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Biomedical subjects

M Fujino

Publications and source records attributed to M Fujino.

At least 145 records · Page 8Linked to original sources
PubMed1993

Effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) on the spontaneous release of acetylcholine from the rat hippocampus by brain microdialysis.

Vasoactive intestinal polypeptide (VIP) has been suggested to have a presynaptic effect on cholinergic terminals in the rat hippocampus, which results in an activation of acetylcholine (ACh) synthesis. Recently, a VIP-related novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from the ovine hypothalamus, and we previously demonstrated in the rat that PACAP binding site densities were high in the hippocampus. In the present study, we investigated the effects of VIP and PACAP on the release of ACh from the rat hippocampus. We succeeded in detecting the spontaneous release of ACh from the dorsal hippocampus in the conscious rat using microdialysis and HPLC-ECD. VIP, PACAP38 and PACAP27 were applied through a microinjection cannula placed in a region adjacent to the tip of a microdialysis tube. Injections of VIP, PACAP38 and PACAP27 (12, 120 pmol) resulted in dose-related increases in ACh release. The ability to enhance ACh release was VIP > PACAP38 > PACAP27. The increased release of ACh caused by these peptides was highly calcium-dependent. Tetrodotoxin (10(-6) M) added to the perfusion medium significantly reduced both the release of ACh enhanced by these peptides and the basal release. The present results suggest that VIP, PACAP38 and PACAP27 presynaptically stimulate cholinergic activity in the hippocampus, which may be reflected by an increase in ACh synthesis to maintain releasable terminal stores of ACh.

Acetylcholine↗
PubMed1993

Studies of a key protein in the mechanism of the excitation-contraction coupling process of frog skeletal muscle, using phenylglyoxal.

The excitation-contraction (E-C) coupling process in single twitch fibres from frog toe muscle was inhibited selectively by phenylglyoxal (PGO), a specific guanidyl modifying reagent. A new protein (31.5 kDa), which has PGO-binding ability and seems to play a key role in the E-C coupling process, was solubilized from transverse tubule membrane-junctional sarcoplasmic reticulum complexes (TTM-JSR) of frog skeletal muscles, using 14C-PGO. The monoclonal antibody against this protein applied extracellularly inhibited the E-C coupling process of the single fibres. This protein appears to constitute the very first step of input for E-C coupling. It is considered to behave as an indispensable part of an 'electrometer' to measure membrane potentials. Therefore, the name 'electrometrin' is suggested for the new protein.

Animals↗
PubMed1993

Regional distribution of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat central nervous system as determined by sandwich-enzyme immunoassay.

We investigated endogenous levels of a novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the rat central nervous system. The amount of PACAP was measured by means of highly specific and sensitive sandwich-enzyme immunoassay. This assay system following HPLC analysis revealed that PACAP38 was a major portion of the total PACAP immunoreactivity and PACAP27 levels were negligibly low in the brain. Therefore, we measured the amount of PACAP38 in 62 regions punched out from frozen tissue sections. High amounts of PACAP38 were found in the lateral septal nucleus (intermediate part), diagonal band, central amygdaloid nucleus, several parts of the hypothalamus (suprachiasmatic, supraoptic, periventricular and arcuate nuclei), central gray, interpeduncular nucleus and dorsal raphe. The suprachiasmatic, paraventricular and periventricular hypothalamic nuclei showed the highest levels. A moderate amount of the peptide was observed in the lateral septal nucleus (dorsal part), medial septal nucleus, medial amygdaloid nucleus, thalamus (paraventricular, paratenial, central medial, ventromedial, reuniens and rhomboid nuclei), hypothalamus (lateral hypothalamic area and mammillary body), ventral tegmental area, interfascicular nucleus and in the locus coeruleus. Such a distribution of endogenous PACAP38 did not parallel the localization of PACAP binding sites which we had demonstrated recently. Moreover, the topographical distribution of PACAP38 observed in the present study differed from that of VIP which had been previously reported. The present results suggest that PACAP38 may have a neurotransmitter/neuromodulator role which is different from that of VIP in the central nervous system.

Animals↗
PubMed1993

Production of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) by human neuroblastoma cells, IMR-32: detection and characterization with monoclonal and polyclonal antibodies against different epitopes of PACAP.

