Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.     

Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species

Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met⁵]-enkephalin-Arg⁶-Gly⁷-Leu⁸. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu⁵]- enkephalin at the C-terminus.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum

An antiserum was generated against a synthetic peptide corresponding to amino acids 95-117 of bovine proenkephalin, and a sensitive radioimmunoassay was developed. Comparison of the reactivities of the synthetic peptide, its specific cleavage products, and other synthetic peptides showed that the important immunological determinant was contained within residues 101-109 of bovine proenkephalin (-Gly-Gly-Glu-Val-Leu-Gly-Lys-Arg-Tyr-). Radioimmunoassay of fractions after gel filtration of bovine adrenal medullary chromaffin granule lysate showed three pools of immunoreactivity: pool 1 (Mr 20,000-30,000), pool 2 (Mr 10,000-20,000), and pool 3 (Mr approximately 5,000). Further characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that the antiserum recognized 27-, 20.5-, 16.5-, and 5.6-kilodalton enkephalin-containing proteins. The radioimmunoassay was also used to detect proenkephalin-like material in extracts of rat adrenal and regions of rat brain and spinal cord following gel filtration. Immunoreactivity from the rat adrenal chromatographed predominantly as high molecular weight material (Mr 31,500-43,500), whereas material in regions of rat brain showed a broader molecular weight distribution (Mr 4,000-43,500). This indicated differences in the processing of proenkephalin between rat adrenal and brain tissue. Differences were also seen in the molecular weight profile of immunoreactivity in different brain regions, most noticeable in the case of striatum and hypothalamus, suggesting regional differences in processing. Based on quantitation of higher molecular weight immunoreactive proenkephalin-like material and free Met-enkephalin immunoreactivity in different brain regions, it was apparent that extensive processing of proenkephalin occurs in brain. We concluded that antisera against proenkephalin-(95-117) recognize a wide range of intermediates in the processing of proenkephalin in both bovine adrenal medulla and rat adrenal, brain, and spinal cord, making it a useful tool for further studies concerned with the expression and post-translational processing of proenkephalin.     

Synenkephalin in bovine and human spinal cord

Summary Synenkephalin, which comprises 70 residues at the aminoterminal of proenkephalin, was studied with immunocytochemical methods in the human and bovine spinal cord. Immunoreactive fibers had the same general distribution as methionine-enkephalin, but not as leucine-enkephalin fibers. They were found in all spinal layers and were most numerous in lamina II (outer zone) and V–VI (lateral portion). Synenkephalin immunoreactivity was overall less dense than that of the enkephalins. These results suggest that proenkephalin is the precursor protein also in enkephalinergic neurons of the human spinal cord.     

Distribution and characterization of synenkephalin immunoreactivity in the bovine brain and pituitary

The distribution of synenkephalin, the N-terminal fragment of proenkephalin, was studied in various parts of the bovine brain (globus pallidus, caudate nucleus, hypothalamus) and in the posterior pituitary by the use of a radioimmunoassay. The distribution of synenkephalin-immunoreactivity (IR) was compared to the distribution of Met-enkephalin-IR. Gel exclusion chromatography was used to examine the molecular forms of the immunoreactivities present in the tissues. The distribution of synenkephalin-IR was similar to the distribution of Met-enkephalin-IR, with a molar ratio of Met-enkephalin/synenkephalin ranging between 2.7 and 5.9. In all regions tested except the hypothalamus the synenkephalin-IR was present as a single species. However, in the hypothalamus a small amount of IR material (3% of the total synenkephalin-IR) was detected in fractions where larger Met-enkephalin-containing peptides eluted. Based on the concordance between the molar ratio of Met-enkephalin to synenkephalin found in the tissues and the molar ratio present in the sequence of adrenal proenkephalin, it is concluded that the brain and adrenal glands utilize a similar precursor for enkephalin biosynthesis.     

Mitogenic activation of the human lymphocytes induce the release of proenkephalin derived peptides

Several reports indicate that enkephalins participate in lymphocyte proliferation and several events of the immune response. It has been proposed that peptides involved in these processes may originate in the nervous system or endocrine glands. We have found that human peripheral blood lymphocytes (PBL) activated with a mitogenic agent contain and release proenkephalin derived peptides. The kinetics of met-enkephalin and cryptic products of proenkephalin in PBL activated with phytohemaglutinin (PHA) were studied. Peptides were released to the supernatant of stimulated PBL, reaching the highest values after 18 to 24 hours. The material secreted corresponds to high, intermediate and low molecular weight peptides derived from proenkephalin, displaying met-enkephalin and synenkephalin (proenkephalin 1-70) immunoreactivity. Therefore, an intrinsic lymphocytic proenkephalin system is induced by PHA and may play an important role in the regulation of the immune response.     

