Inhibitory and excitatory effects of methionine-enkephalin in the isolated rabbit ileum

Methionine-enkephalin first inhibited and then stimulated the spontaneous rhythmic activity of the rabbit isolated ileum. The inhibitory effect of methionine-enkephalin was antagonized by naloxone. On the contrary, atropine did not change significantly either the inhibitory or the stimulatory action of this peptide. Furthermore, methionine-enkephalin inhibited the peristaltic reflex of the rabbit isolated ileum as well. Naloxone completely antagonized the inhibition of peristalsis produced by methionine-enkephalin. On the other hand, acetylcholine reversed only the propulsive activity and the back pressure, but not the peristaltic movements of the longitudinal muscle previously abolished by methionine-enkephalin. It appears, therefore, that only the inhibitory effects of methionine-enkephalin are mediated through opioid receptors in the intestine.     

An orexigenic role for mu-opioid receptors in the lateral parabrachial nucleus

The pontine parabrachial nucleus (PBN) has been implicated in regulating ingestion and contains opioids that promote feeding elsewhere in the brain. We tested the actions of the selective mu-opioid receptor (mu-OR) agonist [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) in the PBN on feeding in male rats with free access to food. Infusing DAMGO (0.5-4.0 nmol/0.5 microl) into the lateral parabrachial region (LPBN) increased food intake. The hyperphagic effect was anatomically specific to infusions within the LPBN, dose and time related, and selective for ingestion of chow compared with (nonnutritive) kaolin. The nonselective opioid antagonist naloxone (0.1-10.0 nmol intra-PBN) antagonized DAMGO-induced feeding, with complete blockade by 1.0 nmol and no effect on baseline. The highly selective mu-opioid antagonist d-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 1.0 nmol) also prevented this action of DAMGO, but the kappa-antagonist nor-binaltorphimine did not. Naloxone and CTAP (10.0 nmol) decreased intake during scheduled feeding. Thus stimulating mu-ORs in the LPBN increases feeding, whereas antagonizing these sites inhibits feeding. Together, our results implicate mu-ORs in the LPBN in the normal regulation of food intake.     

High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.     

Evaluation of peristalsis in multiple segments of the guinea-pig isolated small intestine: optimisation of tissue use by refined in vitro methodology

Peristalsis is the aboral movement by which the intestine propels its contents. Since pharmacological research requires an experimental model with which drug-induced modifications of peristalsis can be reliably quantified, we set out to develop and validate an in vitro method for studying peristalsis in multiple gut segments. In our arrangement, up to four 10cm segments isolated from the guinea-pig jejunum and ileum can be set up in parallel and their lumens perfused. Peristalsis was elicited by pressure-evoked wall distension, and the peristalsis-induced changes in the intraluminal pressure were evaluated with software that determined the peristaltic pressure threshold, the frequency, maximal acceleration and amplitude of the peristaltic waves, and the residual baseline pressure. Validation experiments showed that the peristalsis parameters at baseline and after modification by morphine (0.01-10microM) did not differ between segments from the jejunum and ileum, or between segments examined in a consecutive manner. In conclusion, our work succeeded in optimising the use of the guinea-pig jejunum and ileum for multiple recordings of peristalsis in vitro, and in refining the recording and evaluation of peristaltic motility. This system promises to be particularly useful in the pharmacological screening and testing of drugs which modify peristalsis.     