Sensitive and specific two-side enzyme immunoassays (two-site EIAs) for pituitary adenylate cyclase activating polypeptides, PACAP38, and PACAP27, have been established using six monoclonal antibodies against PACAP38, and a rabbit antibody against a C-terminal portion of PACAP27. In extracts of rat hypothalamus, these EIAs detected not only PACAP38 and PACAP27 but also an immunoreactive (ir-) PACAP lacking an epitope of a monoclonal antibody, PA-1C, which recognizes the C-terminal portion of PACAP38. By the use of these EIAs, it was found that one of the human neuroblastoma cell lines, IMR-32, produced ir-PACAP. In reverse-phase (RP-)HPLC, intracellular and extracellular ir-PACAPs were separated into two peaks, of which one was eluted at a position close to that of PACAP38 and the other in rather hydrophobic fractions. Those ir-PACAPs also lacked PA-1C epitope of PACAP38. SDS-PAGE and immunoblot analysis of the two peaks of the RP-HPLC indicated that they consisted of several components including those with apparent molecular weights of 6.5 k and 10 k for the first peak ir-PACAP, and 14 k and 20 k for the second peak ir-PACAP. These results indicate that IMR-32 produces a precursor of PACAP and related peptides generated in various processing steps. Although the significance of the modification in the C-terminus of PACAP38 is unknown, IMR-32 may be a cell line useful for studying the regulation of the biosynthesis of PACAP.

Animals↗
PubMed1993

Design and synthesis of ETA receptor antagonists and study of ETA receptor distribution.

Many oligopeptides were designed to find ETA receptor antagonists on the hypothesis that an ETA receptor can recognize two hydrophobic parts of ET-1, i.e., Val-Tyr-Phe and Ile-Ile-Trp, over a short distance. They were synthesized from the benzyl ester of the C-terminal amino acid by stepwise chain elongation using the solution method. The binding affinity of the synthetic peptides to the endothelin receptors was examined in porcine cardiac ventricular muscle membrane for ETA receptor and in bovine whole brain membrane for ETB receptor. Hexamethyleneiminocarbonyl-Leu-trp-ala-beta ala-tyr-phe (TTA-386) was selected as an ETA receptor-selective competitive antagonist to ET-1. It competed against ET-1 at ETA receptor sites and showed one-third the binding affinity of ET-1 for ETA receptor and < 1/10,000 the affinity for ETB receptor. It inhibited the ET-1-induced increase of cytosolic free calcium concentration in A-10 cells. The 125I-labeled hexapeptide (125I-TTA-386) was prepared to distinguish the distribution of ETA receptor from ETB receptor. Scatchard plot analysis of saturation binding of 125I-TTA-386 to porcine cardiac membranes showed the same Bmax value as that of 125I-ET-1. Autoradiographic studies showed that ETA receptors are most abundant in the cardiac muscle, intestine, large bowel, spleen, and testis.

Amino Acid Sequence↗
PubMed1993

Dilated cardiomyopathy in a patient receiving continuous ambulatory peritoneal dialysis.

We describe a unique case of a 10 year old girl with dilated cardiomyopathy who has been receiving continuous ambulatory peritoneal dialysis. She responded to treatment with a beta 1-agonist. We note that electrocardiography and echocardiography are effective for early recognition of the onset of dilated cardiomyopathy and that a thallium scan should be performed to assess the severity of dilated cardiomyopathy in patients with chronic renal insufficiency.

Adrenergic beta-Agonists↗
PubMed1993

Pharmacologic profile of endothelinA/B antagonist, [Thr18, gamma methyl Leu19]endothelin-1.

The pharmacologic profile of [Thr18,gamma methyl Leu19]endothelin-1 (TM ET-1) was investigated in several in vitro and in vivo studies. We found that TM ET-1 inhibited 125I-ET-1 binding in porcine cardiac membrane (ETA receptor) and in bovine brain membrane (ETB receptor), with IC50 values of 0.7 and 0.25 nM, respectively. These values were almost comparable to those for ET-1. TM ET-1 had no effect on intracellular Ca2+ concentration ([Ca]i) in A10 cells mediated by ETA receptors, even at 10(-5) M, or in mouse peritoneal macrophages (MPMs) mediated by ETB receptors, even at 10(-6) M. Increases in [CA]i in A10 cells by ET-1 and in MPMs by ET-3 were completely blocked by pretreatment with 10(-7) M of TM ET-1. In porcine right coronary arteries (PCAs) and in great cardiac veins (PCVs), TM ET-1 caused no contraction at concentrations < or = 10(-6) and 10(-7) M, respectively, although it competitively inhibited ET-1-induced contraction of PCAs and Ala1,3,11,15-ET-1-induced constriction of PCVs with pA2 values of 7.0 and 9.2, respectively. Furthermore, TM ET-1 was more potent than BQ123, an ETA-specific antagonist, in inhibiting the rise in perfusion pressure in a hind-limb preparation in vitro and the increase in blood pressure in rats. These results suggest that TM ET-1 is a potent ETA and ETB antagonist without agonistic effects and can be used in future studies to help clarify the physiological role of ET.

