gastrins and cionin

Cholecystokinin may play a role in regulation of interdigestive motility, but this still remains to be investigated. CCK constitutes the major hormonal stimulus for postprandial gallbladder emptying. CCK exerts its contractile effects mainly through interaction directly with receptors on the gallbladder smooth muscle cells in the muscle layer, but also through interaction with cholinergic nerves extrinsic and/or intrinsic in nature. Furthermore, CCK can enhance ongoing nicotinic ganglionic transmission occurring in the serosal layer by release of acetylcholine. CCK interaction with the gallbladder smooth muscle CCKA receptor was studied in further detail. CCK contracts strips of gallbladder muscle in a concentration-dependent way with a potency in the nanomolar range in all tested species. The potency is 1,000-fold better than that of gastrin; thus, the receptor is of type CCKA. CCK binding to this receptor is specific and of high affinity, 1,000-fold better than that of gastrin with no differences between the tested species including bovine, porcine, and human. Also, CCK binding affinity was independent of age, gender, or weight of the person and pathology of the human gallbladder. The biochemistry of the CCKA receptor varies between the tested species (bovine and human). Both CCKA receptors are heavily glycosylated, but of different size and carbohydrate content. The bovine CCKA receptor is of apparent size M(r) = 70-85 kD with N-linked complex carbohydrates and sialic acids. The human CCKA receptor is of M(r) = 85-95 kD, with N-linked complex carbohydrates, but no sialic acids. They both have a protein core of apparent size M(r) = 43 kD, with almost identically sized fragments after enzymatic cleavage. Probably the protein cores contain the receptor binding region, which seems well preserved between species. CCK and the CCKA gallbladder muscularis receptor are main regulators of postprandial gallbladder emptying. The biochemistry of the CCKA gallbladder smooth muscle receptor is in accord with newly generated data of purification and cloning of the rat pancreatic CCKA receptor.

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Digestion; Gallbladder; Gastrins; Humans; In Vitro Techniques; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neuropeptides; Receptors, Cholecystokinin; Sequence Homology, Amino Acid

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Ciona intestinalis; Cloning, Molecular; Gastrins; Mammals; Molecular Sequence Data; Neuropeptides; Sequence Homology, Amino Acid; Vertebrates

Cholecystokinin (CCK) and gastrin are vertebrate brain-gut peptides featured by a sulfated tyrosine residue and a C-terminally amidated tetrapeptide consensus sequence. Cionin, identified in the ascidian, Ciona intestinalis, the closest species to vertebrates, harbors two sulfated tyrosines and the CCK/gastrin consensus tetrapeptide sequence. While a putative cionin receptor, cior, was cloned, the ligand-receptor relationship between cionin and CioR remains unidentified. Here, we identify two cionin receptors, CioR1 and CioR2, which are the aforementioned putative cionin receptor and its novel paralog respectively. Phylogenetic analysis revealed that CioRs are homologous to vertebrate CCK receptors (CCKRs) and diverged from a common ancestor in the Ciona-specific lineage. Cionin activates intracellular calcium mobilization in cultured cells expressing CioR1 or CioR2. Monosulfated and nonsulfated cionin exhibited less potent or no activity, indicating that CioRs possess pharmacological features similar to the vertebrate CCK-specific receptor CCK1R, rather than its subtype CCK2R, given that a sulfated tyrosine in CCK is required for binding to CCK1R, but not to CCK2R. Collectively, the present data reveal that CioRs share a common ancestor with vertebrate CCKRs and indicate that CCK and CCK1R form the ancestral ligand-receptor pair in the vertebrate CCK/gastrin system. Cionin is expressed in the neural complex, digestive organs, oral siphon and atrial siphons, whereas the expression of ciors was detected mainly in these tissues and the ovary. Furthermore, cioninergic neurons innervate both of the siphons. These results suggest that cionin is involved in the regulation of siphonal functions.

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Ciona intestinalis; Consensus Sequence; Evolution, Molecular; Gastrins; Molecular Sequence Data; Neuropeptides; Oligopeptides; Phylogeny; Receptors, Cholecystokinin; Tyrosine