Glutamic acid decarboxylase (GAD)-like immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary A substance immunologically related to vertebrate glutamic acid decarboxylase (GAD) has been visualized in the pedal ganglion of Mytilus with the pre-embedding peroxidase-antiperoxidase method, by use of an antiserum raised in sheep against rat brain GAD. The results show that GAD-like immunoreactivity is present both in neuronal perikarya and in nerve fibers. Positive neurons are located mainly among the fibers of the ganglion neuropil at the commissural level, and more rarely close to unreactive cortical cell bodies. Immunoreactive nerve fibers are observed throughout the neuropil and also in cerebropedal and pedal nerves.Supported by Ministero Pubblica Istruzione (40%)     

Development of region-specific antisera for the C-terminal tetrapeptide of gastrin/cholecystokinin and their use in studies of immunoreactive forms of cholecystokinin in rat brain

Molecular forms of cholecystokinin in rat brain were studied by radioimmunoassay using two new antisera raised against the C-terminal tetrapeptide common to cholecystokinin and gastrin. Evidence is presented to show that one antiserum (L112) reacts at the C-terminus of the tetrapeptide, while the other antiserum (L131) reacts at its N-terminus. With antiserum L112 the predominant immunoreactive form of CCK found in extracts of rat brain corresponded to the C-terminal octapeptide; a minor immunoreactive form eluted from Sephadex G25 between the C-terminal octapeptide and the tetrapeptide. A similar pattern of molecular forms was found using a third antiserum (L48) previously shown to react well with the C-terminal octapeptide and poorly with the C-terminal tetrapeptide. Antisera L112 and L48 also revealed a quantitatively similar distribution of immunoreactive material in different regions of rat and cow brain. In contrast, antiserum L131 failed to demonstrate significant amounts of immunoreactive material in rat brain. It is concluded that the C-terminal octapeptide of cholecystokinin predominates in rat brain and that contrary to findings of previous workers there is little or no free C-terminal tetrapeptide present.     

Immuohistochemical demonstration of alpha-atrial natriretic plypeptide-containing neurons in the rat brain

Using the immunoperoxidase technique in conjunction with specific antisera to alpha-atrial natriuretic polypeptide (alpha-ANP), it was shown that immunoreactive cell bodies and varicose fibers are widely distributed throughout the rat brain. The highest concentrations of alpha-ANP-containing neuronal cell bodies and fibers were found in the hypothalamus and septum. This result confirms the radioimmunological determination of alpha-ANP immunoreactivity in the rat brain.     

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in the rat and mouse pituitary gland.

The distribution of the octapeptide Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephalin A-derived opioid peptide, in the rat and mouse pituitary gland was studied using the indirect immunofluorescence technique and immunoelectron microscopy. The anterior lobe contained a few MEAGL-immunoreactive cells but no nerve fibers. A previously unknown enkephalin-immunoreactive nerve fiber system was revealed in the intermediate lobe. These fibers originated in a dense MEAGL-immunoreactive plexus located along the border between the intermediate and posterior lobes and were distributed throughout the lobe. In the posterior lobe, MEAGL immunoreactivity was found in a very dense network of varicose fibers that was evenly distributed over the entire lobe. These results provide a morphological correlate for previous chemical studies and together with them suggest that MEAGL-immunoreactive innervation regulates endocrine functions of the intermediate and posterior lobes directly at the pituitary level.     

Histamine-immunoreactive cells in the superior cervical ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary Using the immunoperoxidase technique in conjunction with specific antisera to -atrial natriuretic polypeptide (-ANP), it was shown that immunoreactive cell bodies and varicose fibers are widely distributed throughout the rat brain. The highest concentrations of -ANP-containing neuronal cell bodies and fibers were found in the hypothalamus and septum. This result confirms the radioimmunological determination of -ANP immunoreactivity in the rat brain.     

Detection of Synenkephalin, the Amino-Terminal Portion of Proenkephalin, by Antisera Directed Against Its Carboxyl Terminus

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.     

Visualization and quantification of central 5-HT1A receptors with specific antibodies

Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT_1A receptor. Both antisera allowed the immunoprecipitation of 5-HT_1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of 63 kDa, corresponding to the molecular weight of the rat 5-HT_1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT_1A sites labelled by selective radioligands such as [³H]8-OH-DPAT and [¹²⁵I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT_1A receptor binding subunits with G proteins from one brain area to another.     

Localization of Endomorphin-2-Like Immunoreactivity in the Rat Medulla and Spinal Cord

Martin-Schild, S., J. E. Zadina, A. A. Gerall, S. Vigh and A. J. Kastin. Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord. Peptides 18(10) 1641–1649, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are endogenous ligands that have greater affinity and selectivity for the μ-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin–biotin–peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing μ receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.     

Cholecystokinin octapeptide (CCK 26–33), nonsulfated octapeptide and tetrapeptide (CCK 30–33) in rat brain: Analysis by high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA)

Cholecystokinin carboxyterminal octa- and tetrapeptide concentrations have been measured in rat brain by a combination of high pressure liquid chromatography and radioimmunoassay. The sulfated octapeptide is the predominant form of Cholecystokinin in rat brain (approx. 80%). The concentration of the tetrapeptide represents 5–10% of that of the sulfated octapeptide in molar terms, depending upon the brain region. In addition to the tetrapeptide, a peptide with the properties of Cholecystokinin octapeptide in its nonsulfated form could be detected in concentrations similar to those of the tetrapeptide.     

Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the major salivary glands of the rat: evidence for both sympathetic and parasympathetic origin

Summary The localization of the proenkephalin A-derived octapeptide, Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), was studied in the major salivary glands of Sprague-Dawley and Wistar rats with the indirect immunofluorescence method. MEAGL-immunoreactive nerve fibers were found around the acini, along intra-and interlobular salivary ducts and in close contact with blood vessels. In the parotid and submandibular glands tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibers around the acini, in association with intra- and interlobular salivary ducts and around blood vessels, while in the sublingual gland TH-immunoreactive nerve fibers were only seen around blood vessels. Parasympathetic neurons in submandibular ganglia contained MEAGL immunoreactivity. Moderate TH immunoreactivity was seen in some neurons of the submandibular ganglia. A subpopulation of sympathetic principal neurons in the superior cervical ganglion were immunoreactive for both MEAGL and TH. In the trigeminal ganglion, no MEAGL-immunoreactive sensory neurons or nerve fibers were observed. Superior cervical ganglionectomies resulted in a complete disappearance of TH-immunoreactive nerve fibers, while MEAGL-immunoreative nerve fibers were still present in the glands. The presence of MEAGL immunoreactivity in neurons of both sympathetic superior cervical ganglia and parasympathetic submandibular ganglia and the results of superior cervical ganglionectomies suggest, that MEAGL-immunoreactive nerve fibers in the major salivary glands of the rat have both sympathetic and parasympathetic origin.     

Comparative immunocytochemical localization of putative opioid ligands in the central nervous system

We report a detailed comparative immunocytochemical mapping of enkephalin, CCK and ACTH/beta-endorphin immunoreactive nerves in the central nervous system of rat and guinea pig. Enkephalin immunoreactivity was detected in many groups of nerve cell bodies, fibers and terminals in the limbic system, basal ganglia, hypothalamus, thalamus, brain stem and spinal cord. beta-endorphin and ACTH immunoreactivity was limited to a single group of nerve cell bodies in and around the arcuate nucleus and in fibers and terminals in the midline areas of the hypothalamus, thalamus and mesencephalic periaqueductal gray with lateral extensions to the amygdaloid area. Cholecystokinin immunoreactive nerve fibers and terminals displayed a distribution similar to that of enkephalin in many regions; but striking differences were also found. An immunocytochemical doublestaining technique, which allowed simultaneous detection of two different peptides in the same tissue section, showed that enkephalin-, CCK- and ACTH/beta-endorphin-immunoreactive nerves although closely intermingled in many brain areas, occurred separately. The distributions of nerve terminals containing these neuropeptides showed striking overlaps and also paralleled the distribution of opiate receptors. This may suggest that enkephalin, CCK, ACTH and beta-endorphin may interact with each other and with opiate receptors.     

Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system

With the use of an antiserum generated in rabbits against synthetic human calcitonin gene-related peptide (CGRP) the distribution of CGRP-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system. A detailed stereotaxic atlas of CGRP-like neurons was prepared. CGRP-like immunoreactivity was widely distributed in the rat central nervous system. CGRP positive cell bodies were observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, medial forebrain bundle), premamillary nucleus, amygdala medialis, hippocampus and dentate gyrus, central gray and the ventromedial nucleus of the thalamus. In the midbrain a large cluster of cells was contained in the peripeduncular area ventral to the medial geniculate body. In the hindbrain cholinergic motor nuclei (III, IV, V, VI, VII XII) contained CGRP-immunoreactivity. Cell bodies were also observed in the ventral tegmental nucleus, the parabrachial nuclei, superior olive and nucleus ambiguus. The ventral horn cells of the spinal cord, the trigeminal and dorsal root ganglia also contained CGRP-immunoreactivity. Dense accumulations of fibers were observed in the amydala centralis, caudal portion of the caudate putamen, sensory trigeminal area, substantia gelatinosa, dorsal horn of the spinal cord (laminae I and II). Other areas containing CGRP-immunoreactive fibers are the septal area, nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus tractus solitarii, and in the confines of clusters of cell bodies. Some fibers were also noted in the anterior and posterior pituitary and the sensory ganglia. As with other newly described brain neuropeptides it can only be conjectured that CGRP has a neuroregulatory action on a variety of functions throughout the brain and spinal cord.     

Immunoreactive peptides related to dynorphin B (=rimorphin) in the rat brain

We have developed a radioimmunoassay for synthetic dynorphin B, a novel opioid tridecapeptide, which shares a common precursor molecule with dynorphin_1–17 (=dynorphin A) and the neo-endorphins. The levels of immunoreactivity towards this peptide in rat brain and pituitary show a pattern quantitatively and qualitatively similar to those found for dynorphin A and -neo-endorphin in earlier studies. The antiserum used was highly specific with only dynorphin-32 and dynorphin B-29, both of which contain the dynorphin B sequence, showing substantial cross-reactivity. Gel filtration of whole rat brain extracts in combination with HPLC analysis provide strong evidence for the existence of these latter two peptides in rat brain.