Effects of dynorphin1–13 on cardiac rhythm and cyclic adenosine monophosphate (cAMP) levels in the isolated perfused rat heart

We have studied the effects of dynorphin_1–13 on cardiac rhythm and cAMP levels in the isolated perfused rat heart. The standard Langendorff isolated heart preparation was used. The myocardial cAMP levels and the incidence of cardiac arrhythmias were determined after injection of dynorphin_1–13. Dynorphin_1–13 caused simultaneously cardiac arrhythmias and an increase in myocardial cAMP levels in a dose-dependent manner, and both effects were antagonized by naloxone. Further studies are needed to determine whether myocardial cAMP mediates the dynorphin-induced cardiac arrhythmias.     

A strychnine-sensitive site is involved in dynorphin-induced paralysis and loss of the tail-flick reflex

Dynorphin A(1-13) administered intrathecally to rats induces a reversible hindlimb paralysis and permanent loss of the tail-flick reflex in a dose-dependent and all-or-none manner. The loss of the tail-flick reflex has been determined to result from neurotoxicity linked to the N-methyl-D-aspartate (NMDA) receptor. Recently, it has been reported that NMDA antagonists attenuate irreversible paralysis induced by dynorphin A(1-17) and dynorphin A(2-17). In the present studies, we examined whether repeated injections of dynorphin A(1-13) acetate salt could change the characteristics of the reversible paralysis. Injections repeated every 48 h resulted in hindlimb paralysis upon each injection which was not different in terms of magnitude or duration (P greater than 0.60). Injections repeated at 2 h intervals resulted in desensitization of the paralytic effects (P less than 0.05). We also examined if strychnine sulfate, a glycine antagonist would alter the paralytic response to dynorphin. Strychnine protected rats from paralysis (P less than 0.01) and loss of the tail-flick reflex with an ED50 of 7 nmol. We conclude that the reversible paralysis induced by dynorphin A(1-13) is repeatable which suggests that the paralysis results from nontoxic or subtoxic actions of dynorphin. Desensitization to the paralytic effects occurs with closely spaced injections by some unknown mechanism. In addition, we conclude that the spinal glycinergic inhibitory system may participate in the induction of the paralysis because strychnine antagonizes dynorphin-induced paralysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of food deprivation on opioid receptor binding in the brain of lean and fatty Zucker rats

The effect of food deprivation on opioid receptor binding was studied in 6 brain regions of lean and fatty Zucker rats; using [3H]dynorphin A. There was no significant difference between lean and fatty rats fed ad libitum in binding parameters for any regions studied. Food deprivation increased Bmax and/or Kd for cortex, midbrain and striatum of lean rats, and the former two regions of fatty rats. These results suggest that food deprivation may influence opioid receptor binding in lean and fatty Zucker rats.     

Comparative studies of the neuro-excitatory behavioural effects of morphine-3-glucuronide and dynorphin A(2-17) following spinal and supraspinal routes of administration

Morphine-3-glucuronide (M3G) administered centrally produces dose-dependent neuro-excitatory behaviours in rodents via a predominantly non-opioid mechanism. The endogenous opioid peptide, dynorphin A (Dyn A) (1-17), is rapidly cleaved in vivo to the relatively more stable fragment Dyn A(2-17) which also produces excitatory behaviours in rodents via a non-opioid mechanism. This study investigated the possible contribution of Dyn A(2-17) to the neuro-excitatory behaviours evoked by supraspinally and spinally administered M3G in male Sprague–Dawley (SD) rats. Marked qualitative differences in behaviours were apparent following administration of M3G and Dyn A(2-17). Administration of 11 nmol i.c.v. doses of M3G produced intermittent myoclonic jerks, tonic–clonic convulsions, and ataxia, as well as postural changes, whereas i.c.v. Dyn A(2-17) at 15 nmol produced effects on body posture alone. Administration of 11 nmol i.t. doses of M3G produced intermittent explosive motor activity, and touch-evoked agitation, as well as postural changes, whereas i.t. Dyn A(2-17) at 15 nmol produced postural changes, touch-evoked agitation, and paralysis. Pre-treatment with Dyn A antiserum (200 µg) markedly attenuated total behavioural excitation following i.c.v. and i.t. administration of Dyn A(2-17) by  94% and 78%, respectively. However, total behavioural excitation following i.c.v. and i.t. administration of M3G was less markedly attenuated (both  27%) by pre-treatment with Dyn A antiserum, with reductions in tonic–clonic convulsions ( 43%), explosive motor behaviour ( 28%), and touch-evoked agitation ( 22%). The present findings discount a major role for Dyn A in mediating the neuro-excitatory effects of M3G, although it may contribute to maintaining some individual neuro-excitatory behaviours.     

Effects of veratridine and high potassium on micro-opioid receptor internalization in the rat spinal cord: stimulation of opioid release versus inhibition of internalization

Veratridine and high K⁺-induced μ-opioid receptor (MOR) internalization in rat spinal cord slices by evoking opioid release. Veratridine induced up to 75% MOR internalization but showed an atypical concentration–response: its effect increased steeply from 5 μM to 10 μM, and declined thereafter to disappear at 100 μM. At 100 μM, veratridine also inhibited of MOR internalization induced by exogenous endomorphin-2. This inhibition was caused by Na⁺ entry, since the Na⁺ ionophore monensin (50 μM) also inhibited endomorphin-induced MOR internalization. In contrast, veratridine induced neurokinin 1 receptor internalization (by evoking substance P release) without any inhibition at high concentrations. KCl evoked up to 80% MOR internalization, which disappeared in the presence of lidocaine or in the absence of peptidase inhibitors, indicating that it involved neuronal firing and peptide release. Unlike veratridine, KCl did not inhibit MOR internalization at high concentrations. However, both KCl and veratridine evoked more MOR internalization when applied for 2 min than for 20 min because of a direct inhibition of MOR internalization with the longer incubation times. These results show that short incubations with 20 μM veratridine or 30 mM KCl are optimal stimuli to evoke opioid release and MOR internalization in the spinal cord.     

