RESUMEN
In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt.
Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Neuroblastoma/enzimología , Proteínas Quinasas/metabolismo , Línea Celular Tumoral , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Fosforilación/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptores Muscarínicos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Although many perspectives on the impact of the Health Insurance Portability and Accountability Act of 1996 (HIPAA) on health services research have been wide spread, little empirical evidence has been reported about HIPAA-related barriers during the implementation of research projects. Using three cases of health services research projects, this study examined practical barriers created by HIPAA regulation. During the stage of implementation of the three projects, we experienced some HIPAA-related concerns, previously raised in the field of health services research. We found that technically complicated consent forms and privacy protection forms as well as socially-sensitive clinical conditions make patients less willing to participate in research projects; concerns about safety of patient medical information makes health organizations more hesitant to let researchers have access to the patient's information, especially through electronic transfer; more restrictive IRB processes and challenging patient recruiting processes make health services researchers reluctant to go through the process; and as a result, they may compromise with the scientific soundness of the project. Overall, HIPAA complicates the research process and requires more resources and longer time to conduct research.