A study of the effect of B-EP and naloxone on the function of the hypothalamo-pituitary-testicular axis of the rat.

To investigate whether endogenous opioid peptides (EOP) play an important role in intragonadal regulation of testicular function and regulation of the hypothalamic-pituitary-gonadal axis of the male rat, the authors employed two principal methods: culture of testicular Leydig cells and Sertoli cells, and in vitro perifusion of hypothalamo-pituitary Leydig cells of the adult rat. The results demonstrated that incubation of Leydig cells with B-endorphin (B-EP 10(-9) = 10(-6) mol/L) or naloxone (NAL 10(-5) = 10(-8) mol/L) manifested no significant changes of non-stimulated or hCG-stimulated testosterone secretion both in 20 and 60 day-old rats. Similar results were obtained when the cells were treated with B-EP (10(-10) = 10(-7) mol/L) for 48 h during culture. Pretreatment of incubated Leydig cells with B-EP in similar concentrations for 48 h showed no effect on the response to hCG stimulation. In addition, treatment with B-EP in vitro for 24 or 72 h manifested no effects on estradiol production by aromatization of cultured Sertoli cells. Neither NAL 10(-5) given in vitro nor NAL (5 mg/body weight) injected subcutaneously 1 h before decapitation affected LH and testosterone release from the perifused hypothalamo-pituitary Leydig cells system. These results could not support the hypothesis that B-EP is a local regulator of testicular function. The physiological significance of EOP in regulating the function of gonadal axis of adult male rat remains to be investigated further.

[Antagonism of morphine analgesia and electroacupuncture analgesia by cholecystokinin octapeptide (CCK-8) administered in periaqueductal gray (PAG) of the rabbits].

We have reported that intracerebroventricular (i. c. v.) injection of 1-4 ng of CCK-8 to the rat produced a remarkable antagonistic effect on morphine analgesia. In order to study the species specificity and the site of action, CCK-8 was microinjected into the PAG of the rabbit, and its influence on morphine analgesia and electroacupuncture analgesia was observed. The latency of the escape response (ERL) to radiant heat focused on the snout was measured as an index of the pain threshold. Microinjections were made via cannulae chronically implanted into the PAG. The drug solutions were delivered in a volume of 1 microliter, at a speed of 0.125 microliter/min. The ERL was measured for a period of 60 or 70 minutes at 10 min intervals. 1. CCK-8 administered unilaterally to the PAG of the rabbit at a dose of 3 ng antagonized the analgesia induced by morphine (4 mg/kg, i. v.) by 73% (P less than 0.001), and reduced the analgesic effect of electroacupuncture by 67% (P less than 0.001). These effects were dose-dependent within the range from 1.5 ng to 6.0 ng. The effect of CCK-8 was reversed by CCK receptor blocker proglumide (4 microliters, intra-PAG injection). Unsulfated CCK-8 (CCK-us) had no effect in this regard. These results indicate that in the PAG of the rabbit, exogenously administered CCK-8 was capable of antagonizing opioid analgesia by the activation of CCK receptors. 2. Two groups of rabbits were given with morphine (2 mg/kg, i. v.) and simultaneous injection of CCK-8 antiserum (CCK-AS, 1 microliter) or normal rabbit serum (NRS) into the PAG.(ABSTRACT TRUNCATED AT 250 WORDS)

Electroacupuncture and morphine analgesia potentiated by bestatin and thiorphan administered to the nucleus accumbens of the rabbit.

The endogenous opioid peptide enkephalin (EK) is known to be degraded mainly by two enzymes, the dipeptidyl carboxypeptidase 'enkephalinase' and aminopeptidase. Microinjection of the enkephalinase inhibitor thiorphan or the aminopeptidase inhibitor bestatin into the nucleus accumbens of the rabbit produced a dose-dependent analgesic effect. This analgesic effect was totally reversed by the narcotic antagonist naloxone or by antibodies against [Met5]enkephalin (MEK) administered to the same site. Antibodies against [Leu5]enkephalin were not effective. Moreover, microinjection of thiorphan or bestatin into the nucleus accumbens resulted in a marked potentiation of the aftereffect of electroacupuncture (EA) produced analgesia, as well as the analgesia induced by a small dose of morphine. It is concluded that the analgesic effect elicited by EA and morphine is mediated, at least in part, by MEK-like immunoreactive substance(s) in the nucleus accumbens.

Met-enkephalin-Arg6-Phe7-like immunoreactive substances mediate electroacupuncture analgesia in the periaqueductal gray of the rabbit.