Brain levels of neuropeptides in human chronic methamphetamine users

Animal data show that neuropeptide systems in the dopamine-rich brain areas of the striatum (caudate, putamen, and nucleus accumbens) are influenced by exposure to psychostimulants, suggesting that neuropeptides are involved in mediating aspects of behavioral responses to drugs of abuse. To establish in an exploratory study whether levels of neuropeptides are altered in brain of human methamphetamine users, we measured tissue concentrations of dynorphin, metenkephalin, neuropeptide Y, neurotensin, and substance P in autopsied brains of 16 chronic methamphetamine users and 17 matched control subjects. As expected, levels of most neuropeptides were enriched in dopamine-linked brain regions such as the nucleus accumbens and striatum of normal human brain. In contrast to animal findings of increased neuropeptide levels following short-term methamphetamine exposure, striatal neuropeptide concentrations were either normal or moderately decreased in the methamphetamine users. In other examined dopamine-poor cortical and subcortical brain areas, neuropeptide levels were generally either normal or variably reduced. Although the neuropeptide differences might be explained by methamphetamine-induced damage to neuropeptide-containing neurons, our human data are consistent with the possibility that, at least in the human striatum, long-term methamphetamine exposure leads to an adaptive process that is distinct from that which increases neuropeptide levels after acute methamphetamine exposure.     

Control of hormonal stress reactivity by the endogenous opioid system

Regulations of hormonal stress responses entail the initiation, amplitude and termination of the reaction, as well as its integration with other stress response systems. This study investigates the role of endogenous opioids in the regulation and integration of behavioral, thermal and hormonal stress responses, as these neuromodulators and their receptors are expressed in limbic structures responsible for stress responses. For this purpose, we subjected mice with selective deletion of beta-endorphin, enkephalin or dynorphin to the zero-maze test, a mildly stressful situation, and registered behaviors and stress hormone levels. Behavioral stress reactivity was assessed using zero-maze, light-dark and startle-reactivity paradigms. Animals lacking enkephalin displayed increased anxiety-related behavioral responses in each three, dynorphin knockouts in two models, whereas the responses of beta-endorphin knockouts indicated lower anxiety level in the zero-maze test. All knockout strains showed marked changes in hormonal stress reactivity. Increase in ACTH level after zero-maze test situation, unlike in wild type animals, failed to reach the level of significance in Penk1(-/-) and Pdyn(-/-) mice. Corticosterone plasma levels rapidly increased in all strains, with a lower peak response in knockouts. In wild-type and beta-endorphin-deficient mice, corticosterone levels returned to baseline within 60min after stress exposure. In contrast, mice lacking dynorphin and enkephalin showed longer-lasting elevated corticosterone levels, indicating a delayed termination of the stress reaction. Importantly, the behavioral and hormonal responses correlated in wild-type but not in knockout mice. Hyperthermia elicited by stress was reduced in animals lacking dynorphin and absent in Penk1(-/-) mice, despite of the heightened behavioral anxiety level of these strains. These results demonstrate an important role on the endogenous opioid system in the integration of behavioral and hormonal stress responses.     

Characterization of dynorphin-containing neurons on dissociated dentate gyrus cell cultures

In the dentate gyrus, the synthesis of the opioid peptide, dynorphin, is modulated by a variety of stimuli. In order to elucidate the cellular and molecular mechanisms regulating the synthesis of dynorphin in the hippocampus, we have established a routine primary cell culture of dentate granule neurons and identified granule-like neurons by a characteristic marker, dynorphin, in these cultures. Cultures were prepared from 7-day-old rat pups and maintained in medium with 2% fetal bovine serum. These cultures contained approximately 20% neurons and survived for over 4 weeks. After 2 weeks in culture, neurons expressing dynorphin-A and its messenger RNA were detected using immunocytochemistry and in situ hybridization, respectively. In dentate cultures, enkephalin-, cholecystokinin-, neuropeptide Y- and substance P-positive cells were observed in addition to dynorphin-positive cells with immunocytochemistry. The results suggest that dentate gyrus cell cultures provide a valid in vitro model for studying molecular mechanisms regulating prodynorphin gene expression.     

海上氦氧150 m饱和-182 m巡回潜水对潜水员血浆AVP、DynA、ACTH和CRH含量的影响

目的　研究海上大深度饱和潜水对潜水员血浆精氨酸加压素 (AVP)、强啡肽 A(Dyn A)、促肾上腺皮质激素 (ACTH)和促皮质素释放激素 (CRH)含量的影响。方法　用放射免疫法检测 8名潜水员在饱和潜水前后血浆 AVP、Dyn A、ACTH和 CRH的含量。结果　饱和潜水后潜水员血浆 AVP(14 4 8.5± 16 33.6 ) ng/L、Dyn A(4 9.4± 36 .8) ng/L、ACTH(392 .1± 2 4 5 .6 ) ng/L 和 CRH(378.1±4 4 .6 ) ng/L 含量显著或非常显著地高于饱和潜水前 (P<0 .0 5或 <0 .0 1)。结论　大深度饱和潜水能引起潜水员血浆 AVP、Dyn A、ACTH和 CRH含量的改变 ,所观察的 4种神经肽可能参与了大深度饱和潜水引起的机体应激反应