Origin of neurohypophyseal neuropeptide-FF (FLFQPQRF-NH2).

Neuropeptide-FF (FLFQPQRF-NH2), originally isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. Neuropeptide-FF (NPFF) is unevenly distributed in the central nervous system, with the highest concentrations in posterior pituitary and spinal cord. In the rat pituitary, NPFF is found exclusively in the neural lobe, where it is localized in nerve terminals and fibers, indicating the hypothalamus as a possible source of the neural lobe NPFF. In this study the origin of neurohypophyseal NPFF was investigated using various hypothalamic lesions and an anterograde tracing experiment. The results suggest that at least part of the neurohypophyseal NPFF originates from the supraoptic nucleus and may be localized in some of the arginine vasopressin-containing magnocellular neurons.

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.

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.

FMRF-NH2-like peptide is deficient in the pituitary gland of the Brattleboro rat.

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2), isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. In the rat, F-8-F-NH2 immunoreactivity (IR) is highly localized in the neurohypophysis. In this study, F-8-F-NH2-IR was studied in the hypothalamo-neurohypophyseal system of an Arg8-vasopressin (AVP)-deficient animal, the Brattleboro (DI) rat, and the normal control Long-Evans (LE) strain. F-8-F-NH2-IR in the DI pituitary is below the level of detection in contrast to that in the LE (0.50 +/- 0.04 pmol/gland). Neuropeptide Y (NPY) levels are increased two-fold in the DI pituitary while AVP levels are below detection. The content of F-8-F-NH2-IR in the hypothalami and spinal cords of DI and LE rats is not statistically different, suggesting that the absence of F-8-F-NH2-IR in the Brattleboro pituitary is not due to a genetic defect in F-8-F-NH2 biosynthesis. The results of this study raise the question whether AVP could be involved in the regulation of F-8-F-NH2 immunoreactivity in the neurohypophysis.

Immunohistochemical distribution and partial characterization of FLFQPQRFamidelike peptides in the central nervous system of rats.

Two peptides that are structurally related to the molluscan tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRF-NH2) were recently isolated from bovine brain extract (Yang et al.: Proc. Natl. Acad. Sci. USA 82:7757-7761, '85). These peptides have an attenuating effect on morphine-induced analgesia when injected intracerebroventricularly in rats. Antisera against the two peptides--an octapeptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F8F-NH2), and an octadecapeptide, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 (A18F-NH2)--were raised in rabbits and characterized with standard radioimmunoassay and immunohistochemical blocking controls. This study was aimed at localizing neurons in the rat brain that contain immunoreactivity for these peptides. Cryostat sections of normal and colchicine-treated Sprague-Dawley rat brains were incubated with the specific antisera and the immunoreactivity was visualized by the PAP or the FITC method. Immunoreactive neurons were detected in the hypothalamus and nucleus of the solitary tract. Relatively dense networks of fibers and terminals were observed in the lateral parabrachial nucleus and in the nucleus of the solitary tract. Fibers and terminals were also seen in the lateral septum, amygdala, hypothalamus, neurohypophysis, thalamus, periaqueductal gray, and several medullary nuclei. In these areas the immunoreactivity was abolished when the antisera were incubated with the corresponding synthetic peptides F8F-NH2 and A18F-NH2. Preincubation of the antisera with neuropeptide Y (NPY) did not affect the staining. The results suggest that there are neurons in the rat brain that contain F8F-NH2- and A18F-NH2-like peptides. The neuronal system described here may have a role in the regulation of autonomic functions, such as hypothalamic control and respiratory functions. The neuronal connections of the cells containing F8F-NH2- and A18F-NH2-like peptides remain to be studied.

Rat brain regional distribution and spinal cord neuronal pathway of FLFQPQRF-NH2, a mammalian FMRF-NH2-like peptide.

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-NH2) is a peptide, originally detected by FMRF-NH2 antisera, and subsequently isolated from bovine brain. Using a specific radioimmunoassay for F-8-F-NH2, we have examined the regional distribution and characteristics of F-8-F-NH2 immunoreactivity (IR) in rat brain, spinal cord and pituitary gland. In CNS, F-8-F-NH2-IR is highly concentrated in the spinal cord, hypothalamus and pons-medulla (368, 202 and 136 fmol per mg protein, respectively); lowest values are in the cortex and hippocampus. A modest rostrocaudal gradient of F-8-F-NH2-IR was observed; levels in the sacral cord are 50% higher than in the cervical cord. Dorsal cord content is 8 times higher than in the ventral cord. Dorsal rhizotomy failed to change F-8-F-NH2-IR in the affected regions of the spinal cord while significantly reducing substance P levels. F-8-F-NH2-IR was significantly decreased caudal to a spinal transection, indicating the presence of a descending pathway within the spinal cord. The highest concentration of F-8-F-NH2-IR (1008 fmol per mg protein) was found in the neurointermediate lobe of the pituitary, while no F-8-F-NH2-IR could be detected in the anterior lobe. Immunohistochemically, F-8-F-NH2-IR was confined to nerve terminal-like structures in the neural lobe. The anterior and intermediate lobes were devoid of immunoreactive structures. HPLC characterization of F-8-F-NH2-IR in the dorsal spinal cord, medulla-pons and pituitary revealed one major immunoreactive peak which is more hydrophobic than bovine F-8-F-NH2. In addition to this material, the hypothalamus was found to contain another, more abundant F-8-F-NH2-immunoreactive peak. Analysis of F-8-F-NH2-IR from posterior pituitary with various antisera having differing affinities for F-8-F-NH2 and gamma 1-MSH indicates that the F-8-F-NH2-IR of rat pituitary is not due to gamma 1-MSH. The high concentration of F-8-F-NH2-like peptide in the dorsal spinal cord supports a role in mediating nociceptive transmission while the localization of F-8-F-NH2-IR in the posterior pituitary suggests an additional autonomic or endocrine function.

