Hsiu-Ying T. Yang

Projections of substance P containing neurons from neostriatum to substantia nigra.

Substance P has been found to be most concentrated in the substantia nigra in hurnanlo~lz and rat brai+. Subcellular distribution studies have revealed that most of the substance P present in the hypothalamus and substantia nigra can be recovered in the nerve ending fractionss. However, precise information on the location of substance P cell bodies which project to substantia nigra is still lacking. We have attempted to obtain such information by making various types of brain lesion and determining the substance P content in substantia nigra and other brain regions. Sprague-Dawley rats weighing 175-225 g were used (from Allison Park, Pa.). Hemitransections of the brain with procedures similar to those described by McGeer et al.9 were performed at A 4380 pm, A 6860 pm, and A 7600 ym, according to Kanig and Klippel atlas7. Electrolytic lesions of globus pallidus were made stereotaxically by placing a temperature sensing probe at A 6.3; L 2.8; V -0.6 (according to Kiinig and Klippel atlas7) and raising the tissue temperature to 50 “C for 1 min by a radiofrequency lesion generator as described previously 5. Brain tissue was dissected and substance P concentration was determined by radioimmunoassay as reported befores.

Repeated electroconvulsive shocks and the brain content of endorphins.

Repeated electroconvulsive shocks (ECS) cause an increase of [met5]-enkephalin (ME) content in hypothalamus and some limbic areas such as n. accumbens, septum and amygdala. The temporal characteristics of this increase resemble the time course of the clinical effects of electroconvulsive therapy (ECT). Single shock failed to change the ME content in any part of the brain. After 6 daily shocks ME content increases by 60% in hypothalamus and maximal effect (100% increase) was obtained after 10 daily shocks. The increase of ME content persists 6 days after the termination of ECS. Subconvulsive shocks failed to alter the content of ME in all the brain gions determined. When the seizure induced by ECS was prevented by pretreatment with an anesthetic dose of phenobarbital, the ECS-induced increase in hypothalamic and septal ME content was also blocked. beta-Endorphin content of hypothalamus remained unchanged after repeated ECS despite a two-fold increase in the ME content in the same region. These observations indicate that the time constant for the increase in ME content induced by ECS resembles the time constant for the appearance of the clinical benefits by ECT and may give credence to the possibility that the ME increase may participate in the antidepressive action of ECS.

FMRF-NH2-like mammalian octapeptide: Possible role in opiate dependence and abstinence

Yang et al. have isolated from bovine brain an octapeptide, FLFQPQRF-NH2 (F-8-F-NH2), with certain antiopiate properties. Malin et al. previously found that ICV injection of this peptide could precipitate an opiate abstinence syndrome in dependent rats. RIA revealed significantly higher levels of F-8-F-NH2 immunoreactivity in CSF withdrawn from the cisterna magna of morphine-dependent rats as opposed to CSF withdrawn from sham-implanted controls. ICV infusion of IgG from antiserum against F-8-F-NH2 significantly reduced the number of abstinence signs subsequently precipitated by naloxone in morphine-dependent rats.

Proglumide prevents and curtails acute tolerance to morphine in rats

The antagonism of the antinociceptive action of morphine elicited by CCK-8-SO4 can be counteracted by proglumide, a CCK antagonist. The addition of morphine (10(-6)M) to the artificial spinal fluid perfusing the subarachnoidal space of rat spinal cord increases the CCK content of the perfusate. Proglumide can potentiate morphine analgesia without changing the half life of morphine. After seven to eight subcutaneous injections of morphine (4 mg/kg) repeated every two hrs there is tolerance to the antinociceptive action of morphine. Proglumide can partially block or reverse this acute tolerance to morphine.

Enkephalin in bovine adrenal gland: multiple molecular forms of [Met5]-enkephalin immunoreactive peptides.

Abstract The existence and heterogeneity of enkephalin-like substances in bovine adrenal glands has been demonstrated using enkephalin radioimmunoassays. Both [met5]- and [leu5]-enkephalins were found to be highly enriched in the medulla i.e. 6.65 pmol [met5]-enkephalin/mg protein but only a very small amount, 0.041 pmol [met5]-enkephalin/mg protein, was detected in the cortex. In addition to the pentapeptide, [met5]-enkephalin, a large proportion of the total [met5]-enkephalin immunoreactive substance was found by gel filtration chromatography to be present in two high molecular weight forms. Determination of the true concentration of the high molecular weight forms, however, awaits purification and use of a proper standard. The existence of the different molecular forms of [met5]-enkephalin immunoreactive substances was substantiated by immunoabsorption experiment and gel filtration chromatography with 6 M guanidine. The two high molecular weight immunoreactive substances were not assayable by the radioreceptor binding assay; however, when trypsinized, they released a product which was capable of binding to the opiate receptor and was indistinguishable from the pentapeptide. [met5]-enkephalin, with respect to its size and thin layer Chromatographic characteristic. Whether the high molecular weight [met5]-enkephalin immunoreactive substances can function as precursors of [met5]-enkephalin in adrenal glands remains to be determined.