Animals↗
PubMed1993

White pulp reconstitution after human bone marrow transplantation.

To reveal the reconstitution process of the white pulp after bone marrow transplantation (BMT), spleens of 24 marrow recipients whose survival times ranged from 34 to 303 days after BMT, were analyzed at the histopathological and immunohistochemical level. Up to 3 months after BMT, the white pulp was atrophic and consisted mainly of T cells forming periarteriolar lymphatic sheaths (PALS). Approximately 100 days after BMT, B cells aggregated in some of the white pulp, forming primary follicles, whereas marginal zones could not be detected. Beyond 4 months after BMT, the PALS, the lymphoid follicle, and the marginal zone of the white pulp could be seen in most of the recipients' spleens. However, the recovery of the marginal zone was poor up to 10 months after BMT. Thus, the white pulp was reconstituted sequentially, beginning in the PALS, followed by reconstitution in lymphoid follicles, and finally in the marginal zone. The development of the PALS corresponded well with the appearance of interdigitating dendritic cell, as did the development of lymphoid follicles with the appearance of follicular dendritic cell. The sequential reconstitution of the white pulp demonstrated in this study provides the morphological basis for the functional immune recovery of marrow recipients. In particular, the delay of the marginal zone reconstitution seems to be responsible for the functional asplenia of long-term survivors.

Adolescent↗
PubMed1992

Cloning and sequence analysis of a snake, Atractaspis engaddensis gene encoding sarafotoxin S6c.

A 469 base pair genomic DNA, which encodes the mature region of a snake cardiotoxic peptide, sarafotoxin S6c, was isolated from the liver of the burrowing asp, Atractaspis engaddensis. The nucleotide sequence encoding the mature peptide region showed a high sequence homology with those of mammalian vasoconstrictor peptides, endothelin family as expected from the high homology of their amino acid sequences. In contrast, both of the upper and lower flanking sequences of sarafotoxin gene and the deduced amino acid sequence of the sarafotoxin precursor were quite different from those of endothelin family. These results suggest that the ancestral gene and biosynthetic pathway of sarafotoxins are different from those of endothelin.

Amino Acid Sequence↗
PubMed1992

Contribution of the 6-120 disulfide bond of alpha-lactalbumin to the stabilities of its native and molten globule states.

The unfolding and refolding of a derivative of alpha-lactalbumin, in which the disulfide bond between Cys6 and Cys120 is selectively reduced and S-carboxymethylated, are investigated by equilibrium and kinetic circular dichroism measurements. The native conformation of this derivative is known to be essentially identical to that of intact alpha-lactalbumin. The equilibrium unfolding of the derivative involves a stable intermediate, which is also similar to the molten globule state of the disulfide intact protein. The results of stopped-flow circular dichroism experiments show that the same intermediate is formed rapidly as a transient intermediate in kinetic refolding. The conformational stabilities for the native and intermediate states have been estimated and compared with the stabilities for the corresponding states of intact alpha-lactalbumin. The stabilization of the native state by the disulfide has been interpreted in terms of a decrease in chain entropy in the unfolded state and elimination of the strain imposed on the disulfide bond in the native state. The molten globule state is also stabilized by the disulfide bond, although the degree of stabilization of the molten globule state is smaller than of the native state. The results suggest that, in the molten globule state, some ordered structures are present within the loop moiety formed by the 6-120 disulfide.

Animals↗
PubMed1992

Pituitary adenylate cyclase activating polypeptide stimulates gallbladder motility in conscious dogs.

