Biochemical, cytochemical localization and physiological studies on Malpighian tubules of Locusta migratoria L.
Specific inhibitors of protein kinase A (PKA), Rp-cAMP, protein kinase C (PKC), chelerythrine and staurosporine, and protein phosphatase 1 and 2A okadaic acid, inhibited fluid secretion whilst corpora cardiaca (CC) extract and cAMP were found to stimulate fluid secretion. Tubules inhibited by PKC inhibitors, were significantly stimulated when CC extract was included in the bathing medium, but cAMP did not stimulate under these conditions. The inclusion of cAMP in the presence of Rp-cAMP failed to prevent a further reduction in the rate of secretion compared to that in the presence of Rp-cAMP alone. The addition of CC extract in presence of Rp-cAMP failed to stimulate fluid production. The effect of PKA and PKC inhibitors on the cationic composition of "urine" in the presence and absence of CC extract or cAMP were studied. The composition of the secreted fluid was not significantly changed during stimulation by cAMP. Whilst, CC extract significantly increased [Na(^+)], decreased [K(^+)], increased Na(^+)/K(^+) ratio and increased both Na(^+) flux and K(^+) flux. However, cAMP caused an increase of both Na(^+) flux and flux, but did not alter the Na(^+)/K(^+) ratio. PKC inhibitors significantly reduced the [K(^+)] of the secreted fluid, without affecting its [Na(^+)] . CC extract in the presence of chelerythrine or staurosporine increased [Na(^+)] but [K(^+)] was not reduced below the concentration that already observed in presence of these inhibitors alone. The addition of 1mM cAMP in the presence of chelerythrine or staurosporine did not significant change the ionic composition of the secreted fluid compared to that seen in presence of these inhibitors alone. lµM chelerythrine or staurosporine caused a significant increase in the Na(^+)/K(^+) ratio. This ratio increased further when tubules bathed in chelerythrine or staurosporine saline were also exposed to CC extract. However, cAMP did not change the level of Na(^+)/K(^+) ratio from than that observed in the presence of PKC inhibitors alone. 0.1mM Rp-cAMP caused a significant fall in [Na(^+)] and a rise in [K(^+)]. The addition of CC extract appeared to reverse the effects of Rp-cAMP. The Na(^+) content of the secreted fluid significantly rose, and whilst the mean K(^+) content was somewhat lower, the difference from that observed in the presence of Rp-cAMP alone was not significant. The addition of cAMP to the saline containing Rp-cAMP caused a significant increase in [Na(^+)], restoring it to normal control levels. However, cAMP did not significantly change the [K(^+)] compared with that observed in the presence of Rp-cAMP alone. 0.1mM Rp-cAMP caused a significant decrease in the Na(^+)/K(^+) ratio and both Na(^+) flux and K(^+) flux. In contrast, the addition of CC extract or cAMP to Rp-cAMP saline caused a significant increase in the Na(^+)/K(^+) ratio. Inclusion of either CC extract or cAMP plus Rp- cAMP did not reverse the decrease in Na(^+) and K(^+) flux observed in the presence of Rp-cAMP alone. Immunocytochemical localization and cell fractionation methods used in conjunction with biochemical analyses demonstrate the major of Na(^+)/K(^+)-ATPase activity present on basal cell membranes and HCO(_3)(^-) -stimulated and V-type ATPase activities on the apical cell membranes. The biochemical properties of V-type ATPase activity were studied. Immunogold localization study using specific monoclonal antibodies confirmed that V-type ATPase and Na(^+)/K(^+)-ATPase located on the apical and basal cell membranes, respectively. The results are discussed and a hypothetical model is proposed to account for the role of PKA, PKC and V-type ATPase enzymes in the mechanisms of ion and fluid movement across apical and basal cell surfaces.