Northern blot analysis shows that TCA3 is expressed after activation with either PMA/ionophore or high affinity IgE cross-linking in C57 cells. mRNA of TCA3 is induced within 30 min to 1 hour, peaks at 2 to 3 hours, and disappears at 4 to 6 hours. Half-life of mRNA determined by adding actinomycin D is approximately 2 hours. These results suggest the TCA3 is regulated transcriptionally. Nuclear run-on assays show that the rate of TCA3 mRNA transcription is increased after activation consistent with transcriptional regualation. CAT assay show no detectable promotor activity in TCA3 promotor CAT constructs up to 1.3 kb upstream TATA, while HIV-CAT/TATA constructs showed constitutive expression with 3- to 5-fold induction with PMA. Primary bone marrow mast cells as well as various mast cell lines contain and secrete ET-1. ET-1 secretion is not directly related to mast cell degranulatin, but rather synthesized and secreted hours later. Mast cells express a single class of high affinity ETA receptors resembling the ET receptor. Stimulation of mast cell ET-1 receptors does not provoke histamine release or result in a mitogenic response of bone marrow derived cultured mast cells. We examined the effect of thrombin on astrocytic endothelind and found that endothelin-1 is released into the culture fluid in response to thrombin treatment. This increased production of endothelin-1 is not accompanied by an increase in steady state levels of endothelin-1 mRNA as assessed by reverse transcriptase polymerase chain reaction, even though thrombin stimulation leads to increased inositolphospholipid turnover and activation of the nuclear factor AP1. Thus, astocytic production of endothelin-1 may be mainly post-transcriptionally regulated in response to thrombin stimulation. In addition, two endothelin receptor genes (ET/A and ET/B) were found to be transcribed simultaneously in primary astrocyte cultures, and both thrombin and endothelin-1 stimulation result in a temporary decrease in ET/A mRNA. These studies suggest a role for thrombin in the regulation of brain perfusion through endothelin-1 expression.
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