Biochemical characterization of FMRF-NH2-like peptides in spinal cords of various mammalian species using specific radioimmunoassays.

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A-18-F-NH2), originally detected by FMRF-NH2 antiserum and subsequently isolated from bovine brain, were found to be highly localized in the bovine spinal cord. Using specific radioimmunoassays coupled with HPLC, F-8-F-NH2 and A-18-F-NH2 immunoreactivities in spinal cord of bovine, rat, mouse, guinea pig and human were studied. One major F-8-F-NH2 immunoreactivity was detected in the spinal cord of every species except in human, however, the retention time of F-8-F-NH2 immunoreactivity appears to vary from species to species. In the human spinal cord three major F-8-F-NH2 immunoreactivities are detected and one of them was eluted in the position of F-8-F-NH2. Two major A-18-F-NH2 immunoreactivities were detected in every species except guinea pig; one of these immunoreactivities can be identified as F-8-F-NH2 immunoreactivity due to the high affinity of the A-18-F-NH2 antiserum to F-8-F-NH2. F-8-F-NH2 and A-18-F-NH2 immunoreactivities can also be clearly detected by FMRF-NH2 antiserum, however, the quantities of these peptides can be grossly underestimated by the FMRF-NH2 RIA. These results confirm that there is a novel system of FMRF-NH2-like peptides in mammalian CNS and some of them are more closely related to the bovine peptides, F-8-F-NH2 and A-18-F-NH2 than to FMRF-NH2.

Neuropeptide Y release from the adrenal medulla after cholinergic receptor stimulation.

Peripheral nerves as well as chromaffin cells have been shown to contain neuropeptide Y (NPY), a 36 amino acid peptide that exhibits potent stimulatory actions on vascular smooth muscle. Because NPY-like immunoreactive material (NPY-IR) is stored in chromaffin granules we chose to investigate, using the retrogradely perfused bovine adrenal gland, whether nicotinic cholinergic receptor stimulation results in the secretion of this material. In addition we monitored the release of [Met5]-enkephalin-like immunoreactive material (ME-IR), which has been demonstrated to be released from the bovine adrenal medulla. NPY-IR and ME-IR were measured using antisera prepared from hemocyanin conjugates of the peptides. Increasing concentrations of acetylcholine (1 X 10(-6) M-5 X 10(-5) M), infused in the presence of 1 X 10(-5) M physostigmine, produced significant increases in NPY-IR and ME-IR release. Hexamethonium (5 X 10(-4) M) antagonized the release of both of these substances, whereas atropine (5 X 10(-7) M) did not. The role of the nicotinic cholinergic receptor in this process was established further by examining the effects of 1,1-dimethyl-4-phenylpiperazinium. Increasing concentrations of 1,1-dimethyl-4-phenylpiperazinium (1 X 10(-6) M-5 X 10(-5) M) also stimulated the release of NPY-IR and ME-IR. Analysis of the perfusate by reverse phase liquid chromatography revealed the presence of four peaks of NPY-IR. A major peak coeluted with authentic NPY. These data demonstrate that NPY-IR can be released through stimulation of the nicotinic cholinergic receptor. The adrenal medulla appears to be one of the sources for circulating NPY-IR.

Distribution and characterization of two putative endogenous opioid antagonist peptides in bovine brain.

Highly sensitive radioimmunoassays were developed and used in studies of the distribution and chromatographic properties of two mammalian FMRF-NH2-like peptides recently isolated from bovine brain; an octapeptide with the structure Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and on octadecapeptide, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A-18-F-NH2). F-8-F-NH2 and A-18-F-NH2 immunoreactivities are unevenly distributed in bovine brain. The highest concentrations (pmol g-1) of F-8-F-NH2 and A-18-F-NH2 are found in dorsal spinal cord (9.8 and 16.4 respectively), periaqueductal grey (8.6 and 6.8) and pons medulla (7.0 and 8.9); lowest quantities are in cortex, cerebellum and striatum. HPLC analysis coupled with radioimmunoassay reveals that the major immunoreactivities are identical to synthetic F-8-F-NH2 and A-18-F-NH2 while there are additional immunoreactive materials, distinct from NPY, whose structures still remain to be determined. The enrichment of these peptides in dorsal cord and periaqueductal grey, areas important in opioid-mediated pain perception, suggest that they may play a role in mediating antinociception.

