Pro-enkephalin gene derived peptides in the porcine stomach: cellular distribution and molecular forms

Antisera to the enkephalin variants Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 have been used in immunohistochemistry and radioimmunoassay studies of hog stomach. In the mucosa of the antrum, but not fundus, there was identified a population of immunoreactive endocrine-like cells. When extracts of antral mucosa were fractionated by gel filtration on Sephadex G50 the predominant immunoreactive forms of Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 were found to elute before the standards, and were compatible with N-terminally extended variants. In the muscle layers of both antrum and fundus, immunoreactive nerve fibers were found, these were especially numerous in the myenteric plexus. In extracts of the antral muscle, 50-60% of both Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 immunoreactivity eluted in the position of the standards and the remainder had the properties of N-terminally extended variants. In the fundus muscle the variants accounted for 70-80% of total activity. The results indicate that the proenkephalin gene is expressed in neurones and endocrine cells of the hog stomach. The different patterns of molecular forms found in different regions of the stomach suggest that the precursor is processed by different pathways in different populations of endocrine cells and neurones.     

Identification by radioimmunoassay and HPLC of morphine modulatory peptide-immunoreactivity in rat spinal cord and brain

Two so-called morphine modulatory peptides, an octapeptide and an octadecapeptide, have recently been isolated from bovine spinal cord. We have raised antibodies to the octapeptide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH₂: FF-8), which in radioimmunoassay react with peptides terminating in Arg-Phe-NH₂. This dipeptide is common to both the morphine modulatory peptides and the molluscan neuropeptide FMRF amide. The distribution and molecular forms of immunoreactive peptides were examined in the rat central nervous system and gastrointestinal tract. Highest concentrations of FF-8-like immunoreactivity were found in the dorsal spinal cord, brain stem and hypothalamus. The immunoreactive material in central nervous system extracts was resolved by reversed phase HPLC into three peaks of activity, the two largest peaks eluted in similar positions to the standard octapeptide and octadecapeptide. It appears that previously observed FMRF amide-like immunoreactivity in the rat central nervous system corresponds to peptides immunochemically and chromatographically similar to the two bovine spinal cord peptides.     

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.     

Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum

Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.     

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.     

Novel Opioid Peptide Amidorphin: Characterization and Distribution of Amidorphin-Like Immunoreactivity in Bovine, Ovine, and Porcine Brain, Pituitary, and Adrenal Medulla

We have recently isolated from bovine adrenal medulla a novel C-terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species, authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N-terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high-molecular-weight form of amidorphin was detected. These findings are indicative of a tissue-specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C-terminal fragments in the brain and pituitary.     

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.     

Corticotropin releasing factor (CRF)-like immunoreactivity in the hypothalamus and posterior pituitary of the goat, bovine, rat, monkey and human

Goat hypothalamic extract prepared by HCl extraction and chromatographed on a Sephadex G-50 column showed two immunoreactive CRF peaks. Most of the immunoreactivity coeluted with synthetic ovine CRF, and a small peak eluted near the void volume. Bovine, monkey, rat and human hypothalamic extracts prepared by acid-acetone or acid-methanol extraction showed three immunoreactive peaks. Most of the immunoreactivity coeluted with ovine CRF, and other smaller peaks eluted near the void volume and slightly before arginine vasopressin. Goat hypothalamic extract showed the highest cross-reactivity with anti-ovine CRF serum, followed by bovine hypothalamic extract. Less cross-reactivity was found in human, rat and monkey hypothalamic extracts. CRF immunoreactivity in goat hypothalamic extract coeluted with ovine CRF on reversed phase high performance liquid chromatography (HPLC) and main CRF immunoreactivity in human and rat hypothalamic extracts eluted slightly later than ovine CRF. These results suggest that there is a heterogeneity among the CRF molecules in these species and that goat CRF may be more similar to that of sheep CRF and the amino acid sequence or molecular weight of other animals CRF may be different from that of sheep CRF. The monkey posterior pituitary and rat neurointermediate lobe showed similar elution patterns of CRF immunoreactivity to their hypothalamic extracts on Sephadex gel filtration and HPLC. These results indicate that the posterior pituitary contains a similar CRF to hypothalamic CRF.     

