The single kinin receptor signals to separate and independent physiological pathways in Malpighian tubules of the yellow fever mosquito

Abstract

In the past, we have used the kinins of the cockroach Leucophaea (the leucokinins) to evaluate the mechanism of diuretic action of kinin peptides in Malpighian tubules of the yellow fever mosquito Aedes aegypti. Now using the kinins of Aedes (the aedeskinins), we have found that in isolated Aedes Malpighian tubules all three aedeskinins (1 microM) significantly 1) increased the rate of fluid secretion (V(S)), 2) hyperpolarized the basolateral membrane voltage (V(bl)), and 3) decreased the input resistance (R(in)) of principal cells, consistent with the known increase in the Cl(-) conductance of the paracellular pathway in Aedes Malpighian tubules. Aedeskinin-III, studied in further detail, significantly increased V(S) with an EC(50) of 1.5 x 10(-8) M. In parallel, the Na(+) concentration in secreted fluid significantly decreased, and the K(+) concentration significantly increased. The concentration of Cl(-) remained unchanged. While the three aedeskinins triggered effects on V(bl), R(in), and V(S), synthetic kinin analogs, which contain modifications of the COOH-terminal amide pentapeptide core sequence critical for biological activity, displayed variable effects. For example, kinin analog 1578 significantly stimulated V(S) but had no effect on V(bl) and R(in), whereas kinin analog 1708 had no effect on V(S) but significantly affected V(bl) and R(in). These observations suggest separate signaling pathways activated by kinins. One triggers the electrophysiological response, and the other triggers fluid secretion. It remains to be determined whether the two signaling pathways emanate from a single kinin receptor via agonist-directed signaling or from a differentially glycosylated receptor. Occasionally, Malpighian tubules did not exhibit a detectable response to natural and synthetic kinins. Hypothetically, the expression of the kinin receptor may depend on developmental, nutritional, and/or reproductive signals.

Fig. 1.

Experimental methods. Ramsay fluid secretion assay (A), and 2-electrode voltage-clamp method (B) for measurements of the basolateral membrane voltage (V_bl) and the input resistance (R_in) of a principal cell (PC) in an isolated Malpighian tubule [modified from Wu and Beyenbach (64)]. The Ramsay assay requires a minimum of 5 to 10 min before effects of kinins added to the peritubular bath are observed. The 2-electrode voltage-clamp method yields immediate data of voltage and resistance. A scanning electron micrograph of a Malpighian tubule of a female mosquito (Aedes aegypti) is shown in B.

Fig. 2.

Effects of leucokinin-VIII and aedeskinins-I, -II, and -III on the rate of transepithelial fluid secretion and the V_bl and R_in of principal cells in isolated Malpighian tubules of female Aedes aegypti. All kinins were used at a concentration of 10⁻⁶ M. Data are means ± SE with the number of paired tubule experiments in parenthesis. *P < 0.05, **P < 0.001, ***P < 0.0001 paired t-test. Data of the effect of leucokinin-VIII on fluid secretion were taken from a previous study (39) for comparison with the effect of aedeskinins.

Fig. 3.

A: dose-response curve for the effects of aedeskinin-III on transepithelial fluid secretion in isolated Malpighian tubules of Aedes aegypti. The effect of aedeskinin-III (10⁻⁶ M) on the concentrations of ions in secreted fluid and the rates of transepithelial ion secretion are shown in B and C, respectively. Open and shaded bars represent means ± SE with the number of paired tubule experiments in parentheses. *P < 0.02, **P < 0.01, ***P < 0.001, paired t-test.

Fig. 4.

Effects of synthetic kinin analogs on the rate of transepithelial fluid secretion and the V_bl and R_in of principal cells in isolated Malpighian tubules of female Aedes aegypti. Kinin analogs were used at a concentration of 10⁻⁶ M. Analog 1708 stimulated fluid secretion in some tubules and inhibited it in other tubules, such that, on average in 29 tubules, analog 1708 had no significant effect on fluid secretion. Open and shaded bars represent means ± SE with the number of tubule experiments in parentheses; *P < 0.05, **P < 0.01, paired t-test.

Fig. 5.

Representative electrophysiological responses of Malpighian tubules of Aedes aegypti to stimulation with kinins and analogs. Sham Ringer bath change at a Ringer flow rate of 5 ml/min (A); tubule with nonoscillating, high V_bl (B); tubule with the usual oscillating V_bl (C); aedeskinin receptor desensitization (D);and lack of effect of kinin analog 1578 on tubule electrophysiology (E). AK, aedeskinin.

Fig. 6.

Models of ligand/receptor interactions. A: ligand-dependent signaling via a single receptor; [model adapted from Berg and Clarke (2)]. B: glycosylation-dependent signaling, where R₁ and R₂ represent glycosylated and nonglycosylated forms of the same receptor.