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Vasoactive intestinal polypeptide- and peptide histidine isoleucine-like immuno-reactivity co-release by the rat gastric fundus
MOLECULAR CLONING OF THE CANINE GASTRIN/ CCK-B RECEPTOR GENE C- Blandizzi, G. Natale, R. Colucci, D. Carignani and M. Del Tacca Institute of Medical Pharmacology, University of Pisa, Via Roma 55, 1-56126 Pisa, Italy Gastrin/CCK-B receptors mediate important functions and have significant pathophysiological implications. In order to better understand the molecular bases for this receptor family, we have cloned and sequenced the gastrin/CCK-B receptor gene from a canine genomic library constructed into phage k-EMBL~3 as vector. A human gastrin/CCK-B receptor cDNA, labeled with 32p by random priming, was used as probe. Positive hybridization signals with two clones were obtained after screening about 6x 105 plaques. Following digestion of these clones with EcoRI and SstI, restriction fragments were subcloned into M13mp18 vector and sequenced in both directions by dideoxynucleotide method. The canine gene, exceeding 7.5 kb in length, contained a 1359-bp open reading frame encompassing five exons (from 151 to 536 bp) interrupted by four introns (from 129 to more than 1700 bp). The open reading frame encoded a protein of 453 aminoacid residues sharing 100% identity with the gastrin receptor cDNA cloned from canine parietal cells. Consensus sequences for alternative splicing at exon 4, to yield two receptor isoforms differing for a pentapeptide cassette (GGAGP), were found in the canine gene. A computerized analysis of the 5'-flanking region revealed: 1) high G+C content (78%) within a range of 300 bp from the initiation codon (ATG); 2) a 56bp tandem repeat motif (CA+GC, from -345 to -402 bp) which might play an important role in the regulation of the gene expression; 3) recognition sites for transcriptional regulation factors, including SP1, AP2 and GCF. Two signals for polyadenylation (AATAAA) occur in the Y-flanking region 210 and 590 bp downstream from the stop codon (TGA). It appears that the canine gastrin/CCK-B receptor gene has the potential for the molecular expression of at least two receptor isoforms which might imply significant pharmacological differences.
0t)-~:MET~HIST~NE IN~ T O, Mofini D. C,randi *-andG, Bet~ini Phammcology and of* Anatomy, University of Parma+ Parma, Italy.
microscopy after 60 min~xposur¢ +to absolute ethanolwith or i ~ mg/kgby inttagastri¢rct~+
previously shown to neatly completely ptev~ut+-rimoroscopic~y visible lesions induced by.ethanol. In ~atsgiven ~ p ~ ¢ellSa p p ~ to be stimulate, as derived from ~{+~gh mttnb~r .~ n~ithoCondria and tl~ oxpanded, s~rotory canafictdi-i~wd ~ ' numerous ¢nl0ngcdmicrovilil.MUCous cellswere a l S 0 . ~ W [ t l i
deg~nera~onofm.ms~+~/~g ~ns was . o ~ ~ ~ ~ ? mucosa ~ m ~ o l and concomitantlystimulates pari~i i
~e
:
+
....
Vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and its C-terminal-extended forms, i.e. peptide histidine glycine (PHI-Gly) and peptide histidine valine [PHV(142)], are considered the most likely peptidergic mediators of the non-adrenergic non-cholinergic (NANC) relaxation of the rat gastric fundus. We recently showed that high-frequency electrical field stimulation (EFS) of rat gastric fundus strips in vitro induces the release of PHI-like immunoreactivity (LI), which is primarily represented by PHV(1-42) (Br. J. Pharmacol., 113:541, 1994). The aim of the present study was to investigate the relationship between the relative amounts of VIP- and PHI-LI released by the rat gastric fundus in response to EFS. Longitudinal muscle strips from rat gastric fundi were mounted between parallel platinum electrodes and incubated in Krebs solution under NANC conditions. After a 60-min equilibration period, the strips were subjected to EFS (120 mA, 1 ms, trains of 2 rain) at the frequencies of 13 or 16 Hz. EFS evoked a frequency-dependent release of both VIP- and PHI-LI (99.2+18.9 and 271.9+41.0 pg, mean+s.e.m., n=8, respectively, in response to 13-Hz EFS; 141.0-2_i0.8 and 385.1+29.1 pg, n=20, respectively, in response to 16-Hz EFS). On the molar basis, considering the molar weight of PHV(1-42), VIP-LI was 49.3+4.3 % and 50.5+0.2 % of PHI-LI, at 13- and 16-Hz EFS, respectively. In conclusion, our data indicate that VIP- and PHI-LI are co-released by the rat gastric fundus in response to EFS but not in equimolar amounts, PHI-LI being about double the molar amount of VIP-LI. Supported by CNR, grants n ° 94.02848.CT04 and n ° 94.04204.ST74.
