Effects of kappa opiate agonists on neurochemical and neuroendocrine indices: evidence for kappa receptor subtypes

Four kappa opiate agonists, U-50488H, MR-2034, EKC and tifluadom, elevated plasma corticosterone and decreased plasma TSH in a dose-dependent manner. These effects were naloxone-reversible. However, WIN 44441-3, a long acting narcotic antagonist, was unable to reverse the effects of U-50488H and MR-2034 upto doses of 5 mg/kg. U-50488H and MR-2034 but not tifluadom or EKC, also increased levels of DOPAC and HVA in the olfactory tubercle. This effect was also naloxone-reversible but not WIN 44441-3 reversible. Tifluadom and EKC did not increase DOPAC and HVA. The differential responses of the tested kappa agonists to WIN 44441-3 antagonism and dopamine metabolism in A10 neurons suggest that the kappa agonists can be separated into two groups. This is the first physiological evidence suggestive of kappa opioid receptor subtypes.     

TENA, a selective kappa opioid receptor antagonist

A number of opioid antagonists (TENA, naloxone, Mr 2266, WIN 44441) were evaluated for their selectivity in antagonizing the effect of mu, kappa, and delta agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Among these four antagonists, TENA was the most potent and the only ligand which was selective for kappa receptors. In this regard TENA was approximately 27-times more effective in antagonizing the kappa agonist, U-50488H, relative to the mu agonist, morphine, and it was about 5-times more effective against ethylketazocine (EK) relative to morphine. At the same concentration (20 nM) TENA did not significantly antagonize the delta agonist, [D-Ala2,D-Ala5]enkephalin (DADLE), in the MVD. Also, TENA was more effective than naloxone, EK, or U-50488H in protecting kappa receptors from irreversible blockage by beta-CNA. The results of this study indicate that TENA is the most selective kappa antagonist yet reported.     

Cyclic AMP regulates the calcium transients released from IP(3)-sensitive stores by activation of rat kappa-opioid receptors expressed in CHO cells

We analyzed intracellular Ca(2+)and cAMP levels in Chinese hamster ovary cells expressing a cloned rat kappa opioid receptor (CHO-kappa cells). Although expression of kappa(kappa)-opioid receptors was confirmed with a fluorescent dynorphin analog in almost all CHO-kappa cells, the kappa-specific agonists, U50488H or U69593, induced a Ca(2+) transient only in 35% of the cells. The Ca(2+) response occurred in all-or-none fashion and the half-maximal dosage of U50488H (812.1nM) was higher than that (3.2nM) to inhibit forskolin-stimulated cAMP. The kappa-receptors coupled to G(i/o)proteins since pertussis toxin significantly reduced the U50488H actions on intracellular Ca(2+) and cAMP. The Ca(2+) transient originates from IP(3)-sensitive internal stores since the Ca(2+) response was blocked by a PLC inhibitor (U73122) or by thapsigargin depletion of internal stores while removal of extracellular Ca(2+) had no effect. Interestingly, application of dibutyryl cAMP (+ 56.2%) or 8-bromo-cAMP (+ 174.7%) significantly increased the occurrence of U50488H-induced Ca(2+) mobilization while protein kinase A (PKA) inhibitors, Rp-cAMP (-32.3%) or myr-psi PKA (-73.9%) significantly reduced the response. Therefore, it was concluded that cAMP and PKA activity can regulate the Ca(2+) mobilization. These results suggest that the kappa receptor-linked cAMP cascade regulates the occurrence of kappa-opioid-mediated Ca(2+) mobilization.     

