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

Abstract

Secretory processes and their regulation have been extensively studied in mammalian salivary parotid glands. However, little is known regarding the secretory mechanism in the venom glands of snakes, which invariably produce one of the most complex of all animal secretions. The pharmacologically important and toxic components of the Malayan spitting cobra (Naja naja sputatrix) venom include postsynaptic neurotoxins (NTX), presynaptic neurotoxins (phospholipase A2, PLA2), and cardiotoxins (CTX) which, for convenience, have been collectively referred to as 'toxins.' We report here for the first time the mechanism of toxin gene expression by studying the accumulated mRNA level and protein synthesis rates for the three toxins over a period of 8 days after stimulation of venom synthesis by manual 'milking' of the venom gland. Immunofluorescence and in situ hybridization were used to localize the toxins and their mRNAs in venom gland sections. The rate of protein synthesis, as determined by immunofluorescence and liquid chromatography-mass spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting that transcriptional regulation of the toxin genes is involved.