Excretion

The mechanism of action of leucokinin in malpighian tubules of the Yellow Fever mosquito Aedes aegypti: Requirement of intra- and extracellular calcium

M.-J. YU, R. Masia & K. W. Beyenbach

Dept. of Biomedical Sciences, Cornell University, VRT 8014, Ithaca, NY 14853, USA

The insect diuretic neuropeptide leucokinin-VIII (LK-VIII) increases rates of transepithelial electrolyte and fluid secretion in Malpighian tubules of Aedes aegypti by increasing the Cl - conductance of the shunt pathway (Pannabecker et al., J. Membr. Biol. 132, 63, 1993). Upon the addition of 1 µM LK-VIII to the Ca 2+ -containing peritubular Ringer of isolated perfused Malpighian tubules, the transepithelial voltage (Vt) immediately depolarized from 65.4 mV to 1.6 mV in parallel with the drop of the transepithelial resistance (Rt) from 13.5 K?cm to 2.4 K?cm. These voltage and resistance changes reflect the 'self-inflicted' short-circuit that stems from the large increase in the Cl - conductance of apparently septate junctions. Vt and Rt remained low as long as LK-VIII and Ca 2+ (1.7 mM) were present in the peritubular Ringer. In Ca 2+ -free peritubular Ringer (0 mM Ca 2+ and 1 mM EGTA), LK-VIII lowered Vt and Rt only transiently. Thereafter, Vt and Rt oscillated in parallel at a frequency of 3 oscillations/min between short-circuited and open-circuited states. Adding Ca 2+ to the peritubular medium in quantities that exceed the buffer capacity of EGTA, restored the full short-circuit effect of LK-VIII, i.e. permanent low values of Vt and Rt. These results suggest that intracellular Ca 2+ stores serve as the immediate Ca 2+ source for the initial, transient drop of Vt and Rt that is triggered by LK-VIII in Ca 2+ -free media, while extracellular Ca 2+ serves the sustained, steady state opening of the shunt Cl - conductance. In parallel studies using two electrode voltage clamp methods in principal cells, the addition of the Ca 2+ channel blocker verapamil (2 µM) to the peritubular Ca 2+ -containing Ringer bath increased the cell input resistance by 20%, suggesting the presence of Ca 2+ channels in the basolateral membrane of principal cells. However, verapamil did not block the effects of LK-VIII, for which blocker concentrations higher than 2 µM may be required. In summary, these studies support the widely held view that the effects of leucokinin in Malpighian tubules are mediated via G-protein, phospholipase C, inositol tri-phosphate and Ca 2+ (Veenstra, 1994; O'Donnell et al., Am. J. Physiol. 274, R609, 1998; Clark et al., J. Exp. Biol. 201, 1753, 1998), to which our study adds a role of both intra- and extracellular calcium. (NSF IBN 9604394)

Index terms: leucokinin, calcium, EGTA, IP3, G-protein, verapamil.

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