Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.
1992; Elsevier BV; Volume: 267; Issue: 29 Linguagem: Inglês
10.1016/s0021-9258(19)36745-6
ISSN1083-351X
Autores Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoStimulated Increase in Cytosolic Free [Mg?] most cells.We also observed that the Ca2+-mobilizing agonist, carbachol, increases the [M$']i in rat sublingual mucous acini by releasing an intracellular M$+ pool.These results strongly suggest that an agonist-induced rise in the [Mg"]i may be intimately involved in modulating the fluid secretion process in salivary gland acinar cells. EXPERIMENTAL PROCEDURESMaterials-Male 150-250-g Wistar strain rats (Charles River, Kingston facility, NY) were used in all experiments.Mag-fura-2 was from Molecular Probes (Eugene, OR).Hyaluronidase (type lS), forskolin, atropine sulfate, Hepes, BSA (type V; bovine serum albumin), FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and carbachol were obtained from Sigma.Collagenase (type CLSPA) was from Worthington.Basal medium Eagle was from GIBCO.Thapsigargin was from LC Services, Woburn, MA TMB-8 (&(diethylamino)octyl 3,4,5-trimethoxybenzoate) was from Aldrich; and tBuBHQ (2,5-di-(terbutyl)-l,4-benzohydroquinone) was from Fluka, Ronkonkoma, NY.All other chemicals used were of the highest grade available.The digestion medium consisted of BME medium, 1% BSA, 50 units/ml collagenase, and 0.02 mg/ml hyaluronidase.Unless otherwise stated the experiments were carried out in a Modified Earle's-Hank's solution (MEH) containing 110 mM NaCl, 25 mM NaHC03, 20 mM Hepes, 10 mM glucose, 5.4 mM KC1, 1.2 mM CaC12, 0.8 mM MgS04, 0.4 mM KH2P04, and 0.33 mM NaH2P04 adjusted to pH 7.4 at 23 "C with NaOH after bubbling with 95% 0 2 , 5% CO,.For the Na+-free MEH solution, Na+ salts were replaced with N-methyl-Dglucamine.Isolation of Sublingual Acini-After CO, anesthesia, three rats were killed by exsanguination and the glands treated as described previously (Melvin et al., 1991a).Briefly, the sublingual glands were removed and injected with ice-cold digestion medium (0.2 ml/gland).Glands were dissected free of connective tissue and minced in icecold digestion medium.The minced glands were incubated at 37 "C with continuous gassing (95% O,, 5% COP) and shaking in 10 ml of digestion medium.The incubated glands were dispersed by gently pipetting 10 times with a 10-ml pipette at 15-min intervals.After 45 min of digestion, the medium was changed by centrifuging (-500 X g ) for 15 s and replacing the supernatant with fresh digestion medium.After digesting the gland mince for a total of 1.5 h, the well dispersed sublingual acini were washed three times with MEH containing 0.01% BSA at room temperature.The resulting acini were separated into aliquots consisting of small (3-15 acini/clump) and large aggregates by centrifugation at -50 X g for 15 s.The supernatant was recentrifuged at 500 X g for 1 min to obtain the smaller aggregates which were used for the [Mg2+], determinations.Intracellular Mg2+ Measurement-Isolated sublingual acini were loaded with mag-fura-2/AM (2 p ~) at room temperature for 30 min essentially as described previously for rat hepatocytes (Raju et al., 1989).Stock solutions of 2 mM mag-fura-Z/AM were made in dimethyl sulfoxide so that the final concentration of dimethyl sulfoxide during loading was 0.1%.After loading, the acini were washed and centrifuged at 500 X g for 1 min and then resuspended in MEH containing 0.01% BSA.The mag-fura-2-loaded acini were agitated continuously and bubbled with 95% O,, 5% CO, at room temperature until use.An aliquot of mag-fura-2-loaded acini was centrifuged at 500 X g for 15 s, and the supernatant was then decanted.Acini were resuspended in BSA-free MEH to promote attachment and added to a coverslip mounted to the bottom of a perfusion chamber.Solutions were bubbled continuously with 95% 02, 5% COZ.The perfusion rate was -4 ml/min, turning over the solution in the perfusion chamber
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