J.-M. Chemnitius, K. H. Haselmeyer', H. Kreuzer and R. Zech" Departments of Cardiology and Biochemistry', Robert-KochStra2e 40, 37075 G6ttingen, Germany.
(R)-~-methylhistamine (MHA), selective agonist of histamine H3. receptors, effectively prevents gastric lesions induced by-alP~late ethanol in rat. The present S ~ is addressed to. ~ ~ rat gastric mu¢osa by light, s c ~ and trammlission e/ootton
calls+
FUNDUS D. Currb, P. Preziosi and G. Ciabattoni Institute of Pharmacology, Catholic University School of Medicine, Rome, Italy.
M E T O C L O P R A M I D E (MCP): INDIRECT P A R A S Y M P A T H O MIMETIC ACTIVITY BY REVERSIBLE INHIBITION OF HUMAN ACETYLCHOLINESTERASE
HISTOLOGICAL STUDY OF GASTROPROTECTION BY
withOUt'prior treatment with ~
VASOACTIVE INTESTINAL POLYPEPTIDE- AND PEPTIDE HISTIDINE ISOLEUCINE-LIKE IMMUNOREACTIVITY CO-RELEASE BY THE RAT GASTRIC
: 7+i:
--12--
Metoclopramide, in the gastrointestinal tract, enhances smooth muscle motility, and thus accelerates transit time. In addition to its blocking action on dopamine DA2-recep tors, the substance is thought to promote-acetylcholine (ACh) release from mventeric neurons. Adverse extrapyramidal MCP-effects can be treated by anticholinergic s u b stances. We identified human acetylcholinesterase (ACHE; EC 3.1.1.7) as an additional molecular target of MCP. Inhibitory effects of MCP on purified AChE from the human central nervous s_y~tem (caudate nucleus) were studied in a kinetic test (25 C) with acetylthiocholine as substrate. Reversible MCP-inhibition (14 pM, 42 pM, 140 pM MCP) showed kinetics of mixed competitive/non-competitive type (inhibition rate constants: Ki : 8.7 pM (competitive)j and Ki' = 76.9 pM (non-competitive)).We conclude that MCP, in the range of therapeutic plasma cdncentrations (maximum about 300 gM after single oral doses), reversibly inhibits human ACHE. The inhibitory effect can only partially be overcome by increased ACh concentrations. Indirect p a r a sympathomimetic MCP-effects can augment both gastrointestinal and extrapyramidal actions of the substance.
0t)-~:MET~HIST~NE IN~ T O, Mofini D. C,randi *-andG, Bet~ini Phammcology and of* Anatomy, University of Parma+ Parma, Italy.
microscopy after 60 min~xposur¢ +to absolute ethanolwith or i ~ mg/kgby inttagastri¢rct~+
previously shown to neatly completely ptev~ut+-rimoroscopic~y visible lesions induced by.ethanol. In ~atsgiven ~ p ~ ¢ellSa p p ~ to be stimulate, as derived from ~{+~gh mttnb~r .~ n~ithoCondria and tl~ oxpanded, s~rotory canafictdi-i~wd ~ ' numerous ¢nl0ngcdmicrovilil.MUCous cellswere a l S 0 . ~ W [ t l i
deg~nera~onofm.ms~+~/~g ~ns was . o ~ ~ ~ ~ ? mucosa ~ m ~ o l and concomitantlystimulates pari~i i
~e
:
+
....
Vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and its C-terminal-extended forms, i.e. peptide histidine glycine (PHI-Gly) and peptide histidine valine [PHV(142)], are considered the most likely peptidergic mediators of the non-adrenergic non-cholinergic (NANC) relaxation of the rat gastric fundus. We recently showed that high-frequency electrical field stimulation (EFS) of rat gastric fundus strips in vitro induces the release of PHI-like immunoreactivity (LI), which is primarily represented by PHV(1-42) (Br. J. Pharmacol., 113:541, 1994). The aim of the present study was to investigate the relationship between the relative amounts of VIP- and PHI-LI released by the rat gastric fundus in response to EFS. Longitudinal muscle strips from rat gastric fundi were mounted between parallel platinum electrodes and incubated in Krebs solution under NANC conditions. After a 60-min equilibration period, the strips were subjected to EFS (120 mA, 1 ms, trains of 2 rain) at the frequencies of 13 or 16 Hz. EFS evoked a frequency-dependent release of both VIP- and PHI-LI (99.2+18.9 and 271.9+41.0 pg, mean+s.e.m., n=8, respectively, in response to 13-Hz EFS; 141.0-2_i0.8 and 385.1+29.1 pg, n=20, respectively, in response to 16-Hz EFS). On the molar basis, considering the molar weight of PHV(1-42), VIP-LI was 49.3+4.3 % and 50.5+0.2 % of PHI-LI, at 13- and 16-Hz EFS, respectively. In conclusion, our data indicate that VIP- and PHI-LI are co-released by the rat gastric fundus in response to EFS but not in equimolar amounts, PHI-LI being about double the molar amount of VIP-LI. Supported by CNR, grants n ° 94.02848.CT04 and n ° 94.04204.ST74.
J.-M. Chemnitius, K. H. Haselmeyer', H. Kreuzer and R. Zech" Departments of Cardiology and Biochemistry', Robert-KochStra2e 40, 37075 G6ttingen, Germany.
(R)-~-methylhistamine (MHA), selective agonist of histamine H3. receptors, effectively prevents gastric lesions induced by-alP~late ethanol in rat. The present S ~ is addressed to. ~ ~ rat gastric mu¢osa by light, s c ~ and trammlission e/ootton
calls+
FUNDUS D. Currb, P. Preziosi and G. Ciabattoni Institute of Pharmacology, Catholic University School of Medicine, Rome, Italy.
M E T O C L O P R A M I D E (MCP): INDIRECT P A R A S Y M P A T H O MIMETIC ACTIVITY BY REVERSIBLE INHIBITION OF HUMAN ACETYLCHOLINESTERASE
HISTOLOGICAL STUDY OF GASTROPROTECTION BY
withOUt'prior treatment with ~
VASOACTIVE INTESTINAL POLYPEPTIDE- AND PEPTIDE HISTIDINE ISOLEUCINE-LIKE IMMUNOREACTIVITY CO-RELEASE BY THE RAT GASTRIC
: 7+i:
--12--
Metoclopramide, in the gastrointestinal tract, enhances smooth muscle motility, and thus accelerates transit time. In addition to its blocking action on dopamine DA2-recep tors, the substance is thought to promote-acetylcholine (ACh) release from mventeric neurons. Adverse extrapyramidal MCP-effects can be treated by anticholinergic s u b stances. We identified human acetylcholinesterase (ACHE; EC 3.1.1.7) as an additional molecular target of MCP. Inhibitory effects of MCP on purified AChE from the human central nervous s_y~tem (caudate nucleus) were studied in a kinetic test (25 C) with acetylthiocholine as substrate. Reversible MCP-inhibition (14 pM, 42 pM, 140 pM MCP) showed kinetics of mixed competitive/non-competitive type (inhibition rate constants: Ki : 8.7 pM (competitive)j and Ki' = 76.9 pM (non-competitive)).We conclude that MCP, in the range of therapeutic plasma cdncentrations (maximum about 300 gM after single oral doses), reversibly inhibits human ACHE. The inhibitory effect can only partially be overcome by increased ACh concentrations. Indirect p a r a sympathomimetic MCP-effects can augment both gastrointestinal and extrapyramidal actions of the substance.