GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability

Abstract

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5'-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5'-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol.

“…In Swiss 3T3 fibroblasts, the bombesinor vasopressin-stimulated elevation of [Ca2+]i was mediated by an increase in the production of 1,4,5-InsP s, while the platelet-derived growth factor-induced eleva-tion of [Ca~+] i was not (Lopez-Rivas et al, 1987). Other reports demonstrated that (intracellular) GTP can release Ca 2+ from different intracellular pools insensitive to InsP 3 (Henne et al, 1987;Nicchitta et al, 1987;Benedetti et al, 1988), and that elevated concentrations of cytosolic Na + can exchange for mitochondrial Ca ~+. Similar pathways may be activated by ATP, although there is not yet any evidence for a specific mechanism.…”

Effects of CoA and acyl-CoAs on GTP-dependent Ca2+ release and vesicle fusion in rat liver microsomal vesicles

“…To investigate further the effects of CoA and palmitoyl-CoA on microsomal vesicle permeability, we examined the effects of both of these compounds on the GTP-dependent increase in mannose 6-phosphate permeability (Nicchitta et al, 1987). Mannose 6-[32P]phosphate, which was used as substrate, was prepared as described by Nicchitta et al (1987).…”

“…To investigate further the effects of CoA and palmitoyl-CoA on microsomal vesicle permeability, we examined the effects of both of these compounds on the GTP-dependent increase in mannose 6-phosphate permeability (Nicchitta et al, 1987). Mannose 6-[32P]phosphate, which was used as substrate, was prepared as described by Nicchitta et al (1987). Mannose-6-phosphatase activity was assayed by measuring the formation of [32P]Pi, which was determined by extraction of [32P]phosphomolybdate complex into butan-2-ol essentially as described by Arion et al (1972).…”

Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum

“…In the endoplasmic reticulum (ER) a variety of GTPdependent functions have been described, but it is not known whether these functions are subserved by the same GTP-binding protein or by a family of GTPbinding proteins. After the initial demonstration of GTPstimulated core glycosylation in rough microsomes (microsomal fractions) by Godelaine and her Belgian colleagues (Godelaine et al, 1977), GTP was shown to activate CDP-diacylglycerol formation (Sribney et al, 1977), to stimulate membrane fusion (Paiement et al, 1980;Comerford & Dawson, 1988), to induce membrane permeability changes (Godelaine et al, 1983;Nicchitta et al, 1987), to promote calcium release (Dawson, 1985;Gill et al, 1986;Henne et al, 1987;Nicchitta et al, 1987;Comerford & Dawson, 1988) and to assist in the integration of nascent proteins into RER (Wilson et al, 1988). We have looked for GTP-binding proteins in RER with [oa-32P]GTP on nitrocellulose blots of roughmicrosomal proteins separated by one-and two-dimensional polyacrylamide-gel-electrophoretic methods.…”