''Studies on the malpighian tubules of Locusta migratoria miqratorioides (R + F), with particular reference to the role of Na(^+)-K(^+) activated ATPase in fluid secretion

Abstract

The presence of a Na(^+)-K(^+) activated, Mg(^2+) dependent ATPase (E.C. 3.6.1.3.) has been demonstrated in microsomal preparations from Malpighian tubules and hindgutof Locusta miqratoria miqratorioides (R+F) and the conditions for optimal activity determined. The pH optimum was 7.5 and the temperature optima 45ºC for Na(^+)- K(^+)ATPase whilst Mg(^2+)ATPase activity was still rising at 50ºC. Activation energies for the Na(^+)- K(^+) ATPase were 121.6 - (^+) 4.5 and 59.8 (^+)-2.7 Kjoules Mole(^-1) between 6.3-21ºC and 21-42ºC respectively. The Mg(^2+) ATPase had activation energies of 95.8 (^+)- 1.9 and 51.7 - 3.9 Kjoules Mole (-1) over the same ranges. Maximal activation of the Nat(^+)- K(^+)ATPase occured at an ATP : Mg(^2+) ratio of 1:1.3, and at l00mM Na(^+): 20mM K(^+). The Na(^+)- K(^+) ATPase was inhibited by ouabain with a pI(_50) value of 6.1. The Km of Na(^+)-ATPase was 0.15 (^+)- 0.01 mMole 1(^-1) with no significant difference between values obtained at 20ºC, 30ºC and 40ºC. The values for Vmax were 113.6(^+)-42, 213 (^+)- 53 and 373 (^+)- 109 for these temperatures respectively. Physiological studies on fluid secretion by Malpighian tubules in vitro have shown secretion was inhibited by ouabain at concentrations greater than 10(^-6)M, and also in the absence of Na(^+) or K(^+) ions. This strongly suggested the .involvement of Na- K ATPase in secretion which was further implicated by polarographic studies showing a corresponding 35% reduction in oxygen uptake in the presence of ouabain, or absence of K(^+). Similarly the trans-wall potential has been shown to fall in these circumstances. The optimum temperature for secretion was 40ºC, the process having an activation energy of 58.307 Kjoules Mole(^-1) between 5-40ºC. Rate of secretion was inversely proportional to osmotic concentration, and the urine was hyperosmotic to the bathing medium by 22.9 + 2.6m. Osmoles. Fluid secretion was stimulated by cyclic AMP (3 x 10(^-4)M), but 5HT had no effect. Potassium has been shown to be concentrated in the urine, but as electrophysiological studies revealed that the trans- wall potential did not correspond with the values for K(^+) predicted by NERNST, the K(^+) movement must have resulted from active transport. Ultrastructural studies using both scanning and transmission electron microscopy in normal and ouabain Ringer solutions revealed that ouabain had no effect on structure. The results were discussed in terms of the relationship between cell structure, fluid secretion and Na(^+)-K(^+)ATPase activity.