Investigative ophthalmology & visual science · 2025
Pyrimidinergic P2Y1-Like Nucleotide Receptors Are Functional in Rat Conjunctival Goblet Cells.
СтатьяПоддержка исследования: вне правительства СШАПоддержка NIH: внешняя программаНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Fjærvoll Ketil A, Fjærvoll Haakon K, Yang Menglu, Bair Jeffrey, Utheim Tor P, Dartt Darlene A
Аннотация
PURPOSE: To investigate the presence of uridine-5'-triphosphate (UTP)-activated P2Y1-like nucleotide receptors (P2Y2R, P2Y4R, and P2Y6R) in conjunctival goblet cells (CGCs) and determine if they increase intracellular Ca2+ concentration ([Ca2+]i) and induce mucin secretion.
METHODS: Adult, male rat conjunctiva was used for culture of CGCs. To investigate the expression of P2YRs, mRNA was extracted from CGCs and used for reverse transcription PCR (RT-PCR) with commercially obtained primers specific to P2Y2R, P2Y4R, and P2Y6R. Immunofluorescence (IF) and western blot (WB) analyses were performed using first-passage CGCs and stained with antibodies specific to each P2YR. Furthermore, CGCs were incubated with fura-2/AM, and [Ca2+]i was measured after stimulation with the P2YR selective agonists UTP, uridine 5'-diphosphate (UDP), or UDP-glucose and agonists specific to P2Y2R (MRS 2768), P2Y4R (MRS 4062), and P2Y6R (MRS 2693). [Ca2+]i measurements after P2Y2R and P2Y6R siRNA treatment were performed. Mucin secretion was measured after stimulation of P2Y2R, P2Y4R, and P2Y6R.
RESULTS: mRNA for all pyrimidinergic P2Y1-like receptors was found as single bands of expected base pair number with RT-PCR. The presence of these P2YRs was confirmed with IF microscopy and WB analysis. UTP and UDP elicited concentration-dependent increases in [Ca2+]i. The receptor-specific agonists and UDP-glucose increased [Ca2+]i, although these responses were substantially lower than those elicited by UTP and UDP at 10-4 M and 10-3 M and did not show similar dose dependency. P2Y2R- and P2Y6R-depleted CGCs responded with reduced peak [Ca2+]i. UTP, MRS 2768 (P2Y2R), and UDP each stimulated mucin secretion from CGCs.
CONCLUSIONS: P2Y2R, P2Y4R, and P2Y6R are present and functional in rat CGCs and may represent novel therapeutic targets for dry eye treatment and other types of ocular surface disease.
METHODS: Adult, male rat conjunctiva was used for culture of CGCs. To investigate the expression of P2YRs, mRNA was extracted from CGCs and used for reverse transcription PCR (RT-PCR) with commercially obtained primers specific to P2Y2R, P2Y4R, and P2Y6R. Immunofluorescence (IF) and western blot (WB) analyses were performed using first-passage CGCs and stained with antibodies specific to each P2YR. Furthermore, CGCs were incubated with fura-2/AM, and [Ca2+]i was measured after stimulation with the P2YR selective agonists UTP, uridine 5'-diphosphate (UDP), or UDP-glucose and agonists specific to P2Y2R (MRS 2768), P2Y4R (MRS 4062), and P2Y6R (MRS 2693). [Ca2+]i measurements after P2Y2R and P2Y6R siRNA treatment were performed. Mucin secretion was measured after stimulation of P2Y2R, P2Y4R, and P2Y6R.
RESULTS: mRNA for all pyrimidinergic P2Y1-like receptors was found as single bands of expected base pair number with RT-PCR. The presence of these P2YRs was confirmed with IF microscopy and WB analysis. UTP and UDP elicited concentration-dependent increases in [Ca2+]i. The receptor-specific agonists and UDP-glucose increased [Ca2+]i, although these responses were substantially lower than those elicited by UTP and UDP at 10-4 M and 10-3 M and did not show similar dose dependency. P2Y2R- and P2Y6R-depleted CGCs responded with reduced peak [Ca2+]i. UTP, MRS 2768 (P2Y2R), and UDP each stimulated mucin secretion from CGCs.
CONCLUSIONS: P2Y2R, P2Y4R, and P2Y6R are present and functional in rat CGCs and may represent novel therapeutic targets for dry eye treatment and other types of ocular surface disease.