We investigated whether pituitary adenylate cyclase activating polypeptide (PA-CAP27 and PACAP38) had any effect on gallbladder motility in conscious dogs, in which force transducers were chronically implanted in the gastric antrum, duodenum and gallbladder. PACAP27 and PACAP38 were administered intravenously during the digestive and interdigestive states at doses of 30, 100 and 300 pmol/kg. By way of comparison, cholecystokinin octapeptide (CCK-OP) was administrated at doses of 3, 9 and 27 pmol/kg. As a result, each peptide evoked transient and tonic contractions both in the digestive and interdigestive states, and the effect on the motor index was dose dependent. PACAP27 and PACAP38 were 0.11 +/- 0.03 and 0.04 +/- 0.01 as potent as CCK-OP in the digestive state, and 0.18 +/- 0.04 and 0.02 +/- 0.01 in the interdigestive state, respectively, on a molar basis. Although PACAP27 and PACAP38 belong to the vasoactive intestinal polypeptide (VIP) family, intravenous administration of 300 pmol/kg of VIP had no effect on interdigestive gallbladder motility, but on the other hand inhibited gallbladder motility in the digestive state. The contractile effects of PACAP27 and PACAP38 were almost completely abolished by pretreatment with atropine or hexamethonium, but not with L364718. An in vitro study using canine gallbladder strips showed that PACAP27 and PACAP38 had no effect on spontaneous gallbladder motor activity evoked by electric field stimulation, CCK-OP or acetylcholine. It was concluded that PACAP27 and PACAP38 stimulate gallbladder motility in conscious dogs through a preganglionic cholinergic mechanism.

Animals↗
PubMed1992

Binding sites for pituitary adenylate cyclase activating polypeptide (PACAP): comparison with vasoactive intestinal polypeptide (VIP) binding site localization in rat brain sections.

Pituitary adenylate cyclase activating polypeptide (PACAP) is structurally similar to vasoactive intestinal polypeptide (VIP). We investigated the characteristics and topographical distribution of [125I]PACAP binding sites compared with those of [125I]VIP binding sites in the rat brain. Radiolabeled PACAP and VIP showed highly specific binding to sections at the level of the dorsal hippocampus. The specific binding of [125I]PACAP was 10 times higher than that of [125I]VIP in hippocampal sections. [125I]PACAP binding was scarcely displaced by unlabeled VIP, while [125I]VIP binding was effectively displaced by unlabeled PACAP. Therefore, PACAP binding sites may reflect both PACAP specific binding sites and VIP/PACAP binding sites. However, the amount of VIP/PACAP binding sites was negligibly low. Autoradiography revealed that [125I]PACAP binding sites were dense in the piriform cortex, diagonal band, accumbens nucleus, anterior part of the striatum, hippocampal formation, habenular nucleus, lateral hypothalamic area, superior colliculus and dorsal raphe nucleus. Moderate to high labeling was observed in the medial septal nucleus, olfactory tubercle, caudal part of the striatum, most parts of the thalamus, supraoptic and periventricular hypothalamic nuclei, central gray, substantia nigra pars compacta, locus coeruleus, pontine reticular nucleus and cerebellum. Distribution pattern was remarkably different from that of [125I]VIP binding sites in the hippocampal formation, lateral hypothalamic area, substantia nigra pars compacta, pontine reticular nucleus and cerebellum. The present results suggest that PACAP may have a physiological role in the regulation of the central nervous system.

Animals↗
PubMed1992

Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNA in CHO cells and characterization of the products.

cDNA encoding human PACAP precursor was expressed in non-neuroendocrine Chinese hamster ovary cells, CHO-K1, The cells were transfected with expression vector (pTS705) containing the human PACAP cDNA by electroporation. A cell line which produced more than 80 ng/ml of immunoreactive PACAP (ir-PACAP) into the conditioned medium was established. RP-HPLC analysis of culture medium of this established cell line exhibited the presence of two types of PACAP, i.e. PACAP38 and PACAP27. At the same time, it was also revealed that immunoreactive PACAP-related peptide (ir-PRP) was secreted into the cultured medium. The ir-PACAPs were confirmed to ahve biological activities such as induction of cAMP and neurite outgrowth in rat pheochromocytoma PC12h cells.

Adenylyl Cyclases↗
PubMed1992

Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline.

Pituitary adenylate cyclase activating polypeptide (PACAP) provoked the rat chromaffin cells to secrete adrenaline. Within 20 min, the amount of adrenaline secreted by PACAP (10(-8) M) was as much as that caused by acetylcholine (10(-4) M). PACAP, but not acetylcholine, induced a long-term (over 120 min) increase in secretion of adrenaline. PACAP also activated adenylate cyclase and elevated cytosolic Ca2+ concentration. Furthermore, we found immunoreactive PACAP and PACAP binding sites in the rat adrenal medulla. These results suggest that PACAP has an important role in stimulating secretion of adrenaline in the adrenal medulla.

Adrenal Cortex↗