Chizuko Yanaihara

Dose-response comparisons of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide (GRP)

The effect on plasma gastroenteropancreatic hormone levels on infusing the porcine gastrin-releasing peptide and bombesin into dogs demonstrated no qualitative difference in the spectrum of activity of the two peptides. Sustained elevation in plasma immunoreactive gastrin, pancreatic polypeptide, enteroglucagon, gastric inhibitory polypeptide, pancreatic glucagon and transient elevations in plasma insulin were seen during infusions of both peptides. The similar spectrum of activities and the structural homology between the two peptides suggests that the porcine gastrin releasing peptide is the porcine counterpart of the amphibian peptide bombesin.

Immunological Aspects of Secretin. Substance P, and Vip

Secretin, substance P, and vasoactive intestinal peptide (VIP) were studied from the immunological point of view using synthetic hormones and their related peptides which were prepared by the conventional method for peptide synthesis. Immunological properties of these hormones were characterized by radioimmunoassays specific to the respective hormones. Antisecretin antisera (NCC-R-1 and R-801) were generated in rabbits with synthetic porcine secretin absorbed on polyvinylpyrrolidone. Antiserum to substance P (R-400) was produced in a rabbit with synthetic substance P-human alpha-globulin conjugate. Generation of anti-VIP antiserum (R-502) was carried out by immunizing rabbits with synthetic VIP absorbed on polyvinylpyrrolidone. Synthetic polypeptides related to the three hormones that were examined in this study include secretin(4-27), secretin(5-27), secretin(7-27), secretin(11-27), secretin(14-27), secretin(18-27), secretin(1-22)amide, secretin(7-22)amide, Nalpha-tyrosyl-secretin, [1-Tyr]secretin, [4-Ala]secretin, [4-D-Ala]secretin, [4-Ala,5-Val]secretin, [6-Tyr]secretin, substance P(2-11), substance P (3-11), substance P(4-11), substance P(5-11), substance P(6-11), Nalpha-tyrosyl-substance P, [1-Tyr]substance P, [8-Tyr]substance P, [11-Leu]substance P, des-11-Met-substance P, VIP(7-28), VIP(11-28), VIP(18-28), VIP(1-18)amide, and VIP(1-22)AMIDE. The results revealed two antigenic regions at the amino- and carboxylterminal portions of the secretin and VIP molecules. As to substance P, the major antigenic region was located within the 3 to 11 sequence. The proline residue in position 4 and methionine in position 11 seemed to be of special importance. The immunoassays demonstrated the existence of immunoreactivities of these hormones in hot water extracts from various porcine tissues. In the pituitary, VIP and substance P immunoreactivities were detected, whereas secretin was not. Secretin, VIP, and substance P were found in the pancreas, but at low concentrations. Distributions of these hormones in various sites of the gastrointestinal tract were also demonstrated.

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.

Inhibitory effects of galanin on the isolated spinal cord of the newborn rat.

The effects of galanin, a 29-amino-acid peptide, on spinal reflexes were studied. In the isolated hemisected spinal cord of the newborn rat, galanin (0.1-5 microM) depressed the monosynaptic reflex that was induced by dorsal root stimulation and recorded from the corresponding ventral root. In the isolated spinal cord-tail preparation of the newborn rat, galanin (0.3-0.6 microM) depressed the nociceptive reflex that was induced by application of capsaicin to the tail and recorded from a lumbar ventral root. In both preparations the inhibitory effects of galanin were reversible and the full recovery of the reflexes was observed within 3-20 min after removal of the peptide. The mechanisms of action of galanin on the spinal reflexes and the physiological role of the peptide in the spinal cord are discussed.

Coexistence of dopamine and neurotensin in hypothalamic arcuate and periventricular neurons

The coexistence of dopamine and neurotensin in the same neuronal perikarya in the arcuate nucleus of the rat hypothalamus was examined by combined fluorescence histochemistry and immunohistochemistry on the same tissue sections and we obtained the evidence of the coexistence of two substances. The functional significance of those two substances for the prolactin release from the anterior pituitary was also briefly discussed.

Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in the rat hypothalamus, particularly in the arcuate nucleus, the paraventricular nucleus and the median eminence, were investigated by light and electron microscopic immunocytochemistry. The main distributional areas of immunoreactive neuronal perikarya were found to be the arcuate nucleus, the periventricular nucleus and the paraventricular nucleus by light microscopic immunocytochemistry. Immunoreactive neuronal perikarya showed a characteristic distributional pattern in the arcuate nucleus. In the paraventricular nucleus they were distributed in both the magnocellular and parvocellular portions. A large number of immunoreactive terminals were observed throughout the external layer of the median eminence, particularly its lateral portion. A moderate number of immunoreactive terminals were also observed in the internal layer of the median eminence. By electron microscopic immunocytochemistry immunoreactive neuronal perikarya both in the arcuate and paraventricular nuclei showed generally well-developed cell organelles such as mitochondria, r-ER, and Golgi complex. In addition, immunoreactive dense granules were dispersed throughout the perikarya. A large number of immunoreactive terminals containing immunoreactive dense granules, clear vesicles and mitochondria were observed in the vicinity of pericapillary spaces of the external layer of the median eminence. This observation strongly suggests that neurotensin-like immunoreactive substance is released into the portal capillaries.

