In situ hybridization and immunohistochemical analysis of the expression of cardiotoxin and neurotoxin genes in Naja naja sputatrix

Secretory processes and their regulation have been extensively studied in m ammalian salivary parotid glands. However, little is known regarding the se cretory mechanism in the venom glands of snakes, which invariably produce o ne of the most complex of all animal secretions. The pharmacologically impo rtant and toxic components of the Malayan spitting cobra (Naja naja sputatr ix) venom include postsynaptic neurotoxins (NTX), presynaptic neurotoxins ( phospholipase A(2), PLA(2)), and cardiotoxins (CTX) which, for convenience, have been collectively referred to as "toxins." We report here for the fir st time the mechanism of toxin gene expression by studying the accumulated mRNA level and protein synthesis rates for the three toxins over a period o f 8 days after stimulation of venom synthesis by manual "milking" of the Ve nom gland. Immunofluorescence and in situ hybridization were used to locali ze the toxins and their mRNAs in venom gland sections. The rate of protein synthesis, as determined by immunofluorescence and liquid chromatography-ma ss spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting th at transcriptional regulation of the toxin genes is involved.