Naloxone does not influence cardiovascular responses to mild mental stress in postmenopausal women

The interaction between central opioid activity, sex hormones, and the cardiovascular reactivity to stress is unknown. Twenty-eight healthy postmenopausal women, 16 without, and 12 with hormone replacement therapy (HRT) participated in this randomized, double-blind, cross-over study. The opioid receptor antagonist naloxone or placebo was administered intravenously on 2 different days and mild mental stress was induced by the Stroop Color-Word Test. Cardiovascular responses were assessed noninvasively by impedance cardiography. Stress significantly increased stroke volume, cardiac output, blood pressure, and heart rate, which was not influenced by opioid receptor blockade. Whereas naloxone increased cortisol plasma concentrations irrespective of HRT status, luteinizing hormone concentrations, which were higher in non-HRT compared with HRT women, were increased by naloxone in women with HRT only. These data suggest that the opioidergic tone of the hypothalamus-pituitary-adrenal axis persists in postmenopausal women, irrespective of HRT use, while the opioidergic tone on the hypothalamus-pituitary-gonadal axis seems to depend on an estrogenic milieu. Naloxone does not alter cardiovascular mental stress reactions in postmenopausal women independent of their hormone substitution status.     

家兔中枢内源性阿片样物质在紧张性高血糖反应中的作用

本工作观察家兔内源性阿片样物质在紧张性高血糖反应中的作用。通过向家兔侧脑室内注射阿片受体阻断剂纳洛酮或羧基肽酶 A 的抑制剂 D-苯丙氨酸以分别减弱或加强脑内内源性阿片样物质的作用。结果表明,纳洛酮能使由乙醚或2-脱氧葡萄糖所引起的高血糖反应减弱,而使由胰岛素所引起的低血糖反应加强并延搁其回复过程。D-苯丙氨酸表现为相反的效应。在已对吗啡形成耐受的家兔,2-脱氧葡萄糖所引起的高血糖反应也呈减弱。这些结果提示,脑内内源性阿片样物质与紧张性高血糖反应有关。     

Pituitary opioid involvement in ECS-postictal electrogenesis and behavioral depression in rats

The role of pituitary opioids in electroconvulsive shock (ECS)-induced postictal electrogenesis and behavioral depression was investigated in sham-hypophysectomized and hypophysectomized rats. These animals were divided into two subgroups and injected SC with either saline or naloxone (3 mg/kg) 10 min prior to transauricular ECS. Sham-hypophysectomized rats given saline responded to a single ECS with a 65 +/- 18% (s.e.) increase in postictal electrogenesis and a behavioral depression lasting 3840 +/- 530 sec. Naloxone significantly antagonized both the postictal increase in EEG voltage output and behavioral depression. Hypophysectomy by itself was without effect on EEG patterns and only partially attenuated the ECS-induced electrogenesis and postictal depression (31.9 +/- 9% and 2360 +/- 511 sec, respectively). However, in hypophysectomized rats, naloxone did not further antagonize these effects of ECS. Thus, it appears that pituitary opioids may, at least in part, mediate postictal electrogenesis and behavioral depression. Alternatively, since hypophysectomy only partially attenuates these phenomena, central or nonpituitary opioid peptide systems may be involved. In view of the observed decrease in responsiveness to naloxone in hypophysectomized rats, nonopioid systems cannot be ruled out as contributors to the opioid-like effects of ECS in these animals.     

Effects of naloxone infusion on plasma levels of LH, FSH, and in addition TSH and prolactin in males, before and after oestrogen or anti-oestrogen treatment

The inhibitory action of endogenous opioids on gonadotrophin release is now well documented. Since LHRH-producing neurons do not possess oestrogen-receptors, it is likely that some other compound mediates the negative feedback action of oestrogens on the gonadotrophin release in the male. To test the hypothesis that endogenous opioids are implicated in this negative feedback action in the human male, the opioid receptor antagonist naloxone (2 mg/h for 4 h) was infused into 7 normogonadotrophic oligozoospermic men before and after 6 weeks of treatment with the oestrogen-receptor antagonist tamoxifen (TAM) (10 mg twice daily) and 6 eugonadal transsexual males before and after 6 weeks of administration of ethinyloestradiol (EE) (10 micrograms three times a day). The effects of naloxone on TSH and prolactin (PRL) release were also studied. Naloxone administration resulted in a significant release of gonadotrophins, but not of TSH and PRL. Administration of oestrogen and anti-oestrogen did not significantly affect the response of gonadotrophins to naloxone infusion and no evidence of consistently antagonistic effects of oestrogen and anti-oestrogen on the naloxone-induced gonadotrophin release was obtained. This shows that endogenous opioids are probably not intermediary in the negative feedback control of oestrogens on gonadotrophin release in the human male. Surprisingly, in contrast to the eugonadal transsexual males, FSH levels in the oligozoospermic men did not respond to naloxone administration. As naloxone is thought to exert its action on gonadotrophin release via a disinhibition of endogenous LHRH release, this finding is unexpected. Exogenous LHRH administration leads to a normal response of FSH in normogonadotrophic oligozoospermic men. No plausible explanation for this finding can presently be offered.     