Elevation of arterial pressure in rats by two new vertebrate peptides FLFQPQRF-NH2 and AGEGLSSPFWSLAAPQRF-NH2 which are immunoreactive to FMRF-NH2 antiserum.

We found that two recently characterized neuropeptides Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A-18-F-NH2) elevate mean arterial blood pressure (MAP) in conscious, unrestrained rats. The pressor activities of both agents were attenuated, but not abolished, by prior treatment with guanethidine or prazosin. These results suggest that F-8-F-NH2 and A-18-F-NH2 elevate MAP in rats by potentiating the release of catecholamines and by mechanisms independent of catecholamine release.

Neuroanatomy of morphine-modulating peptides.

Antisera against two mammalian peptides related to the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH2 were used to locate immunoreactive neurons in the rat brain, nerve fibres and terminals in the spinal cord, sympathetic ganglion cells and adrenal chromaffin cells. Immunoreactivity for the newly characterised octa- and octadecapeptide was detected in nerve cell bodies in the hypothalamic area, including parts of the dorsomedial, periventricular and paraventricular nuclei, and in the nucleus tractus solitarii. Nerve terminals in the superficial laminae of the spinal cord were also immunoreactive for these peptides, while the sensory ganglia were nonreactive. Some principal ganglion cells in the superior cervical ganglia exhibited bright immunofluorescence for the peptides, and a few adrenal medullary cells were immunoreactive. The presence of these peptides in the substantia gelatinosa of the spinal cord suggests that they may be involved in sensory neurotransmission, especially in the mechanisms mediating pain. In the hypothalamo-hypophysial system these peptides may be involved in the regulation of hormonal systems. They may also act as co-transmitters in the sympathetic nervous system.

Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine.

Two peptides that crossreact with an antiserum raised against Phe-Met-Arg-Phe-NH2 were purified from bovine brain extract. Their structures were determined to be Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 and Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2. The sequences were determined by gas-phase sequencing, except for the COOH-terminal phenylalaninamides. These were assigned on the basis of the reactivity of the peptides with the anti-Phe-Met-Arg-Phe-NH2 antiserum, which appears to recognize the determinant -Arg-Phe-NH2. Both peptides were synthesized, and the synthetic peptides were found to have the same HPLC retention times as the endogenous Phe-Met-Arg-Phe-NH2-immunoreactive peptides, thus confirming the assignment of phenylalaninamide to the COOH-terminal positions. Both of the synthetic peptides were found to decrease tail-flick latency in rats, and the octapeptide was more active than the octadecapeptide. The octapeptide was found also to attenuate the prolongation of the tail-flick latency induced by morphine.

Neuropeptide Y in bovine adrenal glands: distribution and characterization.

Neuropeptide Y (NPY) distribution in bovine adrenal glands was studied by RIA and immunohistochemical technique. NPY content (picomoles per mg protein +/- SEM) of chromaffin cells, medulla, cortex, and whole glands was 4.2 +/- 0.16, 2.7 +/- 0.28, 0.19 +/- 0.02, and 0.94 +/- 0.14, respectively, while the chromaffin granule NPY content was 53. Immunohistochemically, NPY immunoreactivity was detected in norepinephrine containing chromaffin cells and also in nerve fibers crossing through adrenal cortex and medulla. NPY and enkephalin immunoreactivities were found in distinct chromaffin cells. Biochemical characterization by HPLC revealed two major NPY immunoreactive peptides. The most abundant molecular form was identified as authentic NPY.

Release of NPY-like immunoreactive material from primary cultures of chromaffin cells prepared from bovine adrenal medulla.

In primary cultures of bovine chromaffin cells, the cell contents of NPY immunoreactive material was found to be stable for about 4 days. When these cells were transferred to the Locke's solution from the culture medium, NPY immunoreactive material was released spontaneously. Addition of potassium (45 mM) resulted in a 3-fold increase in the rate of NPY release in a calcium dependent fashion. NPY immunoreactivity in chromaffin cells is composed of NPY, a lesser immunoreactive peptide and two very minor immunoreactivities. The same immunoreactive peptides were released by 45 mM potassium.

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

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 immuno-reactivity 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-enkephalin 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 of Met5-enkephalin-Arg6, Phe7 in rat spinal cord.

The distribution of Met5-enkephalin-Arg6, Phe7 (YGGFMRF) in rat spinal cord was determined by a specific RIA and compared with that of Met5-enkephalin. The concentration of YGGFMRF on a per mg protein basis was highest in sacral cord and successively decreased in more rostral segments. A similar rostro-caudal distribution was observed for Met5-enkephalin. Regional microdissection revealed the dorsal grey matter to be highest in YGGFMRF content followed by ventral gray, ventral white and dorsal white matter; a similar pattern was observed for Met5-enkephalin. The ratio of Met5-enkephalin: YGGFMRF concentration was 5.4 +/- 0.15 (S.E.M.) on average in all regions measured, indicating a very close quantitative relationship between the two molecules. Our data suggest that YGGFMRF may act as a precursor of or cotransmitter with Met5-enkephalin in spinal cord tissue.