Regional distribution and in vivo release of tachykinin-like immunoreactivities in rat brain: evidence for regional differences in relative proportions of tachykinins

The regional distribution of various forms of tachykinin-like immunoreactivity (TKLI) was studied in rat brain using radioimmunoassay. TKLI was measured with two different tachykinin-antisera (K12 and E7), which react with neurokinin A (NKA) and neurokinin B (NKB) but not with substance P (SP) and with a specific SP-antiserum. TKLI-K12 and TKLI-E7 were found to have similar regional distributions which were, however, significantly different from that of the substance P-like immunoreactivity (SPLI). Thus, the ratio of the tissue concentrations of TKLI-K12 or TKLI-E7 to that of SPLI was higher in frontal cortex and hippocampus and lower in pons/medulla oblongata than in the other regions studied. Cation-exchange chromatography of neutral water extracts of brain tissue revealed two major immunoreactive components of TKLI-K12 and TKLI-E7, one of which co-eluted with synthetic NKB while the other appeared in the same region as synthetic NKA. The relative quantities of these components varied depending on the brain region studied. No TKLI-K12 or TKLI-E7 co-eluted with synthetic SP. Almost all of the SPLI in acetic acid or water extracts of brain tissue eluted as a single chromatographic component in the same position as synthetic SP. Potassium-stimulated in vivo release of TKLI-K12, TKLI-E7 and SPLI in striatum of rat brain could be demonstrated using intracerebral dialysis. The present results imply that tachykinins, which may serve as neurotransmitters or neuromodulators, are present in different proportions in different regions of rat brain.     

Molecular variants of vasoactive intestinal polypeptide in dog, rat and hog

Molecular forms of vasoactive intestinal polypeptide (VIP) have been examined in the gut and brain of dog, rat and hog. Fractionation of acid extracts on CM-Sephadex revealed three components cross-reacting in a radioimmunoassay using an amino-terminal specific antiserum. One of the components was compatible with standard porcine octacosapeptide VIP, the other two eluted earlier and are so likely to be less positively charged peptides. However, after gel filtration on Sephadex G50, the same peaks of activity eluted in a similar position to porcine VIP indicating similar molecular size. There were marked species differences in the distribution of the different molecular forms. For example, in both muscle and mucosal layers of the rat intestine 50–90% of total immunoreactive VIP was attributable to the molecular variants, while in hog colon the variants were found predominantly in the mucosa and accounted for about 50% of total immunoreactivity. In contrast a form of VIP compatible with the authentic peptide accounted for over 75% of activity in the brain of all three species. The biological activity of the VIP variants is not known but clearly caution needs to be exercised in interpreting the physiological significance of studies on the action, release and metabolism of VIP.     

Corticotropin-releasing factor (CRF)-like immunoreactivity in the adrenal medulla

Bovine adrenal medulla extract prepared by acid-acetone or acid methanol extraction showed two peaks of CRF-like immunoreactivity on Sephadex G-50 chromatography. One eluted near the void volume and another (low molecular weight CRF-like immunoreactivity) eluted slightly before arginine vasopressin (AVP), while most of the immunoreactivity in bovine hypothalamus coeluted with synthetic ovine CRF. When low molecular weight CRF fractions were chromatographed by reversed phase high performance liquid chromatography, three CRF-like immunoreactive peaks appeared. The first peak appeared near TRH, the second one eluted near AVP and the last one eluted near somatostatin. These three peaks of immunoreactivity showed ACTH releasing bioactivity in rat pituitary cells cultures. Therefore, the adrenal medulla-CRF-like substances might be tissue-CRF which may play a role to stimulate ACTH release in the severe stress conditions.     

Distribution and characterisation of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine

Neuromedin U-8 (NMU-8) is a peptide isolated from porcine spinal cord which contracts blood vessels and the uterus. Antisera were raised against NMU-8 and used in a radioimmunoassay (RIA) together with HPLC to characterize NMU-like immunoreactivity (NMU-LI) in tissues extracts of rat brain and gut and guinea pig gut. Samples of duodenum, ileum and distal colon were taken from both species, and processed for detection of NMU-LI by fluorescence immunohistochemistry. In RIA the antiserum had no cross-reactivity with neuropeptide Y, vasoactive intestinal peptide or the C-terminal hexapeptide of pancreatic polypeptide. Preincubation of antiserum with any of these peptides had no effect on the NMU-LI staining. In rats the highest content of NMU-LI was found in the ileum and the lowest in the cerebral cortex and striatum. HPLC studies showed that at least two molecular forms of NMU-LI were present in both species. In rat small intestine, subpopulations of submucous and myenteric neurones were stained; nerve fibres and terminals within these ganglia and in the mucosa were also seen. NMU-LI was sparse in the muscle. In guinea pig ileum small populations of nerve terminals were seen in both myenteric and submucous ganglionated plexuses. No endocrine cells were stained in either species.     

