Injection of bovine parathyroid poly(A)+ RNA into Xenopus oocytes confers sensitivity to high extracellular calcium.
Parathyroid cells detect increments in the extracellular [Ca2+], which lead to substantial increases in intracellular free Ca2+ ([Ca2+]i) and, ultimately, to suppression of parathyroid hormone (PTH) secretion. To determine whether mRNA from parathyroid tissue could confer sensitivity to high extracellular Ca2+, we isolated and injected total bovine parathyroid poly(A)+ RNA into Xenopus laevis oocytes. To assess translational activity of the RNA, PTH released into the media was measured. Intact PTH was detected in the medium for < or = 48 h, and injection of increasing amounts of RNA (approximately 0.5-50 ng/oocyte) led to the release of greater quantities of PTH. We screened for the expression of a putative Ca2+ sensor molecule by measuring 45Ca efflux from preloaded oocytes, in response to raising extracellular [Ca2+] from 0.7 to 5.7 mM. This increment in [Ca2+] stimulated 45Ca efflux by 249 +/- 52 cpm over 20 min from eggs injected with parathyroid poly(A)+ RNA (n = 22). This response was significantly greater than 45Ca efflux from any group of controls exposed to the same change in extracellular Ca2+ (p < 0.02), including oocytes injected with either water, cRNA for the platelet-derived growth factor (PDGF) BB receptor, or T cell poly(A)+ RNA. Size-fractionation of poly(A)+ RNA over sucrose gradients demonstrated that mRNA, which induced responsiveness to high extracellular Ca2+, was present in fractions with transcripts of approximately 5-9 kB. Injection of these fractions also conferred sensitivity to the presence of Ba2+ or Sr2+ (both at 5 mM) in the media.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
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Endocrine Research Unit, Veterans Affairs Medical Center, San Francisco, California.
Pratt
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Shoback
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D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D005260:Female; D015759:Ionomycin / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D010280:Parathyroid Glands / Q000737:chemistry*; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger; D013324:Strontium / Q000494:pharmacology; D014982:Xenopus laevis
D012333:RNA, Messenger; D011061:Poly A; D001464:Barium; D015759:Ionomycin; D012313:RNA; D002118:Calcium; D013324:Strontium