RESUMO
PURPOSE: To examine the expression of prostaglandin (PG) receptors EP2, EP4, and FP in a human lens epithelial cell line (HLE-B3) at molecular and pharmacologic levels. METHODS: Reverse transcription-polymerase chain reactions (RT-PCR) were performed with total RNA preparations from HLE-B3 cells using sense and antisense primers for each of the three prostaglandin receptors. The PCR products were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. At the pharmacologic level, the expression of EP4 receptors was determined by measuring intracellular cyclic adenosine monophosphate (cAMP) formation in response to PGE2 (EP1, EP2, EP3, and EP4 agonist) and the EP4 receptor-selective antagonist AH23848. The expression of FP receptors in HLE-B3 cells was explored by measuring intracellular [Ca2+]i mobilization. RESULTS: RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors. Hybridization of the PCR products with specific 32P-labeled probes and restriction digestion of the PCR products further confirmed that they were generated from the respective EP2, EP4, and FP mRNAs. The EP receptor agonist PGE2 significantly increased the cAMP level in HLE-B3 cells. The formation of cAMP by PGE2 was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. Stimulation of HLE-B3 cells by the FP receptor agonist fluprostenol increased [Ca2+]i in a time-dependent manner. CONCLUSIONS: The results of the molecular biologic and pharmacologic experiments showed conclusively the presence of EP4 and FP receptor messenger RNAs and proteins, respectively, in HLE-B3 cells.
Assuntos
Células Epiteliais/metabolismo , Cristalino/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina/metabolismo , Actinas/genética , Actinas/metabolismo , Compostos de Bifenilo/farmacologia , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , AMP Cíclico/metabolismo , Primers do DNA/química , Sondas de DNA/química , Dinoprostona/farmacologia , Células Epiteliais/efeitos dos fármacos , Humanos , Cristalino/citologia , Cristalino/efeitos dos fármacos , Prostaglandinas F Sintéticas/farmacologia , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To investigate the presence of specific prostaglandin receptors in primary cultures of human ciliary muscle cells by measuring agonist-stimulated intracellular calcium mobilization. METHODS: The ciliary muscle cells, cultured from postmortem human ciliary muscle explants, were first characterized by anti-desmin and anti-smooth muscle alpha-actin antibodies. Increase in intracellular calcium concentrations, in fura 2-AM loaded human ciliary muscle cells stimulated by prostaglandin receptor agonists, were determined with a digital fluorescence imaging system. RESULTS: The resting intracellular calcium concentration in the fura 2-AM loaded cells was 60.0 +/- 6.0 nM. The threshold concentration of PG receptor agonists needed to increase the concentration of intracellular calcium was 10(-8) M. The stimulation of these cells by PGF2 alpha, 17-phenyl trinor PGE2, and U46619, the FP, EP1, and TP receptor agonists, resulted in the dose-dependent increase of intracellular calcium. CONCLUSION: The results of the present study suggest that EP1, FP, and TP receptors are present in human ciliary muscle cells.