Neuropeptides in spinal cord injury: Comparative experimental models

The possible role of endogenous opioids in the pathophysiology of spinal cord injury was evaluated utilizing a variety of experimental models and species. In the cat, we have shown that β-endorphin-like immunoreactivity was increased in plasma following traumatic spinal injury; such injury was associated with a decrease in spinal cord blood flow (SCBF) which was reversed by the opiate receptor antagonist naloxone. Naloxone treatment also significantly improved functional neurological recovery after severe injury. Thyrotropin-releasing hormone (TRH), possibly through its “anti-endorphin” actions, was even more effective than naloxone in improving functional recovery in the cat. In a rat model, utilizing a similar trauma method, TRH proved superior to naloxone in improving SCBF after injury. In addition, naloxone at high doses attenuated the hindlimb paralysis produced by temporary aortic occlusion in the rabbit. The high doses of naloxone required to improve neurological function after spinal injury suggest that naloxone's actions, if opiate receptor mediated, may be mediated by non-μ receptors. Dynorphin, an endogenous opioid with a high affinity for the κ receptor, produced hindlimb paralysis following intrathecal administration in rats. Taken together, these findings suggest that endogenous opioids, possibly acting at κ receptors in the spinal cord, may serve as pathophysiological factors in spinal cord injury.     

Effects of acupuncture on vasopressin-induced emesis in conscious dogs

Although acupuncture has a significant clinical benefit, the mechanism of acupuncture remains unclear. Vasopressin, a posterior pituitary hormone, is involved in nausea and vomiting in humans and dogs. To investigate the antiemetic effects of acupuncture on vasopressin-induced emesis, gastroduodenal motor activity and the frequency of retching and vomiting were simultaneously recorded in conscious dogs. In seven dogs, four force transducers were implanted on the serosal surfaces of the gastric body, antrum, pylorus, and duodenum. Gastroduodenal motility was continuously monitored throughout the experiment. Vasopressin was intravenously infused at a dose of 0.1 U x kg(-1) x min(-1) for 20 min. Electroacupuncture (EA, 1-30 Hz) at pericardium-6 (PC6), bladder-21 (BL21), or stomach-36 (ST36) was performed before, during, and after the vasopressin infusion. To investigate whether the opioid pathway is involved in EA-induced antiemetic effects, naloxone (a central and peripheral opioid receptor antagonist) or naloxone methiodide (a peripheral opioid receptor antagonist) was administered before, during, and after EA and vasopressin infusion. Intravenous infusion of vasopressin induced retching and vomiting in all dogs tested. Retrograde peristaltic contractions occurred before the onset of retching and vomiting. EA (10 Hz) at PC6 significantly reduced the number of episodes of retching and vomiting. EA at PC6 also suppressed retrograde peristaltic contractions. In contrast, EA at BL21 or ST36 had no antiemetic effects. The antiemetic effect of EA was abolished by pretreatment with naloxone but not naloxone methiodide. It is suggested that the antiemetic effect of acupuncture is mediated via the central opioid pathway.     