Co-localization of substance P and Met-enkephalin-Arg6-Gly7-Leu8 in the intraspinal neurons of the rat, with special reference to the neurons in the substantia gelatinosa

A double-labeling immunofluorescence technique was employed to investigate the co-localization of the functionally antagonistic neuropeptides, substance P and enkephalins, within intraspinal neurons of the rat. Anti-Met-enkephalin-Arg6-Gly7-Leu8 (Enk-8) antiserum was used as a marker of the preproenkephalin A neuron system. The observations were focused on the lumbar spinal cord. Co-localization was most prominent within neurons in the substantia gelatinosa, in which more than 95% of substance P-like immunoreactivity neurons showed Enk-8-like immunoreactivity. These double-labeled cells corresponded to 45% of Enk-8-like immunoreactive neurons in the same area. This suggests that SP/Enk-8 interaction occurs at the axon terminals of the substantia gelatinosa neurons. In deeper layers of the dorsal horn (laminae III, IV), only 14% and 6% of SP-like immunoreactive and Enk-8-like immunoreactive neurons were double labeled, respectively. Co-localization was also observed in neurons located in the laminae I, V, VII and X, suggesting concomitant involvement of these peptides in a variety of spinal cord functions.

Immunoreactive helodermin-like peptides in rat: a new class of mammalian neuropeptides related to secretin and VIP.

Helodermin is a peptide from the venom of the lizard Heloderma suspectum (Gila Monster) showing a high degree of sequence similarity with VIP, PHI and secretin in its N-terminal moiety. The present data support the presence of peptide(s) closely related to helodermin in the brain, gut and salivary glands of rat. In our radioimmunoassays, we routinely used one of the three specific antisera obtained from rabbits that were immunized against lizard helodermin coupled to bovine serum albumin with carbodiimide. Heat- and acid-stable immunoreactive helodermin-like material was more abundant in striatum, hippocampus and anterior pituitary than in cerebral cortex and hypothalamus. High levels of helodermin-like material were also present in salivary glands, duodenum and jejunum. When submitted to gel permeation chromatography on a TSK-G 2000 SW column, the apparent molecular radius of most of the immunoreactive material ranged from 6 to 12 KDa.

Luteinizing hormone-releasing hormone (LH-RH) activity of some synthetic polypeptides. I. Fragments shorter than decapeptide

Summary The luteinizing-releasing hormone (LH-RH) activity of II synthetic polypeptides, some of which were speculatively reported as having LH-RH activity, was assayed in vivo and in vitro against pure natural and synthetic LH-RH. In vivo tests showed that (pyro)Glu-Tyr-Arg-Trp-NH 2 had only 1 part in 7800 of the activity of the LH-RH decapeptide. (Pyro) Glu-Val-Ser-NH 2 , (pyro)Glu-Ser-Val-NH 2 and (pyro)Glu-Gln-Ala-NH 2 , were inactive in vivo in doses as high as 5 – 20 μg/rat. Synthetic (pyro)Glu-His-Pro-amide (thyrotropin-releasing hormone) and a synthetic decapeptide proposed as growth-hormone-releasing hormone showed no LH-RH activity in doses up to 100 μg. N-terminal tripeptide and tetrapeptide fragments of LH-RH as well as the C-terminal octapeptide of LH-RH were also inactive. The C-terminal nonapeptide had an extremely low LH-RH activity (about 1 part in 50,000). The structure-activity relationship of LH-RH has been briefly discussed.

The influence of serotonergic inputs on peptide neurons in the rat suprachiasmatic nucleus: An immunocytochemical study

The influence of serotonin (5-hydroxytryptamine; 5-HT) innervation on peptide-containing neurons in the rat suprachiasmatic nucleus (SCN) was investigated by peroxidase-anti-peroxidase (PAP) immunocytochemistry. The 5-HT neuronal system was chemically severed by 5,6-dihydroxytryptamine (5,6-DHT) injection into the medial forebrain bundle bilaterally. After this treatment, a marked decrease of vasoactive intestinal peptide (VIP)-like immunoreactivity in neuronal perikarya occurred in the SCN corresponding to a decrease in number of 5-HT immunoreactive fibers and terminals. However, no alteration of arginine-vasopressin-like immunoreactivity was detected between 5,6-DHT-treated animals and the controls. It is speculated that VIP-like immunoreactive neurons play an important role in the SCN under the influence of strong 5-HT innervation.