Ginsenoside Rf potentiates U-50,488H-induced analgesia and inhibits tolerance to its analgesia in mice

In the present study, the effect of ginsenoside Rf (Rf), a trace component of Panax ginseng on U-50,488H (U50), a selective kappa opioid-induced analgesia and its tolerance to analgesia was studied using the mice tail-flick test. In addition, the possible mechanism by which Rf may affect U50-induced analgesia was investigated. Intraperitoneal administration of U50 (40 mg/kg) produced analgesia. Rf (10(-14)-10(-10) mg/kg) on co treatment dose-dependently potentiated the U50 (40 mg/kg)-induced analgesia. Rf (10(-12)-10(-2) mg/ml) did not alter the binding of [3H] naloxone, a opioid ligand and [3H]PN200-110, a dihydropyridine ligand to mice whole brain membrane. Twice daily administration of U50 (40 mg/kg) for six days induced tolerance to its analgesia. Chronic treatment (day 4-day 6) of Rf (10(-14)-10(-10) mg/kg) to U50-tolerant mice, dose-dependently inhibited the tolerance. The inhibition of tolerance to U50-induced analgesia by Rf was not altered by flumazenil (0.1 mg/kg), a benzodiazepine receptor antagonist and picrotoxin (1 mg/kg), a GABA(A)-gated chloride channel blocker on chronic treatment. In conclusion, these findings for the first time demonstrated that ginsenoside Rf potentiates U50-induced analgesia, inhibits tolerance to its analgesia, and suggests that Rf affects U50-induced analgesia via non-opioid, non-dihydropyridine-sensitive Ca(+2) and non-benzodiazepine-GABA(A)ergic mechanisms in mice.     

Evidence that the hypothermic response of mice to delta-9-tetrahydrocannabinol is not mediated by changes in thermogenesis in brown adipose tissue.

Delta 9-Tetrahydrocannabinol (20 mg/kg i.p.) and propranolol (20 and 50 mg/kg i.p.) produced marked falls in the rectal temperatures of mice kept at an ambient temperature of 22 degrees C. Propranolol (50 mg/kg i.p.) also decreased the thermogenic activity of brown fat, as measured by a decrease in the level of [3H]GDP binding to mitochondria obtained from mouse interscapular brown adipose tissue. In contrast, delta 9-tetrahydrocannabinol (20 mg/kg i.p.) did not affect mitochondrial GDP binding even though the dose used was one shown previously to depress heat production. GDP binding was also unaffected by this cannabinoid in brown adipose tissue taken from mice that had been kept at 13 degrees C instead of 22 degrees C. In mice kept at 34 degrees C, isoprenaline (0.25 and 1.0 mg/kg s.c.) induced a marked rise in rectal temperature and increased the level of GDP binding to brown fat mitochondria. Propranolol (50 mg/kg i.p.) prevented the hyperthermic response to isoprenaline, the mice becoming hypothermic instead. Delta 9-Tetrahydrocannabinol (20 mg/kg i.p.) had no effect on isoprenaline-induced hyperthermia. We conclude from these data that there is no significant involvement of brown adipose tissue in the hypothermic response of mice to delta 9-tetrahydrocannabinol.     

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.     

Role of benzodiazepine-GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice

In the present study, the role of benzodiazepine-GABAA receptor complex in the attenuation of U-50,488H (U50), a selective kappa opioid agonist-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin (GTS) was investigated using the mice tail-flick test. The intraperitoneal (i.p.) treatment of GTS (100 and 200 mg/kg) and diazepam (0.1-1 mg/kg) dose-dependently attenuated the U50 (40 mg/kg, i.p.)-induced analgesia. GTS (0.001-10 microg/ml) did not alter binding of [3H]naloxone to mice whole brain membrane. The attenuation effect of GTS (100 mg/ kg) and diazepam (0.5 mg/kg) on U50-induced analgesia was blocked by flumazenil (0.1 mg/kg, i.p.), a benzodiazepine receptor antagonist, and picrotoxin (1 mg/kg, i.p.), a GABAA-gated chloride channel blocker. However, bicuculline (1 mg/kg, i.p.), a GABAA receptor antagonist blocked the attenuation effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on U50-induced analgesia. Chronic treatment (day 4-day 6) of GTS (50-200 mg/kg) and diazepam (0.1-1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) on chronic treatment blocked the inhibitory effect of GTS (100 mg/kg) and diazepam (0.5 mg/kg) on tolerance to U50-induced analgesia. On the other hand, chronic treatment of bicuculline (1 mg/kg) blocked the inhibitory effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on tolerance to U50-induced analgesia. In conclusion, the findings suggest that GTS attenuates U50-induced analgesia and inhibits tolerance to its analgesia and this action involves benzodiazepine receptors and GABAA-gated chloride channels.     

