Fluid production and cellular elemental composition of Locusta migratoria L. Malpighian tubules : a study using inhibitors and stimulators of fluid production
The in vitro rate and cationic composition of the fluid secreted by the Malpighian tubules of the African migratory locust, Locusta migratoria migratoroides L, was investigated in this study. The concentrations of the elements Na, K, P, S, CI, Mg and Ca within Locusta Malpighian tubule type 1 cells, and the surrounding basement membrane, were quantified. Inhibitors and stimulators of fluid production were used to perturb the normal secretory state of the tubule cells. The rate of fluid secretion under control conditions was between 1.82 and 1.33. nl min(^-1) The fluid [K(^+)] was approximately 126mM, and [Na(^+)] 51 mM. The basement membrane was characterised by high [Na] and [CI] whilst a gradient of [K](_i) was observed. [K](_i) rose from approximately 193 mmol Kg(^-1) d.w. at the basal infoldings to 481 mmol Kg(^-1) d.w. at the apical microvillar border. The central cytoplasmic [K](_i) was 348 mmol Kg(^-1) d.w., estimated as 116mM. [Na](_i) and [Cl](_i) were generally lower, being 57mM and 29mM respectively in the central cytoplasm. Only K assumed a concentration gradient. Intracellular mass dense concretions were observed. Three types were present, the first rich in P and Ca, the second, rich in S, Na and K, and the third, rich in Mg, K and Na. The fluid production inhibitors furosemide (1mM) and bafilomycin A(_1) (1µM) raised the [N(^+)] in the secreted fluid, and altered [K](_i), [Na](_i) and [CI](_i). Furosemide lowered [K](_i) but increased [Na](_i) and [Cl](_i). Bafilomycin lowered [K(^+)] in the secreted fluid, though [K](_i) increased. Both inhibitors abolished the [K](_i) gradient. Replacing K(^+) with Rb(^+) in the bathing saline slowed fluid secretion and lowered [K](_i) and [Cl](_i), though a gradient of [K](_i) was retained. Rb adopted an intracellular gradient which mirrored that of K. Rate of secretion data suggests Rb enters the cell basolaterally primarily via the Na(^+)-K(^+)-ATPase. The fluid secretion stimulator cAMP (1mM) lowered [K](_i), and raised [Na](_i) and [Cl](_i), but corpora cardiaca extract left these elements' concentrations largely unchanged. Stimulation with both corpora cardiaca extract and cAMP maintained the [K](_i) gradient. These stimulators changed the content and number of mass dense concretions present, in manner which suggested that these structures were important in ion transport. These findings support the current model of ionic transport in these cells, including the basolateral presence of an Na(^+)-K(^+)-2Cr cotransporter, and an apical proton-pumping V-type ATPase / K(^+)/nH(^+) antiport complex.