The present study was undertaken to investigate whether the C-terminal extended Met-enkephalin heptapeptide (Met-enkephalin-Arg6-Phe7, MEAP) played a role in mediating the analgesic effect of electroacupuncture in rabbits. MEAP and its degrading enzyme inhibitor captopril as well as antiserum against MEAP were injected into the periaqueductal gray (PAG) via a previously implanted cannula. Their effects on nociception were tested by the escape response latency (ERL) elicited by radiant heat applied on the skin of the snout. (1) Microinjection of MEAP (30-240 nmol) into PAG produced a dose-dependent analgesic effect which was 2.5 times more potent than Met-enkephalin (MEK) and 3 times less potent than morphine. The complete reversal of the analgesia elicited by 240 nmol of MEAP by a small dose of naloxone (0.1 mg/kg, i.v.) indicates that the effect of MEAP is mediated by naloxone sensitive opioid receptors. (2) In rabbits, a dose-dependent analgesia was elicited by an intra-PAG injection of captopril (60-240 nmol). A single dose of 240 nmol captopril increased ERL by more than 100%. This effect could be reversed by 30 nmol of naloxone injected into the same site, or by antiserum recognizing MEAP (1 microliter, titer 1:1500) but not by antiserum recognizing MEK (1 microliter, 1:8000) suggesting that captopril was able to protect MEAP from degradation. (3) Intra-PAG injection of 60 nmol of captopril significantly potentiated the after effect of electroacupuncture (EA) induced analgesia. This effect could be blocked either by 30 nmol (but not 7.5 nmol) of naloxone, or by 1 microliter (but not 0.1 microliter) of MEAP antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Acupuncture mechanisms in rabbits studied with microinjection of antibodies against beta-endorphin, enkephalin and substance P.

Injection of protein-A purified antibodies against Met-enkephalin and beta-endorphin into the periaqueductal gray matter (PAG) was shown to decrease the analgesic effect of electroacupuncture (EA) in rabbits. Met-enkephalin antibodies were more potent than the beta-endorphin antibodies in causing a statistically-significant effect on electroacupuncture analgesia. Antibodies to Met-enkephalin were also active at the spinal level, whereas antibodies against beta-endorphin were without effect: this is in agreement with a rich enkephalinergic innervation and absence of beta-endorphin-containing fibres in the spinal cord. Substance P, the other neuropeptide of this study, also seems to be important in mediating effects of electroacupuncture. Injection of antibodies into the periaqueductal gray caused decrease of the effect of electroacupuncture whereas intrathecal administration of Fab-fragment substance P antibodies caused a marked potentiation. The demonstration of site specificity of the neuropeptides in mediating analgesia induced by electroacupuncture supports the validity of this experimental approach.

Tolerance to electroacupuncture analgesia was reversed by microinjection of 5-hydroxytryptophan into nuclei accumbens in the rabbit.

Electroacupuncture (EA) of the hind limb points of the rabbit for 10 min elicited a significant increase of the latency of aversive responses (ARL) induced by radiant heat stimulation of the snout. This effect of analgesia was found to be gradually diminished when EA was applied repeatedly for 6 hr, implying the development of tolerance. A reversal of EA tolerance was achieved by microinjection of 5-hydroxytryptophan (5-HTP) (the direct precursor of 5-hydroxytryptamine (5-HT)), either intracerebroventricularly (200 micrograms) or into bilateral nuclei accumbens (10 micrograms). Injection of the same amount of 5-HTP into brain structures adjacent to nucleus accumbens, e.g., globus pallidus, corpus callosum, internal capsule, etc., was ineffective. It is concluded that a functional insufficiency of 5-HT in the brain, especially in nucleus accumbens, may constitute one of the mechanisms for the development of EA tolerance.

Effect of intracerebral microinjection of naloxone on acupuncture- and morphine-analgesia in the rabbit.

Although there are unequivocal evidences indicating the participation of endogenous opiate-like substances in acupuncture analgesia, their exact sites of action remain to be elucidated. From the results of localization studies by injecting minute amount of narcotic antagonist naloxone into discrete brain areas and assessing its effect on acupuncture analgesia in rabbits it is concluded that nuclei accumbens, amygdala, habenula and periaquaductal grey are the strategic sites for endogenous opioids to exert their analgesic effect. These brain areas are also of extreme importance for the realization of morphine analgesia.

Central neurotransmitters and acupuncture analgesia.

The role played by central neurotransmitters in acupuncture analgesia was evaluated by correlating neurochemical changes in central nervous system with the acupuncture effect, as well as modification of the acupuncture effects by pharmacological manipulations of central neurotransmitters. The results of experimental studies which were performed mainly on rats and rabbits indicated that central serotonin and endogenous opiate-like substances (OLS) seem to be the most important substrates for mediation of acupuncture analgesia while central catecholamines, especially norepinephrine through alpha receptors, may exert an antagonistic effect. It was also found that prolonged and repeated acupuncture resulted in a gradual decrease of the acupuncture effects. The development of some endogenous anti-opiate substrates (AOS) in central nervous system was tentatively implicated.