Cionin--from the protochordate Ciona intestinalis--is a putative ancestor of cholecystokinin (CCK) and gastrin. Being sulfated on tyrosine in positions 7 and 6 (from the C-terminus), characteristic for CCK and gastrin, respectively, cionin is a structural hybrid of the two peptides. The effects of cionin have previously been characterized in mammalian systems. This study examined a phylogenetically ancient CCK receptor, the rainbow trout (Oncorhynchus mykiss) gallbladder receptor, utilizing cionin, sulfated and nonsulfated CCK and gastrin, and the receptor antagonists L-364,718 and L-365,260. The sulfated peptides induced concentration-dependent contractions of isolated strips of gallbladder with equal efficacy and similar potencies [ED50: 42 (cionin), 23 (CCK-8-s), and 74 nM (gastrin-17-s)], significantly different from the nonsulfated forms [ED50: 1.7 (CCK-8-ns) and 1.9 microM (gastrin-17-ns)]. Ten micromolar L-364,718 and L-365,260 both weakly but significantly inhibited cionin-CCK-8-s, and gastrin-17-s-induced contractions. L-365,260 shifted the concentration response curves 1 1/2 decades to the right and L-364,718 only 1/2 decade. The results confirm that the rainbow trout gallbladder CCK receptor does not distinguish sulfated CCK from sulfated gastrin as do modern CCKA receptors, but does distinguish sulfated from nonsulfated forms of both. However, for optimal effect the receptor does not require a double-sulfated peptide like cionin as might be expected from the lack of selectively between CCK and gastrin. Finally, studies with antagonists known to be specific for either CCK or gastrin receptors in mammalian systems indicate that this ancient receptor behaves more like a mammalian CCKB receptor than as a CCKA receptor.

Topics: Amino Acid Sequence; Animals; Female; Gallbladder; Gastrins; In Vitro Techniques; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neuropeptides; Oncorhynchus mykiss; Sincalide

Topics: Amino Acid Sequence; Animals; Base Sequence; Cholecystokinin; Ciona intestinalis; DNA, Complementary; Gastrins; Gene Expression; Mammals; Molecular Sequence Data; Neuropeptides; Polymerase Chain Reaction; Protein Precursors; Sequence Homology, Amino Acid

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Gallbladder; Gastrins; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neuropeptides; Sequence Homology, Amino Acid; Trout

The protochordean octapeptide cionin is structurally a hybrid of mammalian cholecystokinin (CCK) and gastrin, and thus their possible common ancestor. To determine whether cionin behaves like CCK or gastrin, we examined its effect on canine fundic somatostatin cells and on porcine and bovine gallbladder muscles. Cionin released somatostatin with a potency (ED50 0.15 nM) and efficacy (14.8% of cell content) similar to that of CCK-8 (ED50 0.12 nM, efficacy 16.7%). The efficacies but not the potencies of CCK-8 and cionin differed from those of sulfated gastrin (0.12 nM, 9.7%), nonsulfated gastrin (0.20 nM, 9.4%), and nonsulfated CCK-8 (0.30 nM, 10.4%). CCK and gastrin stimulated contractions of porcine gallbladder muscle strips in a concentration-dependent manner with no differences in efficacy but with characteristic differences in potency. CCK-8 and cionin displayed similar potencies of ED50 2.0 and 2.6 nM; both were significantly different from the ED50 of 0.4 microM for sulfated gastrin and 2.3 microM for nonsulfated gastrin. CCK radioligand binding to membrane-enriched preparations of porcine and bovine gallbladder muscularis was specific and of high affinity. The equilibrium data revealed that binding of CCK and gastrin peptides best fit a single site. CCK-8 and cionin displayed similar affinities [Kd 0.5 nM (porcine), 0.5 nM (bovine, CCK) vs. Kd 0.8 and 0.9 nM (cionin), respectively]. These differed again significantly from Kd 0.6 and 1.5 microM (sulfated gastrin) and 0.7 and 0.2 microM (nonsulfated gastrin). The results show that cionin behaves like CCK rather than gastrin in mammals.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cattle; Cholecystokinin; Dogs; Dose-Response Relationship, Drug; Gallbladder; Gastric Fundus; Gastrins; In Vitro Techniques; Kinetics; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neuropeptides; Receptors, Cell Surface; Sequence Homology, Nucleic Acid; Stomach; Swine

We have purified an acidic octapeptide from the neural ganglion of the protochordate Ciona intestinalis by a three-step procedure including C18 Sep-Pak fractionation, MonoQ ion-exchange chromatography, and C4 reversed-phase high-performance liquid chromatography. The purification was monitored by an immunoassay specific for the alpha-carboxyamidated COOH terminus common to the mammalian brain-gut hormones, cholecystokinin and gastrin. Automated Edman degradation revealed the sequence Asn-Tyr-Tyr-Gly-Trp-Met-Asp-Phe. In accordance with the high acidity of the peptide, amino acid analysis after cleavage with aminopeptidase M showed that both tyrosyl residues are sulfated. Hence, the structure is Asn-Tyr(SO3)-Tyr(SO3)-Gly-Trp-Met-Asp-Phe-NH2, as also confirmed by identity with the synthetic disulfated peptide in different chromatographic systems. The occurrence of two consecutively sulfated tyrosyl residues after a neutral residue challenges present concepts of consensus sites for tyrosyl sulfation. We conclude that the structure of the peptide, named cionin, suits that of a common ancestor for cholecystokinin and gastrin.

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Ciona intestinalis; Gastrins; Indicators and Reagents; Molecular Sequence Data; Neuropeptides; Sequence Homology, Nucleic Acid; Urochordata