Actions of intracerebroventricular administration of kyotorphin and an analog on thermoregulation in the mouse

Intracerebroventricular (ICV) administration of kyotorphin (L-Tyr-L-Arg) and cyclo (N-methyl-L-Tyr-L-Arg), its analog, produced significant dose-dependent hypothermic responses in mice at an ambient temperature of 24°C. The hypothermic action of kyotorphin was much greater than that of Met-enkephalin (Met-ENK) but less than that of cyclo NMTA. This action was slightly but not significantly reversed by intraperitoneally administered naloxone (8 mg/kg), an opioid receptor antagonist. Met-ENK utilized as a control peptide in this study also produced a dose-dependent hypothermia which was slightly antagonized by naloxone (8 mg/kg, IP). Thyrotropin releasing hormone (TRH) injected ICV produced hyperthermia dose-dependently. The hypothermia induced by kyotorphin, its cyclic analog and Met-ENK was prevented by a small dose of TRH (0.18 μg=0.5 nmol/animal) which by itself had little effect on body temperature. A TRH neuronal system in the brain may explain the mechanism of kyotorphin-induced hypothermia. However, there was little evidence of involvement of opioid receptors. The present study demonstrates a potent action of kyotorphin and its analog on thermoregulation.     

Acute effects of the cannabinoid receptor agonist WIN55212-2 on dopamine release in rat: an in vivo electrochemical study

The aim of this study was to investigate the effects of the cannabinoid receptor agonist, WIN55212-2, and the cannabinoid receptor antagonist, SR141716A, on dopamine (DA) release evoked by KC1 (120 mM) microinjected into the striatum. The cannabinoid agonist WIN55212-2 (1 and 5 mg/kg, i.p.) dose-dependently attenuated DA release in the striatum, whereas the cannabinoid receptor antagonist SR141716A (3 mg/kg, i.p.) produced the opposite effect. SR141716A (3 mg/kg, i.p.) blocked the effects on DA release by WIN55212-2 (5 mg/kg, i.p.). Vehicle alone did not change DA release. These results suggest that cannabinoids modulate DA release in the striatum.     

Thermogenic effects of dihydrocodeine in the rat

The object of this study was to assess the effects of dihydrocodeine on thermogenesis and brown adipose tissue activity in the rat from measurements of oxygen consumption and blood flow. Acute injection of dihydrocodeine tartrate (s.c.) stimulated resting oxygen consumption (VO2) in Sprague-Dawley rats in a dose-dependent manner (0.5-50 mg/kg), with a peak response (40-45% increase) occurring at 10-25 mg/kg. This effect was also observed in urethane-anaesthetized rats (although the effect was reduced) and in conscious animals following gastric intubation with the drug. Pretreatment of rats with either a beta-adrenergic antagonist (propranolol, 20 mg/kg), ACTH (4 g/kg), or an opiate antagonist (WIN44441-1, 2 mg/kg) significantly reduced the response to dihydrocodeine, whereas corticosterone injection (5 mg/kg) enhanced the effect. Surgical adrenalectomy or hypophysectomy (HYPX) almost completely abolished the thermogenic effect of dihydrocodeine. Dihydrocodeine also stimulated VO2 in lean (58% increase) and genetically obese Zucker rats (69% increase), and in both Zucker genotypes these responses were only slightly affected by HYPX, but enhanced in HYPX rats treated daily with corticosterone (1 mg/kg). Tissue blood flow, assessed from the distribution of radiolabelled microspheres, was unaffected in white adipose tissue, skeletal muscle, testes, kidney, brain, and liver (arterial supply) after a single injection of dihydrocodeine (25 mg/kg), but flow to interscapular and perirenal brown adipose tissue was increased by 9- to 10-fold. Surgical sympathectomy of brown adipose tissue prevented the increase in blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

