Mechanisms and control of secretion in the Malpighian tubules of Tenebrio molitor : an immunohistochemical and electrophysiological study

Fluid secretion by insect Malpighian tubules is controlled by haemolymph-bome factors. Two corticotropin-releasing-factor (CRF)-related diuretic peptides, Tenmo¬DH37 and Tenmo-DH47. previously isolated from Tenebrio molitor, were found to stimulate in vitro tubule preparations of Tenebrio molitor via the second messenger cyclic AMP. The stimulatory effect of Tenmo-DH37 was reversed on addition of endogenous antidiuretic peptides (Tenmo-ADFa and ADFb) and exogenous cardioacceleratory peptide 2b (CAP2b), both acting via the second messenger cyclic GMP. The immunocytochemical localization of Tenmo-DH37 and the second antidiuretic peptide isolated from Tenebrio molitor, Tenmo-ADFb, was investigated using antisera raised against these hormones. Neurosecretory cells immunoreactive to Tenmo-DH37 were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH37 were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-ADFb immunoreactivity was localized in the brain, in two pairs of bilaterally symmetrical cells in the protocerebrum. Tenebrio tubule secretion appears entirely dependent on the surrounding K+ concentration and intracellular measurements of the basolateral (Vbl) and indirectly apical membrane potentials (Vap) indicate an appreciable sensitivity of both membranes to the bath K+ concentration, but not to Na+. Secretion assay and electrophysiological results indicate that K+ uptake across the basolateral membrane is primarily through barium-sensitive K+ channels, but also implicate a bumetanide-sensitive Na+/K+/2CI cotransporter, an ouabain-sensitive Na+/KV+-ATPase and glibenclamide-sensitive KATP channels. Furthermore, electrophysiological evidence suggests that fluid secretion/inhibition by endogenous factors is achieved by influencing at least three parameters simultaneously: the rate of H+ extrusion by the V-ATPase, basolateral K+ conductance, and possibly CI- conductance. The effect of amiloride on fluid secretion and pH indicates the presence of a cationic H+ exchanger in Malpighian tubules of Tenebrio. To our knowledge the mealworm Tenebrio molitor provides the first known example of antagonistic interactions between endogenous neuropeptides acting on Malpighian tubules and this study is the first to demonstrate the presence of KATP channels in an insect epithelium.