Naloxone and TRH affect regional blood flows in the anesthetized rabbit

The cardiovascular effects of IV naloxone and a subsequent administration of TRH IV were studied in the rabbit. Naloxone caused a vasodilation in the myocardium and adrenal glands. Naloxone elicited an increment in cerebral blood flow in several regions which attenuated the cerebrovasodilating effect of TRH in a few regions. The blockade of endogenous opioids with naloxone did not modify the peripheral vasoconstricting effect of TRH or affect the vascular effects of TRH mediated by the peripheral sympathetic nerves. The results indicate that naloxone has a vasodilating effect in the myocardium and CNS in anesthetized rabbits. The major part of the cardiovascular effect of TRH is not dependent on mechanisms sensitive to naloxone.     

Unexpected reversal effects of naloxone on the guinea pig ileum.

Distension of the guinea pig ileum segment elicits peristaltic activity. If the distension is maintained the peristaltic activity disappears gradually; naloxone restores normal activity in such “fatigued” preparations. The bath solution surrounding a fatigued preparation inhibits peristaltic reflex activity in non-fatigued segments; this inhibitory effect is reversed by naloxone. The latter also antagonizes the inhibitory effects of adenine-nucleotides. These results indicate that during fatigue a substance is liberated which blocks peristalsis. They further suggest that naloxone-induced reversal of inhibition in the guinea pig ileum does not necessarily demonstrate that the inhibition is caused by a direct action on morphine receptors.     

Differential role of opioid receptors in tibial nerve inhibition of nociceptive and nonnociceptive bladder reflexes in cats

Naloxone (an opioid receptor antagonist) was used to examine the role of opioid mechanisms in bladder reflexes and in somatic afferent inhibition of these reflexes by tibial nerve stimulation (TNS). Experiments were conducted in α-chloralose-anesthetized cats when the bladder was infused with saline or 0.25% acetic acid (AA). The bladder volume was measured at the first large-amplitude (>30 cmH(2)O) contraction during a cystometrogram and termed "estimated bladder capacity" (EBC). AA irritated the bladder, induced bladder overactivity, and significantly (P < 0.0001) reduced EBC to 14.3 ± 1.9% of the saline control. TNS (5 Hz, 0.2 ms) at 4 and 8 times the threshold (T) intensity for inducing an observable toe movement suppressed AA-induced bladder overactivity and significantly increased EBC to 41.5 ± 9.9% (4T, P < 0.05) and 46.1 ± 7.9% (8T, P < 0.01) of the saline control. Naloxone (1 mg/kg iv) completely eliminated TNS inhibition of bladder overactivity. Naloxone (0.001-1 mg/kg iv) did not change EBC during AA irritation. However, during saline infusion naloxone (1 mg/kg iv) significantly (P < 0.01) reduced EBC to 66.5 ± 8.1% of the control EBC. During saline infusion, TNS induced an acute increase in EBC and an increase that persisted following the stimulation. Naloxone (1 mg/kg) did not alter either type of inhibition. However, naloxone administered during the poststimulation inhibition decreased EBC. These results indicate that opioid receptors have different roles in modulation of nociceptive and nonnociceptive bladder reflexes and in somatic afferent inhibition of these reflexes, raising the possibility that opioid receptors may be a target for pharmacological treatment of lower urinary tract disorders.     

The effect of naloxone on blood pressure, heart rate, plasma catecholamines, renin activity and aldosterone following exercise in healthy males

There is evidence that endogenous opioids are involved in blood pressure regulation. In the present study the effect of naloxone on the cardiovascular, sympathoadrenomedullary and renin-aldosterone response to physical exercise was investigated in 8 healthy males. Each subject performed a submaximal work test twice, i.e. with and without naloxone. The test consisted of ergometer bicycling for 10 minutes on 50% of the maximal working capacity (MWC), immediately followed by 10 min on 80% of MWC. Ten minutes before exercise the subjects received in a single blind randomized order a bolus dose of naloxone (100 micrograms/kg) or a corresponding volume of the preservatives of the naloxone preparation (control) followed by a slow infusion of naloxone (50 micrograms/kg/h) or preservatives, respectively. Naloxone was without effect on the exercise-induced changes in systolic blood pressure, heart rate, plasma noradrenaline, renin activity and aldosterone, but the adrenaline response increased markedly. The present results indicate that opioid receptors are involved in the plasma adrenaline response to submaximal exercise, but not in the regulation of systolic blood pressure, heart rate, plasma noradrenaline, renin activity and plasma aldosterone.     