CB1-independent inhibition of dopamine transporter activity by cannabinoids in mouse dorsal striatum

Cannabinoid drugs are known to affect dopaminergic neurotransmission in the basal ganglia circuitry. In this study, we used in vitro and in vivo techniques to investigate whether cannabinoid agonists and antagonist could affect dopaminergic transmission in the striatum by acting at the dopamine transporter. Incubation of striatal synaptosomes with the cannabinoid agonists WIN55,212-2 or methanandamide decreased dopamine uptake (IC(50) = 2.0 micromol/L and 3.1 micromol/L, respectively). A similar inhibitory effect was observed after application of the inactive WIN55,212-2 isomer, S(-)WIN55,212-3. The CB(1) antagonist AM251 did not reverse WIN55,212-2 effect but rather mimicked it. WIN55,212-2 and AM251 partially displaced the binding of the cocaine analog [(3)H]WIN35,428, thus acting as dopamine transporter pseudo-substrates in the high micromolar range. High-speed chronoamperometry measurements showed that WIN55,212-2 (4 mg/kg, i.p.) caused significant release of endogenous dopamine via activation of CB(1) receptors, followed by a reduction of dopamine clearance. This reduction was CB(1)-independent, as it was mimicked by S(-)WIN55,212-3. Administration of AM251 (1 and 4 mg/kg, i.p.) increased the signal amplitude and reduced the clearance of dopamine pressure ejected into the striatum. These results indicate that both cannabinoid agonists and antagonists inhibit dopamine transporter activity via molecular targets other than CB(1) receptors.     

Itch-scratch responses induced by opioids through central Mu opioid receptors in mice

We examined scratch-inducing effects of intracisternal, intrathecal and intradermal injections of morphine and some opioid agonists in mice. Intracisternal injection of morphine (3 nmol/animal) and the mu-receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO; 0.2 nmol/animal) elicited scratching of the face, with little effect on scratching of the trunk. Intracisternal injection of the delta-receptor agonist [D-Pen(2,5)]enkephalin (DPDPE) and the kappa-receptor agonist U50488 were without effects. Intrathecal injection of morphine (0.1-3 nmol/animal) produced a dose-dependent increase in body scratching, with little effects on face scratching. Face scratching induced by intrathecal morphine (3 nmol/animal) was almost abolished by subcutaneous pretreatment with naloxone (1 mg/kg). Intradermal injections of morphine (3-100 nmol/site), DAMGO (1-100 nmol/site), DPDPE (10 and 100 nmol/site) and U50488 (10-100 nmol/site) did not elicit scratching of the site of injection. Intradermal injection of histamine (100 nmol/site) induced the scratching in ICR, but not ddY, mice and serotonin (30 and 50 nmol/site) elicited the scratching in either strain of mice. The results suggest that opioids induce scratching, and probably itching, through central mu-opioid receptors in the mouse.     

P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

Background

In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral ??-opioid receptor (MOR) activation are able to direct block PGE₂-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE₂-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated.

Results

Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE₂-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K??/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K?? null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K?? (? 43%).

Conclusions

The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K??/AKT signaling. This study extends a previously study of our group suggesting that PI3K??/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.     

Role of noradrenergic neurons in ethanol-induced elevation of free fatty acids and corticosterone

The effects of ethanol (2 or 3 g/kg) on plasma corticosterone and free fatty acids were examined in rats with bilateral electrolytic lesions of locus coeruleus or pretreated with the noradrenergic neurotoxin DSP 4 (50 mg/kg). Both lesions and DSP 4 significantly attenuated ethanol induced elevation of free fatty acids. No changes in basal levels of free fatty acids or corticosterone were observed nor did either treatment alter the elevating effect of ethanol on corticosterone. The results indicate that ethanol alters plasma free fatty acids through mechanisms involving noradrenergic neurons in the central nervous system.     

