Summary.We have found 5 profilin cDNAs in cultured parsley cells, representing a small gene family of about 5 members in parsley. Specific antibodies were produced using heterologously expressed parsley profilin as antigen. Western blot analysis revealed the occurrence of similar amounts of profilin in roots and green parts of parsley plants. Immunocytochemical staining of parsley cells infected with the oomycetous plant pathogen Phytophthora infestans clearly revealed that profilin accumulates at the site on the plasma membrane subtending the oomycetous appressorium, where the actin cables focus. We also observed the accumulation of Rop GTPases around this site, which might point to a potential function in signaling to the cytoskeleton.
Based on their bronchodilatory effect, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and COPD. As treatment with β2-adrenoceptor agonists has been associated with worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function, molecular mechanism of the regulation of β2-adrenoceptor expression were studied. MRC-5 human lung fibroblasts were cultured in absence or presence of test substances followed by β2-adrenoceptor messenger RNA (mRNA) determination by qPCR. After inhibition of mRNA synthesis by actinomycin D, β2-adrenoceptor mRNA decreased with a half-life of 23 min, whereas inhibition of protein synthesis by cycloheximide caused an about 5- and 6-fold increase within 1.5 and 4 h, respectively. β2-Adrenoceptor mRNA was increased by about 100 % after 1 h exposure to formoterol or olodaterol but decreased by about 60 % after 4 h agonist exposure. Both effects of β2-adrenoceptor agonists were mimicked by forskolin, a direct activator of adenylyl cyclase and cholera toxin, which stimulates adenylyl cyclase by permanent activation of Gs. β2-Adrenoceptor agonist-induced upregulation of β2-adrenoceptor mRNA was blocked by the β2-adrenoceptor antagonist ICI 118551 and prevented by actinomycin D, but not by cycloheximide. Moreover, in presence of cycloheximide, β2-adrenoceptor agonist-induced reduction in β2-adrenoceptor mRNA was converted into stimulation, resulting in a more than 10-fold increase. In conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. The β2-adrenoceptor gene is under strong inhibitory control of short-living suppressor proteins. β2-Adrenoceptor activation induces via adenylyl cyclase - cyclic adenosine monophosphate (cAMP) signaling a rapid in onset direct stimulation of the β2-adrenoceptor gene transcription, an effect opposed by a delayed upregulation of inhibitory factors.
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