Inhibition of peristaltic activity in the guinea-pig ileum by specific stress stimulus; its reversal by naloxone and indomethacin

During continuous peristaltic reflex activity of the isolated guinea-pig ileum a model stress stimulus, elevated intraluminal pressure (120 mm H₂O) plus increased longitudinal tension (3 g) was applied for 2 min. The resulting inhibition of peristalsis outlasted the initial stimulus by several min. The inhibitory interval was shortened or abolished in the presence of naloxone (0.5 μM), an opiate receptor antagonist, or in the preparations made acutely tolerant to morphine. This seems to suggest an involvement of endorphins. An inhibition of endogenous prostaglandin synthesis by indomethacin (5 μM) decreased the amplitude of peristaltic longitudinal muscle contractions, and these contractions were increased in response to the stress stimulus in the presence of naloxone. Thus the response of the guinea-pig ileum to stress stimulation could be profoundly modified by an interference with endorphin and prostaglandin systems.     

Effects of naloxone on the mechanical activity of isolated rat hearts perfused with morphine or opioid peptides

In isolated rat hearts, the infusion for 10 min of 10(-10), 10(-8) or 10(-6) M (-)naloxone affected the cardiac function by markedly increasing the coronary pressure and by reducing both the heart rate and the developed tension. A lower dose of (-)naloxone (10(-11) M) or a dose of 10(-6) M (+)naloxone, did not modify the cardiac function. Morphine (10(-6) or 10(-5) M) and 10(-10), 10(-8) or 10(-6) M methionine-enkephalin or leucine-enkephalin, both significantly reduced the coronary pressure of the isolated rat hearts, during the first 4-6 min of perfusion, but the coronary pressure progressively increased above the control value in the last 4 min of perfusion. Each opioid also influenced the mechanical activity of the isolated rat heart, by significantly lowering both the heart rate and the developed tension. (-)Naloxone, at all the doses tested, was only able to antagonise the hypotensive effect induced by the opioids on the coronary pressure and was ineffective in counteracting the negative inotropic and chronotropic effects produced by each opioid. The perfusion in the presence of (+)naloxone (even at a dose of 10(-6) M) did not affect the opioid-induced changes on both the coronary pressure and the mechanical performance of the isolated heart.     

Similarities and differences in the propagation of slow waves and peristaltic waves

The relationship between slow waves and peristaltic reflexes has not been well analyzed. In this study, we have recorded the electrical activity of slow waves together with that generated by spontaneous peristaltic contractions at 240 extracellular sites simultaneously. Recordings were made from five isolated tubular and six sheet segments of feline duodenum superfused in vitro. In all preparations, slow waves propagated as broad wave fronts along the longitudinal axis of the preparation in either the aborad or the orad direction. Electrical potentials recorded during peristalsis (peristaltic waves) also propagated as broad wave fronts in either directions. Peristaltic waves often spontaneously stopped conducting (46%), in contrast to slow waves that never did. Peristaltic waves propagated at a lower velocity than the slow waves (0.98 +/- 0.25 and 1.29 +/- 0.28 cm/s, respectively; P < 0.001; n = 24) and in a direction independent of the preceding slow wave direction (64% in the same direction, 46% in the opposite direction). In conclusion, slow waves and peristaltic waves in the isolated feline duodenum seem to constitute two separate electrical events that may drive two different mechanisms of contraction in the small intestine.     