Effects of beta-carboline-ethyl ester on plasma corticosterone--a parallel with antagonist-precipitated diazepam withdrawal

Diazepam has been shown to produce physical dependence based on observations of behavioral stimulation or, in our laboratory, by increases in plasma corticosterone (CS) during antagonist-precipitated withdrawal. The behavioral excitation appears similar to that observed following the administration of beta-carboline esters--agents reported to interact with benzodiazepine receptors and termed "inverse agonists." The focus of the present study is to correlate the occurrence of changes in CS with behavioral excitation previously observed by others. Further, these studies are designed to show a parallel between the manifestations of benzodiazepine withdrawal and the pharmacologic effects of beta-carboline ethyl ester. Experiments were done in conscious unrestrained male Sprague-Dawley rats, with chronic i.v. catheters, using sound-attenuated one-way vision boxes. These studies compared the hormonal and behavioral changes induced by beta-carboline ethyl ester (beta CCE) with CGS-8216-precipitated withdrawal in rats treated with diazepam for 8 days. Rats treated chronically with diazepam (5 mg/kg/day), showed a significant increase in plasma (CS) following CGS-8216. Behavioral abstinence scores were also significantly elevated. beta CCE (0.5-5.0 mg/kg) showed a significant dose-related increase in plasma CS. Behavioral scores were also increased at doses of 0.5 and 2.0 mg/kg. beta CCE-induced plasma CS increases were antagonized by CGS-8216 at doses of 1.0 and 2.0 mg/kg but not by 0.5 mg/kg. In animals chronically treated with diazepam, beta CCE evoked a more prolonged plasma CS elevation than in vehicle-treated animals suggesting a dual agonist/antagonist effect. These data suggest a parallel between CS elevations and behavioral effects during withdrawal as well as similarities between the action of beta CCE and the manifestations of this withdrawal.     

Hypotensive response of ethanol in rats pretreated with disulfiram or nitrefazole

The effect of disulfiram or nitrefazole pretreatment on ethanol induced hypotension was examined in urethane anesthetized rats. A relatively low dose of ethanol (150 mg/kg; i.p.) produced a characteristic hypotensive response in rats pretreated for various periods with disulfiram or nitrefazole. This hypotensive episode started 5-10 minutes following ethanol administration and lasted 40-60 minutes. The hypotensive response was not seen unless disulfiram or nitrefazole treatment preceded ethanol administration by a least 6-8 hours. The low dose of ethanol produced a plasma ethanol concentration of 10mg/100ml or less. One treatment with nitrefazole (200 mg/kg) rendered rats vulnerable to ethanol-induced hypotension for 6 but not 8 days. One treatment with disulfiram (200 mg/kg) lasted 4 but not 6 days. In addition, the hypotensive response was greater in rats treated with nitrefazole than in rats treated with an equal dose (200 mg/kg) of disulfiram.     

Role of adrenals in morphine-induced hyperthermia in restrained rats

Role of adrenals in morphine-induced hyperthermia was studied in normal, neurotransmitter antagonist-pretreated, chemical-sympathectomized, adrenalectomized or adrenal-demedullated rats. In restrained female rats, 5 mg/kg morphine produced hyperthermia whereas 20 mg/kg and 40 mg/kg produced hypothermia. Pretreatment with either phenoxybenzamine, propranolol, pentolinium or scopolamine inhibited the hyperthermia. After adrenalectomy, neither 5 mg/kg nor chronic administration of 20 mg/kg morphine produced previously demonstrated hyperthermia. After adrenal-demedullation, a dose of 5 mg/kg morphine also did not produce hyperthermia. In contrast to female rats, restrained male rats showed no significant effect on body temperature after 5 mg/kg morphine, requiring 20 mg/kg and 40 mg/kg morphine to produce hyperthermia. In adrenalectomized male rats, 20 mg/kg morphine did not produce the usual hyperthermia. The results suggest that male rats are more resistant to the hyperthermic effects of morphine than female rats and that in the rat, the adrenals, likely the medulla, play an important role in morphine-induced hyperthermia.     