Chromatographic characterization of dynorphin and [Leu5]enkephalin immunoreactivity in guinea pig and rat testis

Tissues of the reproductive tract have been shown to contain mRNAs coding for pro-opiomelanocortin (POMC), pro-enkephalin and pro-dynorphin. However, the amounts of immunoreactive opioid peptides in these tissues are low, and in the case of the enkephalins and dynorphin, the molecular species responsible for the immunoreactivities have not been characterized. The chromatographic properties of dynorphin and enkephalin immunoreactivities in extracts of guinea pig and rat testis have therefore been determined. Dynorphin A and dynorphin B immunoreactivity was heterogeneous, with a significant amount attributable to high-molecular-weight forms. About 20% of the dynorphin A immunoreactivity, and about 40% of the dynorphin B immunoreactivity, in guinea pig testis extracts behaved as authentic dynorphin A or B, respectively during fractionation by ion exchange, gel filtration and high-performance liquid chromatography. Both high- and low-molecular-weight forms of [Leu5]enkephalin immunoreactivity were also present, with roughly 50-70% of the immunoreactivity attributable to low-molecular-weight forms. In extracts of guinea pig testis only a small part of this immunoreactivity eluted as authentic [Leu5]enkephalin during high-performance liquid chromatography. In rat testis most of the low-molecular-weight [Leu5]enkephalin immunoreactivity behaved as the authentic peptide. These results confirm that opioid peptides are produced in guinea pig and rat testis, and demonstrate that immunoreactive forms of the peptides similar to those found in brain and pituitary are present in the tissue.     

N-terminally extended substance P is released together with substance P from rat spinal cord.

The release of different forms of substance P-like immunoreactivity (SP-LI) from superfused slices of rat spinal cord was studied. The released SP-LI was characterized by reverse-phase high-performance liquid chromatography and radioimmunoassay with two antisera directed to the C- and N-terminal parts of SP, respectively. The SP-LI detected in the superfusates with the C-terminally directed antiserum was found to consist of (undeca) SP, SP-sulfoxide and a late eluting component which was not detectable with the N-terminally directed antiserum. This component was also found in neutral extracts of the spinal cord. Upon trypsin digestion, it produced SP-LI detectable with both C- and N-terminally directed antiserum which also coeluted with SP. From these results we conclude that this form of SP-LI most likely corresponds to an N-terminally extended form of SP. An increase of the potassium concentration in the superfusion fluid from 5 to 50 mM evoked an increased overflow of both SP and the N-terminally extended SP. The present results indicate that N-terminally extended SP is released by a calcium-dependent mechanism together with SP from terminals in the spinal cord in response to potassium stimulation.     

Chromatographic evidence for high-molecular-mass galanin immunoreactivity in pig and cat adrenal glands

Galanin was measured by radioimmunoassay in extracts of pig, cat and rat adrenals using non-C- and mid to C-terminally directed antibodies. The extracts were fractioned by gel chromatography and HPLC. The non-C-terminal galanin immunoreactivity in pig was 92.8 +/- 11.7 pmol/g, in cat 9.1 +/- 0.9 pmol/g and in rat less than 1 pmol/g. Two higher molecular forms of galanin have been identified in both pig and cat adrenal. One major large form behaves as if it was N-terminally extended (Kav pig 0.58, cat 0.48) and the other, a very high-molecular-mass form (Kav pig 0.10, 0.24, cat 0.10), as if it had both N- and C-terminal extensions.     

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons). Using HPLC techniques, one main peak of immunoreactivity was present in brain extracts, whereas two peaks were present in pituitary extracts, one of which coeluted with thymosin alpha 1. The discrete regional distribution of thymosin alpha 1-like peptides was investigated and the highest densities of immunoreactive thymosin were present in the median eminence and arcuate nucleus of the hypothalamus, as well as the neurointermediate lobe of the pituitary. Due to the anatomical proximity of immunoreactive thymosin to loci containing known releasing factors and hormones, thymosin alpha 1-like peptides may function as neuroendocrine regulatory agents.     

