Rubidium reduces potassium permeability and fluid secretion in Malpighian tubules of Locusta migratoria, L.

The basal membrane potential (V(b)) of Locusta Malpighian tubule cells in control saline results from its relatively high permeability to potassium. In the presence of 1 mM barium added to the control saline V(b) hyperpolarized from a mean resting potential of -72.1 mV to -90.1 mV. On substituting rubidium for potassium in the control saline, V(b) also hyperpolarized to a value of -91.4 mV. Rubidium was also similarly effective in hyperpolarizing the basal membrane even in the presence of control concentrations of potassium in the bathing medium. Substitution of rubidium for potassium also effected a approximately 50% reduction in the rate of fluid secretion. The action of inhibitors on V(b) in the presence of rubidium showed that V(b) under these conditions probably originated from the bafilomycin-sensitive electrogenic potential generated across the apical membrane by a V-type ATPase. The responses of V(b) to potassium, barium and rubidium and their inhibition of fluid secretion suggest the presence of a substantial rubidium-blockable potassium conductance located on the basal membrane of Locusta Malpighian tubule cells.

An investigation into the effects of inhibitors of fluid production by Locusta Malpighian tubule Type I cells on their secretion and elemental composition.

The intracellular elemental concentrations of K, Na, Cl, P, Mg and Ca within Type I cells of the Malpighian tubules of Locusta migratoria have been measured using electron probe X-ray microanalysis. The distribution of Na, K and Cl was not homogeneous within the cells and concentration gradients exist from basal to apical surfaces. The rate of secretion and the cationic composition of the secreted tubule fluid have also been determined. Furosemide (1 mM) inhibited fluid secretion by about 60%, raised the [Na(+)] but did not significantly alter the [K(+)] of the secreted tubule fluid. When Rb(+) replaced K(+) in the saline fluid secretion was also inhibited by about 60%, but no additional inhibition occurred by the simultaneous inclusion of furosemide. Thus, Rb(+) and furosemide probably act at the same transport site, and Rb(+) cannot substitute for K(+) at the basal membrane cotransporter. Bafilomycin (1 µM) dramatically inhibited fluid production by 85%, the [K(+)] of the secreted fluid was reduced by about 30% but the [Na(+)] was almost doubled. Furosemide, in common with other inhibitors of fluid secretion acting at the basal surface (ouabain and Rb(+)), caused a fall in intracellular [K] and a rise in [Na]. Bafilomycin, in common with N-ethyl maleimide, which acts at the apical surface, increased the intracellular [K] but did not affect the [Na].

Characterization of ATPases of apical membrane fractions from Locusta migratoria Malpighian tubules.

Apical and basal membrane fractions from Locusta Malpighian tubules were prepared and were characterized by marker enzyme analysis. The apical membranes contained an azide- and orthovanadate-insensitive ATPase activity that was inhibited by bafilomycin A1 (IC50 = 0.44 nM) and NEM (IC50 = 2.15 microM), and thus was characterized as putative V-type ATPase. The enzyme was stimulated by a variety of monovalent cations (Tris > K = Na > choline > Li = Rb) maximal stimulation occurring at 30-40 mM. It was also stimulated by a variety of monovalent anions (maximal activation 30-40 mM), but was strongly inhibited by nitrate and thiocyanate. SDS-PAGE separation of proteins present in the various membrane fractions was carried out. The apical membrane fraction alone contained a 28 kDa protein band that bound a monoclonal antibody specific for a 28 kDa peptide which was a component of the V-type ATPase from midgut of Manduca sexta and, in native gels, possessed ATPase activity which was also sensitive to both bafilomycin and NEM but not to azide or orthovanadate. Binding of the fluorescent monoclonal antibody was located at the apical boundary of the tubule cells. It was concluded that a V-type ATPase is present at the apical surface of Locusta Malpighian tubule cells and that it is involved in their secretory functioning.

The action of inhibitors of protein kinases on fluid and ion secretion by Malpighian tubules of Locusta migratoria, L.

