaglandin synthesis. We therefore investigated the rate limiting mechanisms of PGE₂ release in organ cultured gastric mucosa of the rabbit, determining PGE₂ secretion from organ cultured mucosal biopsies by radioimmunoassay and prostaglandin synthesizing capacity by in vitro incubation of mucosal homogenate or microsomes with [¹⁴C]-arachidonic acid.Freshly taken biopsies secreted PGE₂ at an initial high rate, that decreased during the following 4 hrs of culture. This PGE₂ release was dose dependently reduced by inhibitors of the prostaglandin cyclooxygenase. 5mM acetylsalicylic acid (ASA) maximally suppressed PGE₂ secretion to 7% of controls, and the inhibition by ASA was quantitatively similar at every given culture period. PGE₂ release was markedly increased by carbenoxolone but was only slightly activated by extracellular calcium and the Ca⁺⁺-ionophore A23187. However, Ca⁺⁺/A23187 were unable to maintain PGE₂ secretion at the initial rate.PGE₂ secretion was undisturbed in calcium-free medium but was reduced to 50–60% of controls by excess EDTA. The intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′,-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) similarly inhibited PGE₂ release to 72% of controls. In contrast, PGE₂ release was unaffected by the intracellular calcium antagonist 3,4,5-trimethylene-bis(4-formylpyridinium bromide) dioxime (TMB-8), the calmodulin antagonists N-(6-aminohexyl)-1-5-chloro-1-naphthalenesulfonamide (W-7) and calmidazolium (compound R24571) or various direct inhibitors of endogenous arachidonic acid release like tetracaine, bromophenacyl bromid, neomycine or low dose quinacrine, indicating that the reduction of PGE₂ release by EDTA or BAPTA may be mediated by mechanisms different from substrate release. In contrast, an inhibition of PGE₂ secretion by quinacrine at high concentrations (≥ 0.8mM) was attributed to a direct inhibition of the prostaglandin cyclooxygenase, similar to ASA. Finally, the reduction of the prostaglandin synthesizing capacity by ASA was strongly correlated with the inhibition of PGE₂ secretion, also at low concentrations and minor degrees of inhibition.From these data we conclude, that the activity of the prostaglandin cyclooxygenase is rate limiting for PGE₂ secretion from organ cultured mucosal biopsies rather than arachidonic acid release by a phospholipase A₂. This should be considered for interpretation of studies based on prostaglandin release from cultured mucosa.     

Stimulatory effect of prostaglandin E1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated $\text{in vitro}$ using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE₁, PGE₂, PGF_1α or PGF_2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10^?6M PGE₁. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10^?8M to 1 x 10^?6M.     

Quantitation of luekotrienes C4, D4 amd E4 released by pathophysiological stimuli

In order to examine the modulation of leukotriene (LT) release, the PAF-acether-mediated stimulation of these compounds in rat lung was studied. Release of LTC₄, LTD₄ and LTE₄ in both perfused and chopped lung preparations was measured using HPLC and radioimmunoassay. Pre-incubation or pre-infusion of the tissue with indomethacin and PGE₂ was conducted to investigate the effect of cyclooxygenase inhibitors and products on the lipoxygenase pathway. In addition, the effects of LT levels of pre-incubation with vasoactive intenstinal polypeptide (VIP) in chopped lung were observed.In perfused rat lung, indomethacin reduced the levels of LTC₄ relative to LTD₄ as measured in the first 2 min after stimulation of the lung by PAF-acether. Chopped lung preparations, incubated for 15 min. exhibited higher levels of LTC₄ and LTD₄ in indomethacin-treated samples, this increases being effectively reversed by PGE₂.In the VIP pre-incubation experiments clear inhibition of peptido -leukotriene synthesis was observed, with no LTC₄ and only low levels of LTD₄ and LTE₄ observed in VIP-incubated samples. In preliminary experiments using rabbit C5a des arg and PAF-acether on rabbit lung parenchyma strips to stimulaet LT release, disodium cromoglycate pre-incubation was observed to inhibit this release.Inhibition of the 5-lipoxygenase pathway of PGE₂ is supported by these experiments. VIP appears to act as an inhibitor of LTC₄ and LTD₄ biosynthesis or release in this model. Too little is known that peptidergic actions to postulate a mechanism by which a neuroendocrine peptide exerts control of release of arachidonate metabolites; however, VIP is associated with muscarinic stimulation (1) and has been found in mast cells (2).     

Influence of opiates on the levels of adenosine 3':5'-cyclic monophosphate in neuroblastoma X glioma hybrid cells.

In neuroblastoma x glioma hybrid cells prostaglanddin E₁ (PGE₁) increases the level of adenosine 3′: 5′-cyclic monophosphate. This response to PGE₁ is strongly enhanced in cells that were incubated with morphine, methadon, noradrenaline or carbamylcholine for several hours. All these compounds increase the level of cyclic GMP in the cells. As in untreated cells, the effect of PGE₁ can be inhibited by morphine or noradrenaline. The development of the increased response to PGE₁ is dependent on protein synthesis. The increased response to PGE₁ is discussed in connection with morphine tolerance and withdrawal.     

