Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell.

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C-peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.

Syntheses of C-peptides and human proinsulin.

Syntheses of human, dog, rat, and duck C-peptides and their analogues and preliminary results on the total synthesis of human proinsulin are described. In the syntheses of the C-peptides, chain elongation was performed exclusively by the azide-fragment condensation method in solution. The synthetic human, dog, rat, and duck C-peptides and their analogues were proved to be homogeneous by several analytic means. With these synthetic peptides, radioimmunoassay systems for dog, rat, and duck C-peptides were developed. For the total synthesis of human proinsulin, 10 protected peptide hydrazides were prepared, and the linearly protected hexaoctacontapeptide having the proposed sequence of human proinsulin was constructed by the azide-fragment condensation method in solution starting from the C-terminal undecapeptide (HP 75-86). After deblocking of the alpha-amino protection, the partially protected hexaoctacontapeptide was treated with sodium in liquid ammonia. The ensuing sulfhydryl form was converted to the S-sulfonate form, which was reduced and then air-oxidized. The oxidized material was purified by gel filtration on Sephadex G-50 (fine) followed by ion-exchange chromatography on DEAE-cellulose. The cross-reactivity in the insulin radioimmunoassay of the ensuing product was 62.5 per cent of porcine proinsulin on a weight basis at B/Bo = 60 per cent. Acid hydrolysis and amino acid analysis of this product gave the theoretically expected ratios. In addition, this peptide, as well as the S-sulfonate form of the hexaoctacontapeptide, showed displacement curves superimposable on that of synthetic human C-peptide on an equimolar basis in the human C-peptide radioimmunoassay (antiserum 527). These results confirm the synthesis of human proinsulin.

Distribution of urocortin 2 in various tissues of the rat.

Urocortin (Ucn) 2 is a new member of the corticotrophin-releasing hormone (CRH) neuropeptide family that is expressed in the central nervous system and peripheral tissues. However, the expression levels of Ucn 2 in various tissues of the rat remains unclear. Thus, the aim of the present study was to characterise the expression of Ucn 2 in the various tissues of the rat. Reverse transcriptase-polymerase chain reaction analysis demonstrated that Ucn 2 mRNA is expressed in the hypothalamus, pituitary, adrenal, stomach, skin, ovary, uterus and skeletal muscle. Histologically, Ucn 2 mRNA and Ucn 2-like immunoreactivity (LI) were demonstrated in both the anterior and intermediate lobes of the pituitary, but not detected in the posterior lobe. Furthermore, all Ucn 2-positive cells in the anterior and intermediate lobes were also positive for beta-endorphin. Ucn 2 mRNA was detected in the adrenal cortex and medulla although Ucn 2-LI was only found in the adrenal medulla. High-performance liquid chromatography analysis of hypothalamic, pituitary, and adrenal extracts showed that the main Ucn 2-LI peak occurred at the same molecular size as that of synthetic Ucn 2. These results suggest that Ucn 2 is synthesised in various tissues, including the anterior and intermediate lobes of the pituitary and the adrenal.

Rat pancreas contains the proglucagon(64-69) fragment and arginine stimulates its release.

Rat proglucagon(64-69) corresponding to the C-terminal hexapeptide of putative rat glicentin sequence in the precursor was synthesized. A glicentin C-terminal hexapeptide specific radioimmunoassay, using the synthetic hexapeptide as standard, demonstrated the presence in rat pancreas of a peptide identified with the synthetic rat proglucagon(64-69): H-Asn-Arg-Asn-Asn-Ile-Ala-OH. The hexapeptide was released concomitantly with glucagon by arginine stimulation from the isolated perfused rat pancreas. The results indicate that the pancreas co-stores and possibly co-releases the hexapeptide with glucagon as one of the processing products of proglucagon.

Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas.

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64-69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4-91.7%. The final yield of the hexapeptide was 22 micrograms (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon.

Primary structure of helodermin, a VIP-secretin-like peptide isolated from Gila monster venom.

The complete amino acid sequence of helodermin isolated from the venom of Gila monster was elucidated. The peptide was shown to be a basic pentatriacontapeptide amide: His-Ser-Asp-Ala-Ile-Phe-Thr-Gln-Gln-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala- Leu-Gln-Lys- Tyr-Leu-Ala-Ser-Ile-Leu-Gly-Ser-Arg-Thr-Ser-Pro-Pro-Pro-NH2. A high degree of sequence similarities to secretin/VIP/PHI/(PHM)/GRF from mammal and bird was observed over the entire N-terminal 1-27 sequence. In particular, the amino acid residues in positions 3, 6 and 7 were found to be common to 9 peptides of the family. Another interesting feature of the structure of helodermin was its C-terminal -Pro-Pro-Pro-NH2 sequence. Isolation of helodermin was the first demonstration of the existence of a secretin/VIP-related peptide in an animal that is neither mammal nor bird.

Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva.

Helodermin is a biologically active peptide isolated from the venom of the Gila monster lizard (Heloderma suspectum) whose structure is related to that of vasoactive intestinal peptide and secretin. Using a specific radioimmunoassay based on antisera prepared by immunizing rabbits with natural helodermin, we demonstrated the presence of helodermin-like material in mammalian salivary glands, including parotid, submaxillary and sublingual glands from rat and dog, and parotid and submaxillary glands from man. All helodermin-like materials had an apparent molecular mass of 4-12 kDa. Dog saliva, collected after pilocarpine stimulation, revealed similar immunoreactivity with a major component around 6 kDa.

Immunocytochemical distribution of met-enkephalin-Arg6-Gly7-Leu8 in the rat lower brainstem.

The distribution of methionine-enkephalin-Arg6-Gly7-Leu8, a unique peptide derived from proenkephalin A in the rat brainstem, was studied immunocytochemically by using a highly specific antiserum to this octapeptide sequence. Immunoreactive perikarya with various shapes and sizes were detected in many regions of the rat brainstem. Dense accumulation of immunoreactive perikarya and fibers was seen in the nuclei associated with special sensory and visceral functions, such as the interpeduncular nucleus, the parabrachial nucleus, the nucleus of the solitary tract, and the nucleus of the spinal tract of the trigeminal nerve. Clusters of methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactive perikarya and fibers were observed in certain areas considered to play a role in nociception and analgesia, such as the central gray of the midbrain central gray and the raphe magnus nucleus. Some methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactive perikarya were distributed in the lateral reticular nucleus, the nucleus of the solitary tract, and the raphe magnus nucleus, where monoaminergic neurons were also detected. In addition to the previously reported enkephalinergic cells, we found many methionine-enkephalin-Arg6-Gly7-Leu8 containing neurons; the rostral and caudal linear nucleus of raphe, the median raphe nucleus, entire length of the raphe magnus nucleus, the medial longitudinal fasciculus, the cuneate nucleus, the external cuneate nucleus, the gracile nucleus, and the area postrema. The wide distribution of this octapeptide-like immunoreactivity reflected neurons expressing the preproenkephalin A gene distributed more widely than previously reported and that innervated many regions.

A novel immunization procedure for production of anti-cholecystokinin-specific antiserum of low cross-reactivity.

An anti-cholecystokinin octapeptide (CCK-8) antibody without cross-reactivity with pentagastrin, whose amino acid sequence is identical with the carboxy-terminal portion of CCK-8, was produced by immunization with CCK-8-keyhole limpet hemocyanin and injection of a conjugate of pentagastrin and a copolymer of D-glutamic acid and D-lysine to inhibit production of antibody cross-reacting with pentagastrin.

Presence of epidermal growth factor in human tears.

Epidermal growth factor (EGF) is a polypeptide that stimulates the growth of various tissues, including the cornea. The presence of EGF in tears from normal volunteers and in aqueous humor from cataract patients was investigated via human EGF (hEGF)-specific radioimmunoassay. Immunoreactive hEGF was found to be present at similar concentrations in both reflex (ranging from 0.7 to 8.1 ng/ml) and non-reflex tears (ranging from 1.9 to 9.7 ng/ml), but was undetectable in aqueous humor. Immunoreactive EGF in human tears was indistinguishable immunologically, biologically and biochemically from urine EGF and standard hEGF.

Localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats: light and electron microscopic immunocytochemical studies.

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1-38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.

VIP- and PACAP-induced salivary chromogranin A secretion in the isolated perfused submandibular gland of rats.

In the study reported in this paper, sensitive ELISA for rat CgA was developed using synthetic rat CgA(359-389) as antigen, N alpha-biotinylated glycylglycyl rat CgA(359-389), and antirat CgA(359-389) serum for the measurement of CgA-LI in rat saliva. CgA-LI in rat submandibular tissues and saliva was characterized by both immunohistochemical and immunochemical methods. Using isolated perfused rat submandibular gland. VIP at 0.1-1.0 nM in the presence of 0.1 microM ACh was found to cause CgA-LI secretion, whereas neither PACAP-27 nor PACAP-38 showed any effect on CgA secretion.

Immunohistochemical localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the adrenal medulla of the rat.