Endocrine cells with gastrin/cholecystokinin-like immunoreactivity in the pancreas of the spiny dogfish,Squalus acanthias

Endocrine cells containing gastrin/cholecystokinin (CCK)-like immunoreactivity were localized to the islet tissue in the pancreas of the spiny dogfish. Most of these cells were located in the 'intestinal' lobe of the pancreas; only occasional cells were observed in the 'splenic' lobe. The gastrin/CCK-like immunoreactive cells were often co-localized with the 'classical' pancreas hormones (insulin, glucagon and somatostatin). Radioimmunoassay of water extracts with a C-terminally directed antiserum revealed high levels of immunoreactive material in the intestinal part (48.6 +/- 19.9 pmol/g) and lower levels (4.5 +/- 0.6 pmol/g) in the splenic part. Acetic acid extracts of the intestinal lobe contained low levels (6.8 +/- 3.3 pmol/g) of gastrin/CCK-like immunoreactivity, whereas corresponding extracts of the splenic part showed no immunoreactivity. When the extracts were subjected to DEAE ion-exchange chromatography the gastrin/CCK-like peptides eluted as a major peak. After Sephadex gel filtration, pooled immunoreactive material from the main DEAE chromatographic peak eluted at a position close to that of CCK4. Further characterization by ion-exchange and reversed-phase HPLC showed that, in general, the immunoreactive material behaved like the shorter forms of the gastrin/CCK family (CCK4/G5 and CCK8/Cae 3-10).     

Ontogeny of immunoreactive CCK and VIP in pig brain and gut

The concentrations and hormonal forms of CCK and VIP have been determined in extracts of the brain and duodenum of the developing and adult pig. In methanol extracts of the brain cortex, the single hormone form, CCK8, increased from 130 +/- 20 (Mean +/- SEM) pmol/g at birth to an adult level of 300 +/- 50 pmol/g. In acid extracts of brain, the predominant immunoreactive form had N-terminal immunoreactivity and increased from 240 +/- 20 pmol/g at birth to an adult level 490 +/- 30 pmol/g; the C-terminal immunoreactivity was about 10-fold lower. The concentrations and hormonal forms of immunoreactive CCK in duodenal extracts did not appear to be age-related. C-terminal immunoreactivity in methanol extracts averaged 140 +/- 20 pmol/g and in acid extracts 240 +/- 60 pmol/g. The concentration of N-terminal immunoreactivity in acid extracts averaged 490 +/- 70 pmol/g. The VIP concentrations in acid extracts of the brain cortex was 13.5 +/- 2 pmol/g at birth and rose gradually to 30 +/- 9 pmol/g in the adult; in duodenal extracts it was 240 +/- 18 pmol/g at birth and 195 +/- 38 pmol/g in the adult. These results are in marked contrast with the ontogeny of these hormones in the rat in which brain concentrations of CCK and VIP in the neonate are less than 10% of adult levels and in which there are age-related changes in the content of these hormones in the duodenum as well.     

Gastrointestinal peptides in the brain.

Both immunoreactive intact cholecystokinin (CCK33) and its COOH-terminal octapeptide (CCK8) are detected in brain and gut extracts of monkey, dog, and pig using an antiserum with equivalent sensitivities for detecting CCK8 in the free form or when incorporated in the intact molecule. The failure to detect intact cholecystokinin in extracts from monkey or dog by using an antiserum developed by immunization with porcine CCK33 is due to marked species differences in the NH2-terminal portion of the molecule. Immunohistochemical staining reveals the presence of CCK peptides in rabbit cerebral cortical tissue neurons. Subcellular fractionation of rat cerebral cortical tissue demonstrates that CCK immunoreactivity is concentrated in the pellet identified by electron microscopy to contain a high proportion of synaptic vesicles. A converting enzyme that differs from trypsin has been partially purified from canine and porcine cerebral cortical extracts. It converts porcine CCK to smaller immunoreactive forms, but fails to convert big gastrin to heptadecapeptide gastrin. This enzyme differs from trypsin not only in substrate specificity but also in several physicochemical properties. Cerebral cortical extracts from hyperphagic ob/ob mice have strikingly lower contents of CCK than those from their lean littermates and other normal mice. These studies taken together are consistent with a role for CCK as a neurotransmitter involved in the overall regulation of appetite.     

Existence of different forms of adrenocorticotropin in rat hypothalamus

Adrenocorticotropin (ACTH)-like immunoreactivity and bioactivity were extracted from rat hypothalamus and fractionated by high pressure liquid chromatography. Analysis of the fractions either by radioimmunoassay or bioassay (corticosteroid production from rat adrenal cells) revealed several peaks of immunoreactivity and bioactivity. Only 20-25% of total ACTH-like immunoreactivity and bioactivity eluted with the same retention time as authentic ACTH 1-39. The results suggest that different forms of ACTH exist in rat hypothalamus.