Fluid production in Locusta Malpighian tubules was stimulated by corpora cardiaca extract (c. 100%) and dibutyryl cAMP (c. 50%). Chelerythrine and staurosporine (Protein kinase C, PKC inhibitors) inhibited it in the range 0.07-60&mgr;M (IC(50)3&mgr;M), whereas Rp-cAMP (Protein kinase A, PKA inhibitor) caused inhibition over the concentration range 10-1000&mgr;M (IC(50)264&mgr;M). The protein phosphatase inhibitor, okadaic acid, was also inhibitory over the concentration range 0.1-1000nM (IC(50) 91nM). CC extract stimulation increased fluid [Na(+)] from 41 to 59mM and decreased [K(+)] from 127 to 107mM; stimulation with cAMP had no such effect. The PKC inhibitors reduced the [K(+)] in the secreted fluid from 126 to 107mM but had no effect on the [Na(+)]. Subsequent addition of CC extract stimulated fluid production and caused an increase in [Na(+)] from 41 to about 50mM. The addition of Rp-cAMP reduced fluid production but caused a decrease in [Na(+)] from 37 to 28mM and an increase in its [K(+)] from 124 to 148mM. Fluid production by Rp-cAMP inhibited tubules was not stimulated by corpora cardiaca extract or cAMP, but [Na(+)] rose to 36mM. Protein phosphorylation plays a role in the regulation of fluid production probably via the apical and basal membrane cation transporters.

An X-ray microanalysis study of Locusta Malpighian tubule cell function using rubidium.

Intracellular distributions of Na, K, Mg, Ca, Cl, P, and S were determined in type 1 Malpighian tubule cells of Locusta using X-ray microanalysis. K showed a gradient of increasing concentration from basal to apical surfaces. No other element showed this distribution. Na was below the detection limit. Three types of dark body were present in cytoplasm; one rich in Ca and P and two rich in K and P. Incubation in Rb-Ringer solution resulted in a dramatic fall in cellular K that was not completely replaced by Rb. The distribution of Rb mimicked that of K. Na levels were significantly increased, but the total intracellular monovalent metal concentration was less than in controls. Other elements were little affected. Rb replaced K in dark bodies. Tubules continued to secrete K-rich urine in Rb-Ringer solution even though intracellular [K] was low. Little Rb+ was secreted and Na+ secretion was unchanged. The possible role of dark bodies as a source of secreted K+ is discussed.

An X-ray microanalytical study on the effects of ouabain and N-ethyl maleimide on the elemental concentrations in Malpighian tubule cells of Locusta.

X-ray microanalysis was used to study elemental distribution in Malpighian tubule cells of Locusta migratoria and how these are affected by the replacement of bathing medium K+ with Rb+ and by inclusion of the transport inhibitors ouabain and n-ethyl maleimide (NEM) in standard (K+-containing) and Rb+-Ringer (K+-free) solutions. Incubation of tubules in standard Ringer containing 1mM ouabain dramatically affected the intracellular levels of K and Na. The intracellular K concentration fell and Na concentration increased in all regions studied. Despite this, a gradient of increasing K concentration from basal to apical cell surface was maintained. Ouabain also reduced the intracellular levels of Rb when applied in Rb+-Ringer. Cl and P levels were unaffected by ouabain treatment. Incubation in standard and Rb+-Ringer solutions containing 1 microM NEM caused a significant increase in intracellular K levels in all regions of the cell compared with that observed in the absence of NEM. Rb levels were little affected by NEM except in the apical cytoplasm and microvillar regions where they were significantly reduced compared with Rb+-Ringer controls. NEM effected a significant increase in cellular levels of Na under Rb+-Ringer conditions. Intracellular Cl and P were not significantly affected by NEM. These results are discussed in relation to proposed mechanisms for the transport of ions and water across this secretory epithelium, with particular emphasis on the role of K+ as the 'prime mover' in this process.

Some factors affecting Malpighian tubule fluid secretion and transepithelial potential in Locusta migratoria L.

Both fluid secretion and transepithelial potential were stimulated by cAMP. Fluid secretion was unaffected by 5-HT over the concentration range 10(-8)-10(-4) M. The presence of ouabain in the bathing medium effected a decrease in transepithelial potential.

The stridulatory apparatus of two species of Tettigoniid.

A scanning electron microscopical examination has been made of the stridulatory apparatus of Homorocoryphus nitidulus vicinus (Wlk.) and Jamaiciana flava (Wlk.). Its structure is discussed in relation to function, and the probable affinities of the two species within the Tettigoniidae.