Localization of PACAP in rat adrenal glands was examined by light and electron microscopic immunohistochemistry using a specific antiserum to PACAP 38, R0831. In the light microscopic study, PACAP immunoreactivity was observed in some cell groups in the medulla, but not in the cortex. In comparison with adjacent sections stained with antisera to catecholamine synthesizing enzymes, PACAP-positive cells were immunoreactive to tyrosine hydroxylase and dopamine beta-hydroxylase, but not to phenylethanolamine-N-methyltransferase, suggesting that they were coincident with noradrenaline secreting cells. In the electron microscopic study using the ABC method, DAB reaction products were diffusely distributed in the cytoplasmic matrix of PACAP-positive cells, without intense accumulation on the secretory granules. The splanchnic nerve terminals were PACAP negative. In postembedding immunohistochemistry, gold particles were localized diffusely in the cytoplasma, but not aggregated on the secretory granules. It was suggested that PACAP would localize in the cytoplasmic matrix of noradrenaline cells and stimulate the catecholamine synthesis and release in the adrenal medulla.

Identification of two pro-VIP forms in a human neuroblastoma cell line.

The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0.1-0.3 pmol per mm2 with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB-OK-1 cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit anti-PHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of human corticotropin releasing factor (CRF) on gastric and pancreatic secretion in vivo and in vitro.

Human CRF given IV inhibited dose-dependently pentagastrin- but not histamine-induced gastric acid secretion. When added to the incubation medium of the isolated gastric glands, CRF did not alter the formation of HCl under basal conditions or after stimulation with histamine or DBcAMP. CRF caused a small but significant increase in pancreatic HCO3 and protein secretion. It augmented CCK-induced pancreatic protein and secretin-induced HCO3 secretion in vivo but failed to affect basal or stimulated (CCK and urecholine) amylase release by the in vitro dispersed pancreatic acini. This study indicates that CRF inhibits gastric and stimulates pancreatic secretion in vivo but not in vitro and these effects are indirect involving, at least in part, alterations in the pancreatic circulation.

Immunochemical study on PHI/PHM with use of synthetic peptides.

We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1-6), PHI (1-15), PHI (14-19), PHI (14-27), PHI (20-27), PHM (1-15) and PHM (13-27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recognition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells.

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.

Immunoreactive GRP.

Radioimmunoassay specific for GRP was developed using C-terminal specific anti-synthetic porcine GRP sera. Measurement of the porcine gastrointestine and brain tissue extracts with GRP and with bombesin radioimmunoassays indicates that the bombesin-like immunoreactivity found in the porcine tissues is attributable to GRP or GRP-like materials. GRP-like immunoreactivity in extracts of porcine gastrointestinal tissues and pancreas was shown, by gel filtration, to comprise mainly two components: the first-eluting component corresponding to synthetic GRP and the second-eluting one to synthetic GRP(14--27) in their elution volumes. An extract of the hypothalamus also contained two similar components, while extracts of the medulla oblongata and cerebral cortex contained mainly the second-eluting immunoreactivity. Immunostaining with anti-GRP sera revealed positive nerve fibers in the myenteric nerve plexus of the porcine duodenum and in intrapancreatic ganglia, but no immunoreactivity endocrine cells in the mucosa of the porcine stomach and intestine. The results gave support to GRP as a new brain-gut peptide and suggested physiological significant of GRP as a neurotransmitter or neuromodulator.

Actions of galanin fragments on rat, guinea-pig, and canine intestinal motility.

The 1-20 fragment of synthetic porcine galanin, prepared by tryptic digestion of the intact molecule, was equipotent to synthetic porcine galanin 1-29 in the smooth muscle actions of exciting the rat jejunal longitudinal muscle in vitro and inhibiting circular muscle contractions of the canine small intestine in vitro and in vivo, but was less potent in inhibiting nerve-stimulated contractions of the guinea-pig taenia coli. Fragment 21-29 was effective at high doses only in the canine ileum. Activity of galanin 1-11 was greatly reduced in the dog in vivo. These results may reflect species or cell type differences.

Helodermin has a VIP-like effect upon canine blood flow.

The effect of helodermin on vascular physiology was studied in anesthetized dogs using a synthetic replicate of helodermin and helodermin related peptides. Intraarterial infusion of helodermin caused a dose-dependent increase in femoral blood flow. Helodermin was 16 times less potent than VIP and 5 times more potent than PHM (human PHI). The helodermin effect lasted significantly longer; the half-life of the helodermin effect was 6.5 times longer than VIP. Synthetic helodermin (Hd) N-terminal fragment Hd(1-27)NH2 retained substantial activity similar to the full helodermin molecule but the prolonged effect was lost. Hd(7-35) and Hd(22-35) were inactive in this system. Intravenous injection of synthetic helodermin produced prolonged systemic hypotension and tachycardia; and, similar to VIP, it increased the common carotid arterial blood flow while those of the superior mesenteric and femoral arteries were decreased. The results demonstrate the VIP-like vasodilating activity and cardiovascular effects of helodermin in anesthetized dogs.