Opioid modulation of LH secretion in the ewe

Administration of opioid agonists and antagonists and measurement of resulting hormone changes were used to study the possible effects of opioids on reproductive function in the ewe. Intravenous administration of the long-acting methionine-enkephalin analogue FK33-824 (250 micrograms/h for 12 h) to 3 ewes during the follicular phase of the oestrous cycle depressed episodic LH secretion. This effect was reversed by administration of the opiate antagonist naloxone (25 mg/h) in combination with the FK33-824 treatment; in fact LH secretion was enhanced by the combined regimen. Naloxone (25 mg/h for 12 h) administered alone to 3 ewes in the follicular phase also enhanced LH secretion. In 3 animals treated with FK33-824 during the follicular phase, progesterone remained basal for 14 days after treatment, suggesting that ovulation was blocked. Jugular venous infusion of naloxone (25, 50 or 100 mg/h for 8h) into 5 ewes during the early and mid-luteal phase of the cycle resulted overall in a significant increase in mean plasma LH concentrations and LH episode frequency. To investigate whether endogenous opioids suppress LH release in seasonally anoestrous sheep, naloxone was infused intravenously into mature (25, 50 or 100 mg/h for 8 h) and yearling ewes (12 . 5, 25 or 50 mg/h for 8 h) during early, mid- and late anoestrus and plasma LH concentrations were measured. In the mature ewes, there was a trend for naloxone to increase LH values during the early anoestrous period but naloxone was without effect during mid- and late anoestrus. In the yearlings, naloxone infusion consistently increased plasma LH concentrations as a result of a significant increase in LH episode frequency. These experiments indicate that endogenous opioid peptides probably modulate gonadotrophin secretion during both the follicular and luteal phases of the oestrous cycle. However, the follicular phase of the sheep cycle is of short duration, and there may be residual effects of luteal-phase progesterone during this period. Secondly, there may be an age-dependent effect of naloxone on LH secretion during seasonal anoestrus in the ewe, with opioids playing a part in the suppression of LH in young but not in mature animals.     

Opiate receptor blockade in man reduces 2-deoxy-d-glucose-induced food intake but not hunger,thirst, and hypothermia

Opioid peptides may act as neuromodulators in the central nervous system to conserve energy stores and water in mammals. To examine this hypothesis in man, the effect of opiate receptor blockade with naloxone on the hunger, thirst, and hypothermic response to 2-deoxy-D-glucose-induced glucoprivic stress was assessed. Opiate receptor blockade decreased stress-induced food intake but did not reduce marked increases in hunger produced by glucoprivation. Naloxone infusions did not change the hypercortisolemic, polydipsic, hypothermic, and thermogenic response to 2-deoxy-D-glucose. While these results do not suggest a major role for a β-endorphin modulation of stress-induced hunger, hypothermia and water conservation, the reduction of food intake could be due to augmented satiety, perhaps associated with retardation of gastric emptying during opiate receptor blockade.     

In vitro effects of beta-endorphin on testicular release of androgens in the lizard podarcis sicula raf

The effects of the proopiomelanocortin-derived opioid peptide, beta-endorphin (β-EP), and of the opioid antagonist, naloxone (NAL), on both basal and pituitary-stimulated androgen secretion from superfused quiescent and active testes were assessed in the adult lizard, Podarcis sicula. In the absence of the homologous pituitary, in vitro treatment with β-EP and/or NAL did not affect basal secretion of androgens from quiescent and active testes. Conversely, in the presence of the homologous pituitary, treatment with β-EP brought about a decrease in androgen secretion in active testes, but no effect on quiescent ones Naloxone counteracted the inhibitory effect of β-EP in active testes, and enhanced maximal pituitary-stimulated secretion of androgens in quiescent but not in active testes. The effects produces by β-endorphin and naloxone were reversible. These results suggest that, in this lizard, opioids might be involved in the control of androgen release. The lack of effect of β-EP and naloxone when added directly to the testes seems to suggest that the opioid agonist and antagonist act on androgen release by modulating pituitary gonadotrophin output. © 1996 Wiley-Liss, Inc.