Differential role of the opioid μ and δ receptors in the activation of prolactin (PRL) and growth hormone (GH) secretion by morphine in the male rat

Administration of naloxazone (50 mg/kg i.v.), an irreversible, selective and long acting antagonist of the μ₁ subclass of the opioid receptors, strongly reduced stimulation of PRL secretion by morphine (5.0 mg/kg i.v.) injected 24 hours later into conscious, unrestrained rats. In contrast, the effect of morphine on PRL release was unimpaired in rats treated 24 hours beforehand with either the reversible opioid antagonist naloxone (50 mg/kg i.v.), or the vehicle for naloxazone. A complete suppression of the PRL response to morphine (3.0 mg/kg i.v.) was observed in animals given intraventricular (IVT) injection of β-funaltrexamine (β-FNA, 2.5 μg), another selective, irreversible and long acting antagonist of the μ receptors, 24 hours beforehand. Neither naloxazone nor β-FNA had any effect on the activation of GH secretion by morphine, which, however, was conspiciously reduced by ICI 154, 129, a preferential δ receptor antagonist, injected IVT (50 μg) 5 minutes before morphine. It is concluded that the PRL stimulating effect of morphine is mediated by the μ receptors, wherease activation of GH probably involves the δ sites.     

Somatostatin,prostaglandin E2 and atropine inhibition of the gastric actions of bombesin in the dog

Bombesin, acetylcholine, prostaglandins and somatostatin are all thought to be involved in the regulation of gastrin release and gastric secretion. We have studied the effects of low doses of atropine, 16-16(Me)₂-prostaglandin E₂ (PGE₂) and somatostatin-14 on bombesin-stimulated gastrin release and gastric acid and pepsin secretion in conscious fistula dogs. For reference, synthetic gastrin G-17 was studied with and without somatostatin. Bombesin, in a dose-related manner, increased serum gastrin, which in turn stimulated gastric acid and pepsin secretion in a serum gastrin, concentration-dependent manner. Somatostatin inhibited gastrin release by bombesin as well as the secretory stimulation by G-17; the combination of sequential effects resulted in a marked inhibition of bombesin-stimulated gastric acid and pepsin secretion. PGE₂ also strongly inhibited gastrin release and acid and pepsin secretion. Atropine had no significant effect on gastrin release, but greatly inhibited gastric secretion. Thus somatostatin and PGE₂ inhibited at two sites, gastrin release and gastrin effects, while atropine affected only the latter.     

Stimulatory effect of PACAP on gastroduodenal bicarbonate secretion in rats

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO₃⁻ secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO₃⁻ secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO₃⁻ secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO₃⁻ secretion after intracisternal administration (1 nmol/rat). PGE₂ (300 μg/kg, iv) significantly increased HCO₃⁻ secretion in both the stomach and the duodenum. The potency of duodenal HCO₃⁻ secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE₂. The duodenal HCO₃⁻ secretory action of PACAP-27 as well as PGE₂ was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 μg/kg, iv), the stimulator of adenylate cyclase, also increased HCO₃⁻ secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO₃⁻ secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO₃⁻ secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO₃⁻ secretion.     

In vivo insulin secretion: Prostaglandin and adrenergic interrelationships

Infusion of prostaglandin (PG) E₁ in anesthetized dogs significantly lowered circulating insulin levels and inhibited insulin responses following intravenous glucose. A similar trend was observed with PGE₂. Alpha adrenergic blockade did not reverse the PGE₁ effect. Epinephrine infusion also inhibited glucose-stimulated insulin secretion, an effect that was not reversed by indomethacin. Therefore, in this investigative model, PGE₁ inhibited insulin secretion but no interdependency of PGE₁ and alpha adrenergic effects were found.     

Stimulation of dopamine synthesis and release by morphine and D-Ala2-D-Leu5-enkephalin in the mouse striatum in vivo

The effects of opiates on dopamine (DA) release and synthesis were assessed in the mouse striatum $\text{in vivo}$ by simultaneously measuring 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels after inhibition of aromatic amino acid decarboxylase. This method was developed to assess stimulus-coupled changes in DA synthesis and release. Peripheral injections of morphine and intraventrcular injections of D-Ala²-Leu⁵-enkephalin elevated DOPAC levels, indicating that “opiates” stimulated DA release. Concomitantly, the rate of DA synthesis was increased. The effects were dose-dependent, saturable and antagonized by naloxone. When morphine and the enkephalin analog were given together in saturating doses, the effects of the two agents were not additive. Thus, the involvement of different receptors in the mediation of the effects of morphine and enkephalins could not be demonstrated.