The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study

The Malpighian tubules of Tenebrio molitor provide a model system for interpreting the actions of endogenous diuretic and antidiuretic peptides. The effects of diuretic (Tenmo-DH(37)) and antidiuretic (Tenmo-ADFa) peptides and their respective second messengers (cyclic AMP and cyclic GMP) on basolateral (V(bl)) and transepithelial (V(te)) potentials of Tenebrio Malpighian tubules were determined using conventional microelectrodes. In the presence of 6 mmol l(-1) Ba(2+), Tenmo-DH(37) (100 nmol l(-1)) reversibly hyperpolarized V(bl) and depolarized V(te). A similar response was seen with the addition of 1 mmol l(-1) cyclic AMP; however, the apical membrane potential (V(ap)) then showed a hyperpolarization, whereas a depolarization of V(ap) was observed with Tenmo-DH(37). Bafilomycin A(1) (5 micromol l(-1)) inhibited fluid secretion of stimulated tubules and reversed the hyperpolarization of V(bl) in response to Tenmo-DH(37). In response to 100 nmol l(-1) Tenmo-ADFa or 1 mmol l(-1) cyclic GMP, V(bl) and V(te) depolarized, although cyclic GMP affected membrane potentials somewhat differently by causing an initial hyperpolarization of V(bl) and V(te). In high [K(+)]-low [Na(+)] Ringer, 1 mmol l(-1) amiloride decreased fluid secretion rates, and depolarized both V(bl) and V(te). Amiloride significantly decreased luminal pH in paired experiments, indicating the presence of a K(+)/nH(+) exchanger in tubule cells of Tenebrio. The results suggest that the endogenous factors and their second messengers stimulate/inhibit fluid secretion by acting on the apical V-ATPase, basolateral K(+) transport, and possibly Cl(-) transport.     

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro‐CT

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro‐computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a ‘glandular band’ by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio‐opaque concretions. Moreover, the anatomy of O. ovis third‐instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a ‘band’ and anterior Malpighian tubules narrowly uniform throughout their entire length.     

Ouabain-insensitive Na-ATPase activity of Malpighian tubules from Rhodnius prolixus

In the present paper, we show the existence of a furosemide-sensitive Na⁺-stimulated, Mg²⁺-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na⁺-pump. The main characteristics of this activity are: (1) K_0.5 for Na⁺=1.49±0.18 mM, (2) V_max=2.8±0.1 nmol inorganic orthophosphate (Pi)·mg prot⁻¹·min⁻¹, (3) it is fully abolished by 2 mM furosemide, (4) it is insensitive to ouabain concentrations up to 10⁻² M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca²⁺ in the incubation medium.     

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca²⁺ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca²⁺ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca²⁺ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca²⁺ regulation. Basolateral Ca²⁺ transport across distal tubules of Drosophila varied throughout the life cycle; Ca²⁺ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca²⁺ uptake and Ca²⁺ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca²⁺ transport is bidirectional. Ca²⁺ uptake across the distal segment of tubules of young adults and Ca²⁺ release across the distal segment of tubules of older adults was also suggestive of reversible Ca²⁺ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca²⁺ sequestration during periods of high dietary calcium intake and passive Ca²⁺ release during periods of calcium deficiency.     

A method for studying histochemical reactions in intact human dentine with a polarizing incident light microscope

Localization and distribution of non-specific esterases has been studied in intact human dentine, by reflected light microscopy. The method of specimen preparation described here permits the visualization of optical sections in depth within the specimen at high optical resolution. Non-specific esterase was found deposited as discrete bands across the tubules. or as droplets, or as a diffuse microsomal variety in the dentinal tubules and in the interglobular spaces. It was possible to distinguish the droplet variety from the microsomal variety, of esterase within the same tubule, by means of a novel optical method using antiflex and differential interference contrast systems of reflected light microscopy. It was found that the coefficient of reflection of dentine diminished gradually from the enamel to the pre-dentine and was inversely related to the scattering of light in dentine. This scattering plays an important role in the formation of the image with reflected light microscopy. The reflected light microscope offers an economically attractive alternative or a supplementary mode of microscopy to the confocal scanning microscopes for studying intact dentine at varying depths.     

The fine structure of the type 2 cells in the Malpighian tubules of the stick insect,Carausius morosus

Summary Type 2 cells, or mucocytes, are present in both inferior and superior Malpighian tubules ofCarausius morosus and are concentrated towards the distal ends of the main urine-secreting parts. They are absent from the proximal few millimetres of the main part and from the distal specialised regions of the tubules. They possess numerous Golgi bodies and abundant granular E. R., which is consistent with the hypothesis that they secrete mucus. However, they possess basal infoldings and apical microvilli suggesting that they may transport substances across the tubule wall. It is suggested that they perform both functions. Reabsorption of ions or water could precipitate solid components of the urine (e.g., uric acid). Mucus may be important in nucleation of crystalline material and also prevent abrasion of the brush border.This investigation formed part of a thesis for the degree of Ph.D. in the University of Newcastle upon Tyne. It is a pleasure to thank Professor J. Shaw for his advice and encouragement and the Science Research Council for financial support.     

Intracellular calcium chelators and oxidant-induced renal proximal tubule cell death

The effect of intracellular calcium chelators on rabbit renal proximal tubule (RPT) cell death induced by t-butyl hydroperoxide (TBHP) and H₂O₂ was examined. Preincubation of RPT suspensions with 50 μM QUIN 2/AM completely prevented TBHP (0.5 mM) and H₂O₂ (2 mM) induced cell death [i.e., release of lactate dehydrogenase (LDH)]. QUIN 2/AM, BAPTA/AM, EGTA/AM, and FURA 2/AM, at 5 μM, decreased LDH release (at 6 hr) from 41% to 4%, 21%, 26%, and 33%, and decreased lipid peroxidation (at 1 hr) from 1.0 to 0.1, 0.4, 0.6, and 0.8 nmol MDA/mg protein, respectively, after TBHP exposure. Since oxidant-induced lipid peroxidation and cell death are iron-dependent in this model, these results suggest that the intracellular calcium chelators inhibit cell death by chelating iron.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules of Drosophila melanogaster larvae

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance–associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically‐obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph. © 2010 Wiley Periodicals, Inc.