1,25-Dihydroxyvitamin D3-mediated alterations in microtubule proteins isolated from chick intestinal epithelium: analyses by isoelectric focusing.
Recent work has indicated that vectorial Ca2+ transport across the intestinal epithelium occurs in vesicles and may involve the participation of microtubules [Nemere et al., 1986]. Since 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulates this Ca2+ transport process, microtubule (MT) isotypes were studied as a potential regulatory point. The effect of 1,25(OH)2D3 status on tubulin isotypes was analyzed by isoelectric focusing (IEF) gels of taxol stabilized MTs prepared from intestinal epithelium of vitamin D-deficient chicks dosed with vehicle (-D) or 1.3 nmoles of 1,25(OH)2D3 (+D) 2.5, 5, 10, 15, or 43 h prior to sacrifice. Four bands, one of which was identified as alpha-tubulin on the basis of Western analysis, increased in Coomassie Blue staining intensity 5-15 h after 1,25(OH)2D3, corresponding to the time course of augmented vesicular Ca2+ transport. Dose-response studies revealed similar changes in tubulin isotype profiles in IEF gels, again corresponding to doses known to elicit enhanced Ca2+ absorption (52-6,500 pmoles of hormone). The role of Ca2+ transport was also examined. Isoelectrically focused intestinal epithelial tubulin from -D chicks allowed to transport Ca2+ for 30 min revealed increased staining of bands relative to nonabsorbing -D controls. By comparison, Ca2+ transport in +D chicks resulted in fainter bands relative to nonabsorbing, +D controls. MTs prepared from fasted or fed chicks revealed similar changes upon IEF, but of much smaller magnitude. Enhanced phosphorylation did not account for the appearance of the more acidic bands, although 1,25(OH)2D3 treatment resulted in decreased 32P content of a presumptive non-tubulin component, relative to preparations from -D controls. Glucocorticoids, which are known to suppress 1,25(OH)2D3-stimulated Ca2+ transport, led to severely diminished levels of total tubulin, as judged by SDS-PAGE, rather than altered tubulin isotypes. Thus, MTs of intestine are subject to regulation by hormonal status, as well as by the amount of Ca2+ available for transepithelial transport.
Nemere
I
I
Department of Biochemistry, University of California, Riverside 92521.
Feld
C
C
Norman
A W
AW
D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004386:Duodenum / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008868:Microtubule Proteins / Q000378:metabolism*; D010766:Phosphorylation; D013997:Time Factors
D008868:Microtubule Proteins; D003907:Dexamethasone; D002117:Calcitriol; D002118:Calcium