Using an expression cloning strategy, a highanit meltonin receptor cDNA has been isolated from Xenopus laei dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in h-affinity 2-[125I-odomneatonn binding (Kd = 6.3 ± 0.3 x 10-11 M). In addition, six ligands exhibited a rank order of inhibition of specific 2-uI has revealed the presence of high-affinity melatonin receptors (Kd < 2 x 10-10 M) (4-6). Receptor affinity is sensitive to guanine nucleotides and activation of the receptors consistently leads to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism (7-11). High-affinity melatonin receptors thus appear to belong to the superfamily of guanine nucleotide binding protein (G protein)-coupled receptors. To better understand how melatonin acts at a cellular and molecular level, it is important to determine melatonin receptor structure.One of the earliest described actions of melatonin is its ability to cause melanin aggregation in dermal melanophores of amphibians (for review, see ref. 12). This action is mediated through a high-affinity melatonin receptor that is coupled to inhibitory G protein (Gi) (13,14). Using a cDNA library constructed from an immortalized cell line ofXenopus dermal melanophores and a mammalian cell expression cloning strategy, we have succeeded in cloning a high-affinity melatonin receptor. The cDNA encodes a protein that is a newly discovered member of the G protein-coupled receptor family. 11 MATERIALS AND METHODS Expression Cloning. Xenopus laevis dermal melanophores were cultured as described (15). The melanophores were derived from a clonal line that was isolated from a primary culture. Total cellular RNA was isolated from melanophores by extraction with guanidinium thiocyanate and separation in cesium chloride. Melanosomes were removed as described before centrifugation on the cesium chloride gradient (16). Poly(A)+ RNA was isolated by established methods (17).A random-primed cDNA library was constructed with a kit from Pharmacia. Double-stranded cDNA was ligated with nonpalindromic BstXI/EcoRI adaptors (Invitrogen). The cDNA was size-fractionated on an agarose gel, and cDNA>2 2 kb was recovered by electroelution. The size-selected cDNA was ligated into the expression vector pcDNAI (Invitrogen) and introduced into Escherichia coli MC1061/P3 by electroporation.A total of 4 x 105 recombinants were obtained from 5 pg of poly(A)+ RNA and divided into 54 pools of "7400 clones each. Plasmid DNA was prepared from each pool by the alkaline lysis method and transfected into COS-7 cells by the DEAE-dextran method (18). Three days after transfection, cells were incubated with 90 pM 125I-Mel in 50 mM Tris-HCl (pH 7.4) containing 100 mM NaCI, 5 mM KCl, 2 mM CaCl2, and 5% Nu-Serum I (Collaborative Biomedical Products, Bedford, MA) for 2 hr at room temperature. Cells were washed, air dried, and exposed to x-ray film for 14 days. A pool of clones that showed positive signals was subdivided, and the transfection procedure was repeated. This ...
Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause zymogen activation and pancreatitis. The effects of the CCK analog, caerulein, and alcohol on trypsin and chymotrypsin activation in isolated pancreatic acini were examined. Caerulein increased markers of zymogen activation in a time- and concentration-dependent manner. Notably, trypsin activity reached a peak value within 30 min, then diminished with time, whereas chymotrypsin activity increased with time. Ethanol (35 mM) sensitized the acinar cells to the effects of caerulein (10(-10) to 10(-7) M) on zymogen activation but had no effect alone. The effects of ethanol were concentration dependent. Alcohols with a chain length of >or=2 also sensitized the acinar cell to caerulein; the most potent was butanol. Branched alcohols (2-propanol and 2-butanol) were less potent than aliphatic alcohols (1-propanol and 1-butanol). The structure of an alcohol is related to its ability to sensitize acinar cells to the effects of caerulein on zymogen activation.
The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 μM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.
Keywordscaerulein; chymotrypsin; secretin; trypsin THE PREMATURE ACTIVATION of digestive zymogens within the pancreatic acinar cell contributes to the initiation of pancreatitis. In the rodent pancreas, ligands such as CCK or the muscarinic agonist carbachol can cause zymogen activation in isolated pancreatic acini. These ligands activate specific G protein-linked receptors that stimulate phospholipase C and increase cytosolic Ca 2+ . Several reports (7,9) have observed that zymogen activation within the acinar cell requires an increase in cytosolic Ca 2+ . CCK-induced activation depends on the presence of extracellular Ca 2+ and an increase in cytosolic Ca 2+ (9). Removal of extracellular Ca 2+ or chelation of intracellular Ca 2+ blocks caerulein (a CCK homolog)-induced trypsin activation (7,9). Whether an increase in intracellular Ca 2+ alone is sufficient to cause activation is controversial. Increasing cytosolic Ca 2+ by thapsigargin was found to cause zymogen activation in one study but not in another (7,9). Thus additional signaling pathways may act independently or with Ca 2+ to cause zymogen activation.The pancreatic acinar cell has receptors for pathways that are coupled to increasing both cytosolic Ca 2+ and cAMP. Stimulation of acinar cell CCK or muscarinic receptors is linked to increased cytosolic Ca 2+ . Secretin, pituitary adenylate cyclase activating peptide (PACAP), and VIP stimulate receptors that are primarily coupled to cAMP generation. However, some 2+ and cAMP (5). Moreover, although physiological concentrations of CCK do not cause a measurable increase in cAMP, they do activate cAMP-dependent protein kinase (5). Similarly, secretin has been reported in some but not all studies to not only increase cellular cAMP, but to also stimulate phospholipase C activity causing an increase in cytosolic Ca 2+ in the acinar cell (10,12). Because ligands that increase both acinar cell Ca 2+ and cAMP are released in...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.