Vagotonic effects of enkephalin are not mediated by sympatholytic mechanisms

This study examined the hypothesis that vagotonic and sympatholytic effects of cardiac enkephalins are independently mediated by different receptors. A dose-response was constructed by administering the delta-receptor opioid methionine-enkephalin-arginine-phenylalanine (MEAP) by microdialysis into the interstitium of the canine sinoatrial node during vagal and sympathetic stimulation. The right cardiac sympathetic nerves were stimulated as they exited the stellate ganglion at frequencies selected to increase heart rate approximately 35 bpm. The right cervical vagus was stimulated at frequencies selected to produce a two-step decline in heart rate of 25 and 50 bpm. A six-step dose-response was constructed by recording heart rates during nerve stimulation as the dose of MEAP was increased between 0.05 pmol/min and 1.5 nmol/min. Vagal transmission improved during MEAP at 0.5 pmol/min. However, sympathetically mediated tachycardia was unaltered with any dose of MEAP. In Study 2, a similar dose-response was constructed with the kappa-opioid receptor agonist trans(+/-)-3-4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide-HCl (U-50488H) to illustrate an independent sympatholytic effect and to verify its kappa-receptor character. U-50488H gradually suppressed the sympathetic tachycardia, with a significant effect obtained only at the highest dose (1.5 nmol/min). U-50488H had no effect on vagally mediated bradycardia. Surprisingly, the sympatholytic effect was not reversed by withdrawing U-50488H or by the subsequent addition of the kappa-antagonist 17,17'-(dichloropropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetroldi-hydrochloride (norBNI). Study 3 was conducted to determine whether the sympatholytic effect of U-50488H could be prevented by norBNI. NorBNI blocked the sympatholytic effect of the U50488H for 90 mins. When norBNI was discontinued afterward and U-50488H was continued alone, a sympatholytic effect emerged within 30 mins. Collectively these observations support the hypothesis that the vagotonic influence of MEAP is not dependent on a sympatholytic influence. Furthermore, the sympatholytic effect is mediated independently by kappa-receptors. The sympatholytic effect of sustained kappa-receptor stimulation appears to evolve gradually into a functional state not easily reversed.     

Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for -receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic -selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific -antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, -opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.     

3H]U-69593 labels a subtype of kappa opiate receptor with characteristics different from that labeled by [3H]ethylketocyclazocine

[3H]U-69593 is an opiate agonist that has been reported to bind in vitro with high affinity and selectivity to the kappa receptor subtype. The studies reported here were designed to determine the optimal conditions for labeling kappa receptors with [3H]U-69593 and to further characterize the binding site. The effects of temperature and NaCl on [3H]U-69593 binding were of particular interest because previous studies reported that [3H]ethylketocyclazocine ([3H]EKC) and [3H]bremazocine binding to kappa receptors was optimal at 4 degrees C in the presence of NaCl. Those conditions were not found to be optimal for [3H]U-69593 binding. Although the pharmacological specificity and Bmax of [3H]U-69593 binding was similar at room temperature and at 4 degrees C, the binding affinity was approximately three times lower at 4 degrees C than at room temperature. In addition, NaCl had an effect on [3H]U-69593 binding that was opposite that on [3H]EKC binding at 4 degrees C (100 nM DAGO and 100 nM DADLE were included in all [3H]EKC assays to prevent binding to mu and delta receptors), i.e. NaCl decreased, rather than increased, [3H]U-69593 binding at 4 degrees C. These differences between [3H]U-69593 and [3H]EKC binding at 4 degrees C were accentuated by a vast difference in the density of the binding sites [Bmax approximately equal to 12 fmol/mg protein for [3H]U-69593 vs approximately equal to 375 fmol/mg protein for [3H]EKC at 4 degrees C in the presence of NaCl) and suggested that [3H]U-69593 might bind selectively to a kappa receptor subtype. This concept was supported by competition experiments. In particular, the site labeled by [3H]EKC at 4 degrees C was found to be relatively insensitive (compared to [3H]U-69593 and [3H]EKC binding at room temperature) to the kappa agonist U-50488H, a close analog to U-69593. Based on these findings, we propose that [3H]U-69593 (and U-50488H) labels a kappa receptor subtype which differs from that labeled by [3H]EKC at 4 degrees C.