Substance P-induced release of Met5-Enkephalin from striatal and periaqueductal gray slices

Substance P(SP), the heptapeptide SP and the stable analogue (p-Glu5-MePhe8-MeGly9) SP (DiMe-C7) induce a Ca2+-dependent release of Met5-enkephalin (MET) from slices of periaqueductal gray matter (PAG) and striatum of rats. The MET release from striatal slices is greater than that from PAG slices because of the higher MET content of striatum. Intraventricular injection of SP and of the two related peptides induce analgesia in the rat, and their analgesic potency is in line with their capacity to release MET. Other neuropeptides which possess antinociceptive activity such as bombesin, neurotensin, vasopressin and somatostatin fail to release MET from PAG slices.

Mammalian FMRF-NH2-like peptide in rat pituitary: Decrease by osmotic stimulus

Previous studies with the Brattleboro rat suggested a possible interaction at the pituitary level between AVP and the neuropeptide, F-8-F-NH2. In order to test this hypothesis, we studied the effect of various osmotic stimuli on neurohypophyseal F-8-F-NH2. In rats drinking 2% NaCl solution for two days, neural lobe AVP and F-8-F-NH2 levels were equally reduced by 87%. After maximal depletion, pituitary levels of F-8-F-NH2 and AVP rebounded in parallel when normal drinking water was reintroduced. Pituitary stalk transection depleted neurohypophyseal F-8-F-NH2. The results of this study suggest that neurohypophyseal F-8-F-NH2 originates from the hypothalamus and, furthermore, is coreleased along with AVP in response to hyperosmotic stimuli.

Conversion of [met5]-enkephalin-Arg6-Phe7 to [met5]-enkephalin by dipeptidyl carboxypeptidase.

Abstract [Met5]-Enkephalin-Arg6-Phe7 can be hydrolyzed by a microsomal preparation of bovine striatum, which contains dipeptidyl carboxypeptidase, to release [met5]enkephalin. This reaction was inhibited by 10−6 M SQ 20881 (PGlu-Gly-Leu-Pro-Pro-Gly-Pro-Pro-Ileu Pro-Prol) which has little or no effect in inhibiting the production of Tyr-Gly-Gly from [met5]-enkephalin. The conversion of [met5]-enkephalin-Arg-Phe7 to [met5]-enkephalin is not enhanced by 100 mM chloride ion.

The opioid octapeptide Met5-enkephalin-Arg6-Gly7-Leu8: characterization and distribution in rat spinal cord.

Abstract The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met5-enkephalin-Arg6-Gly7-Leu8 were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met5-enkephalin-Arg6-Gly7-Leu8 content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immunoreactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met5-enkephalin-Arg6-Gly7-Leu8 was further identified to be authentic Met5-enkephalin-Arg6-Gly7-Leu8 on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met5-enkephaline and Met5-enkephalin-Arg6-Phe7 indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met5-enkephalin-Arg6-Gly7-Leu8 in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.

Distribution and Characterization of Neuropeptide FF-like Immunoreactivity in the Rat Nervous System with a Monoclonal Antibody

Monoclonal antibodies against neuropeptide FF were produced and characterized. The antibodies are directed and highly specific to neuropeptide FF, and reactivity requires the C‐terminal dipeptide of neuropeptide FF (Arg‐Phe‐NH2). Tissue extracts from bovine spinal cord, rat spinal cord and hypothalamus were analysed by high‐pressure liquid chromatography coupled with radioimmunoassay using the characterized monoclonal antibody. Only one immunoreactive peptide was detected and it coeluted with authentic neuropeptide FF. Using this highly specific monoclonal antibody, the distribution of neuropeptide FF‐like immunoreactivity was further studied by indirect immunohistochemistry. Immunoreactivity was seen in two major cell groups in the rat brain. The largest cell group was located in the medial hypothalamus between the dorsomedial and ventromedial nuclei. The other one was found in the nucleus of the solitary tract. Fibres immunoreactive for neuropeptide FF were located in the lateral septal nucleus, amygdala, different hypothalamic areas, nucleus of the solitary tract, ventral medulla, trigeminal complex and the dorsal horn of the spinal cord. Spinal and sympathetic ganglia were non‐reactive. No neuropeptide FF immunoreactivity was seen in the gut autonomic nervous system or endocrine cells. The results show that neuropeptide FF‐like immunoreactivity has a clearly more limited distribution in the nervous system than typical brain–gut peptides.