From structure-activity relationship studies of rat growth hormone-releasing factor (rGFR) on the vasoactive intestinal peptide (VIP) receptor in an in vitro preparation of exocrine pancreas, we predicted that [4Cl-D-Phe6, Leu17]VIP would be a competitive antagonist for the action of VIP. Micromolar concentrations of synthetic [4Cl-D-Phe6, Leu17]VIP competitively antagonized VIP-stimulated amylase release in the pancreatic preparation and VIP-stimulated short-circuit current changes in a colonic tumor cell line. In addition, [4Cl-D-Phe6, Leu17]VIP inhibited amylase release stimulated by rGRF, high concentrations of secretin (agents that act through the VIP receptor), and peptide contaminants in a preparation of natural glucagon. Finally, [4Cl-D-Phe6, Leu17]VIP did not inhibit the action of agonists for the secretin, GRF, or glucagon receptors.
The ion transport properties of the rough endoplasmic reticulum (RER) from liver have been defined by using measurements of active and potential gradient-driven transport. The Ca21 pump is shown to be electrogenic, and both ATP and potential difference is able to drive vanadate- Accumulation of Ca2+ in a cellular compartment and its release requires cooperation between a primary or secondary active transport system and cognate ion pathways within the same membrane. This paper attempts to define the transport properties of the endoplasmic reticulum fraction associated with Ca2+ signaling in hepatocytes. METHODSA fraction enriched in RER was prepared by differential and sucrose step-gradient centrifugation from rat liver homogenate as described (7). The vesicles of a density greater than 44.5% sucrose were washed and suspended in a medium containing 250 mM sucrose, 5 mM Hepes/Tris (pH 7.0), 0.02% NaN3, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (solution A). The ion transport properties of the relevant membrane vesicles were determined by defining the conditions required for the uptake or efflux of 45Ca2+ and 86Rb+. The radioactivity of the vesicles was determined by passing 50 ,ul of the vesicle suspension through a Dowex 50X8 cation-exchange column with two 0.75-ml aliquots of 250 mM sucrose as eluent (8).The ionic requirements for ATP-dependent Ca2+ uptake were determined by measuring 45Ca2' trapped after the addition ofvesicles to a medium containing 5 The presence of a monovalent cation conductance was monitored by preloading vesicles with a K salt of an impermeant anion, removing the extravesicular salt by passing the suspension over a cation-exchange column and adding the K gluconate-containing vesicles to a solution containing 250 mM sucrose, 5 mM Hepes/Tris (pH 7.0), and 0.4 ,ACi of 86Rb+. As necessary, Ins-P3 or tetraethylammonium chloride (Et4NCl) were added, and the uptake of radioactivity was followed over a period of 30 min. Since the addition of a proton conductance in the form of 5 ,M TCS blocked 86Rb+ uptake, this method defines the presence of a K+ conductance (9).The net uptake of 86Rb+ was followed after addition of sucrose-equilibrated vesicles to a medium containing 250 mM sucrose, 5 mM Hepes/Tris, 0.2 mM EGTA, 10 mM KCl or K gluconate, and 0.4 ,Ci of 86Rb+ in the absence or presence of bumetanide or furosemide at 1.5 mM or of Et4N+ at 5 mM. 4433The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
45Ca2+ fluxes and free cytosolic Ca2+ [( Ca2+]i) were used to describe the Ca2+ permeability and Ca2+ reloading of the agonist-sensitive pool at rest, during stimulation, and at termination of stimulation. A sequence of stimulation with carbachol, inhibition with atropine (cycling), and restimulation with cholecystokinin octapeptide (CCK-8) was used to follow Ca2+ reloading. Reloading of the pool required extracellular Ca2+ and was measured as an increased rate and extent of 45Ca2+ uptake into the acini. The 45Ca2+ incorporated into cycled acini could be completely released with CCK-8. The dose-response curves for 45Ca uptake and release were identical to those of the hormonally evoked [Ca2+]i increase. The increased 45Ca2+ uptake during reloading was not due to an expansion of any intracellular pool size but reflects the labeling of the pool to isotopic equilibrium in cycled acini. The rate constant of Ca2+ efflux from the pool of resting cells was approximately 0.67 +/- 0.01/h. With stimulation, the Ca2+ permeability of the pool membrane rapidly increased, resulting in Ca2+ release into the cytosol and an increase in [Ca2+]i. With termination of stimulation, the Ca2+ permeability of the pool membrane rapidly decreased while the pool continued to reload with extracellular Ca2+. Labeling of the pool to isotopic equilibrium allowed determination of the amount of Ca2+ released from the pool, which was 2.94 +/- 0.06 nmol/mg protein. This indicates that total Ca2+ concentration in the pool is in the millimolar range.
45Ca2+ fluxes and free cytosolic Ca2+ measurements in guinea pig pancreatic acini indicated that after agonist stimulation and the release of Ca2+ from the agonist-sensitive pool at least part of the Ca2+ is extruded from the cell, resulting in 45Ca2+ efflux. In the continued presence of agonist, the pool remains permeable to Ca2+ but partially refills with Ca2+. This reloading is dependent on the concentration of extracellular Ca2+. In the absence of extracellular Ca2+, the pool is completely depleted of Ca2+. However, with increasing concentrations of CaCl2 in the incubation solution (from 0.5 to 2.0 mM) there is increasing repletion of the pool with Ca2+ during agonist stimulation. With termination of agonist stimulation, the Ca2+ permeability of the agonist-sensitive pool is rapidly reduced to that measured in the unstimulated cell. As a result, the Ca2+ incorporated into the pool during the stimulation period is rapidly trapped within the pool and exchanges poorly with medium Ca2+. Subsequently, the pool completely refills with Ca2+. The rate of Ca2+ reloading at the termination of agonist stimulation is slower than the conversion of the pool to the impermeable state. In incubation media containing 1.3 mM CaCl2, the half-time for reloading at the termination of stimulation is 5 min. These observations demonstrate the characteristics of Ca2+ reloading of the agonist-sensitive pool both during stimulation and at the termination of stimulation.
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.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.