Leucokinin VIII (Leucokinin 8)

Effects of leucokinin-VIII on Aedes Malpighian tubule segments lacking stellate cells

The diuretic peptide leucokinin is known to increase fluid secretion in Malpighian tubules of the yellow fever mosquito Aedes aegypti by increasing a transepithelial Cl(-) conductance. The present study sought to examine whether stellate cells provided this transepithelial conductance in Aedes Malpighian tubules as they do in Drosophila Malpighian tubules. Aedes Malpighian tubule segments with and without stellate cells were perfused in vitro for measurements of the transepithelial voltage (V(t)), resistance (R(t)) and Cl(-) diffusion potentials (DP(Cl)). In 11 tubule segments containing both principal cells and stellate cells, 1 micro mol l(-1) leucokinin-VIII added to the peritubular bath immediately and significantly decreased V(t) from 39.3+/-14.3 mV to 2.3+/-0.7 mV, decreased R(t) from 12.4+/-2.6 kOmegacm to 2.4+/-0.3 kOmegacm, and increased DP(Cl) from 8.2+/-1.2 mV to 42.1+/-5.4 mV. These effects of leucokinin-VIII were qualitatively and quantitatively similar in six tubule segments containing no stellate cells; V(t) decreased from 37.8+/-7.0 mV to 3.4+/-0.6 mV, R(t) decreased from 8.8+/-2.1 kOmegacm to 1.7+/-0.2 kOmegacm, and DP(Cl) increased from 5.8+/-2.6 mV to 50.0+/-2.1 mV. Thus, stellate cells are not required for signaling or mediating the effects of leucokinin in Malpighian tubules of Aedes aegypti. The results further support previous observations that principal cells signal the effects of leucokinin to increase the Cl(-) conductance of the paracellular pathway through septate (or tight) junctions.

Leucokinin activates Ca(2+)-dependent signal pathway in principal cells of Aedes aegypti Malpighian tubules

The role of Ca(2+) in mediating the diuretic effects of leucokinin-VIII was studied in isolated perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Peritubular leucokinin-VIII (1 microM) decreased the transepithelial resistance from 11.2 to 2.6 kOmega. cm, lowered the transepithelial voltage from 42.8 to 2.7 mV, and increased transepithelial Cl(-) diffusion potentials 5.1-fold. In principal cells of the tubules, leucokinin-VIII decreased the fractional resistance of the basolateral membrane from 0.733 to 0.518. These effects were reversed by the peritubular Ca(2+)-channel blocker nifedipine, suggesting a role of peritubular Ca(2+) and basolateral Ca(2+) channels in signal transduction. In Ca(2+)-free Ringer bath, the effects of leucokinin-VIII were partial and transient but were fully restored after the bath Ca(2+) concentration was restored. Increasing intracellular Ca(2+) with thapsigargin duplicated the effects of leucokinin-VIII, provided that peritubular Ca(2+) was present. The kinetics of the effects of leucokinin-VIII is faster than that of thapsigargin, suggesting the activation of inositol-1,4,5-trisphosphate-receptor channels of intracellular stores. Store depletion may then bring about Ca(2+) entry into principal cells via nifedipine-sensitive Ca(2+) channels in the basolateral membrane.

Regulation of epithelial shunt conductance by the peptide leucokinin

Isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti spontaneously secrete NaCl, KCl and water across an epithelium of modest transepithelial resistance (40-80 omega cm2) and high transepithelial voltage (30-70 mV, lumen positive). Transepithelial electrochemical potentials indicate that Na and K are secreted by active and Cl by passive transport mechanisms. The addition of synthetic leucokinin-VIII (LK-VIII, insect myotropic peptide) to the peritubular bath significantly increases the rates of transepithelial NaCl, KCl and water secretion. In parallel, LK-VIII depolarizes the transepithelial voltage from 59.3 to 5.7 mV, decreases the transepithelial resistance from 57.7 to 9.9 omega cm2, and renders the basolateral and apical membrane voltages nearly equipotential (approximately -90 mV). Unilateral step changes of the [Cl] in the peritubular bath or tubule lumen elicit small transepithelial Cl diffusion potentials in the absence of LK-VIII but large transepithelial Cl diffusion potentials, up to 85% of Nernst equilibrium potentials, in the presence of LK-VIII. In Malpighian tubules treated with dinitrophenol for estimates of the shunt resistance Rsh, LK-VIII reduces Rsh from 52.5 to 5.8 omega cm2. Bilateral reductions of the Cl concentration in tubule lumen and peritubular bath fully restore Rsh to 55.8 omega cm2 in the presence of LK-VIII. LK-VIII has no effects when presented from the luminal side. These results suggest that LK-VIII increases the Cl conductance of the epithelial shunt via a receptor located at the basolateral side of the epithelium.

Leucokinin increases paracellular permeability in insect Malpighian tubules

There are two major transport pathways across epithelia: a transcellular pathway through cells and a paracellular pathway between cells. Previous electrophysiological studies in mosquito Malpighian tubules suggested that the neuropeptide leucokinin-VIII (LK-VIII) increases the chloride permeability of the paracellular pathway. To test the effect of LK-VIII on the paracellular pathway further, we measured transepithelial permeabilities of inulin and sucrose in isolated Malpighian tubules from the mosquito Aedes aegypti. Cell membranes are impermeable to inulin and sucrose, leaving the paracellular pathway as the only route for their transepithelial permeation. LK-VIII (10(-6) mol l-1) significantly increased transepithelial permeability to both inulin (by 73.8 %) and sucrose (by 32.4 %) in parallel with a significant increase in rates of transepithelial fluid secretion (by 75­90 %). Cyclic adenosine monophosphate (cyclic AMP, 10(-4) mol l-1), which is known to stimulate transcellular transport, also increased rates of transepithelial fluid secretion (by 57­59 %), but it did so without increasing the permeability to sucrose and inulin. Thus, LK-VIII increases the permeability of the paracellular pathway whereas cyclic AMP does not.

Voltage clamping single cells in intact malpighian tubules of mosquitoes

Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways.