Intracellular ion activities in Malpighian tubule cells of Rhodnius prolixus: evaluation of Na+-K+-2Cl- cotransport across the basolateral membrane.
(33/268)
Intracellular ion activities (a(ion)) and basolateral membrane potential (Vbl) were measured in Malpighian tubule cells of Rhodnius prolixus using double-barrelled ion-selective microelectrodes. In saline containing 103 mmol l(-1) Na+, 6 mmol l(-1) K+ and 93 mmol l(-1) Cl-, intracellular ion activities in unstimulated upper Malpighian tubules were 21, 86 and 32 mmol l(-1), respectively. In serotonin-stimulated tubules, aCl was unchanged, whereas aNa increased to 33 mmol l(-1) and aK declined to 71 mmol l(-1). Vbl was -59 mV and -63 mV for unstimulated and stimulated tubules, respectively. Calculated electrochemical potentials (deltamuF) favour passive movement of Na+ into the cell and passive movement of Cl- out of the cell in both unstimulated and serotonin-stimulated tubules. Passive movement of K+ out of the cell is favoured in unstimulated tubules. In stimulated tubules, deltamuF for K+ is close to 0 mV. The thermodynamic feasibilities of Na+-K+-2Cl-, Na+-Cl- and K+-Cl- cotransporters were evaluated by calculating the net electrochemical potential (deltamu(net)/F) for each transporter. Our results show that a Na+-K+-2Cl- or a Na+-Cl- cotransporter but not a K+-Cl- cotransporter would permit the movement of ions into the cell in stimulated tubules. The effects of Ba2+ and ouabain on Vbl and rates of fluid and ion secretion show that net entry of K+ through ion channels or the Na+/K+-ATPase can be ruled out in stimulated tubules. Maintenance of intracellular Cl- activity was dependent upon the presence of both Na+ and K+ in the bathing saline. Bumetanide reduced the fluxes of both Na+ and K+. Taken together, the results support the involvement of a basolateral Na+-K+-2Cl- cotransporter in serotonin-stimulated fluid secretion by Rhodnius prolixus Malpighian tubules. (+info)
The control of diuresis in the tsetse fly Glossina austeni: a preliminary investigation of the diuretic hormone.
(34/268)
The rate of secretion of the Malpighian tubules of Glossina austeni is controlled by a diuretic hormone. This hormone is present in the nervous tissue of the fly together with a degradative enzyme that can be activated by boiling. It is demonstrated that the Malpighian tubules are able to destroy the diuretic hormone; they may therefore participate in the control of diuresis. The diuretic hormone appears to be a heat-stable, non-dialysable, alcohol-soluble molecule, containing amino acid, glucose and sialic acid residues. (+info)
Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta.
(35/268)
The actions of various peptides and other compounds on fluid secretion by Malpighian tubules in the tobacco hawkmoth Manduca sexta sexta are investigated in this study. Using a newly developed pharate adult Malpighian tubule bioassay, we show that three tachykinin-related peptides (TRPs), leucokinin I, serotonin (5-HT), octopamine, the cardioacceleratory peptides 1a, 1b and 2c, cGMP and cAMP each cause an increase in the rate of fluid secretion in pharate adult tubules. Whereas the possible hormonal sources of biogenic amines and some of the peptides are known, the distribution of TRPs has not been investigated previously in M. sexta. Thus we performed immunocytochemistry using an anti-TRP antiserum. We show the presence of TRP-like material in a small subset of cells in the M. sexta central nervous system (CNS). The larval brain contains approximately 60 TRP-immunopositive cells and there are approximately 100 such cells in the adult brain including the optic lobes. Every ganglion of the ventral nerve cord also contains TRP-like immunoreactive cells. No TRP-containing neurosecretory cells were seen in the CNS, but endocrine cells of the midgut reacted with the antiserum. We propose the hypothesis that the control in insects of physiological systems by hormones may not always involve tissue-specific hormones that force stereotypical responses in their target systems. Instead, there may exist in the extracellular fluid a continuous broadcast of information in the form of a chemical language to which some or all parts of the body continuously respond on a moment-to-moment basis, and which ensures a more effective and efficient coordination of function than could be achieved otherwise. (+info)
Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles.
(36/268)
Leucokinins are insect neuropeptides that stimulate hindgut motility and renal fluid secretion. Drosophila has a single leucokinin gene, pp, encoding the longest known leucokinin, Drosokinin. To identify its receptor, a genome-wide scan for G-protein-coupled receptors was performed in silico and candidate receptors identified by similarity to known tachykinin receptors. The deduced peptides were expressed, with a transgene for the calcium reporter aequorin, in S2 cells and only one gene (CG10626) encoded a protein that responded to Drosokinin. The properties of the heterologously expressed receptor (action through intracellular calcium with an EC(50) of 4 x 10(-11) m and a t(1/2) <1 s) match closely those reported for the action of Drosokinin on Malpighian (renal) tubules. Antibodies raised against the receptor identified known sites of leucokinin action: stellate cells of the Malpighian tubule, two triplets of cells in the pars intercerebralis of the adult central nervous system, and additional cells in larval central nervous system. Western blots and reverse transcription-PCR confirmed these locations, but also identified expression in male and female gonads. These tissues also displayed elevated calcium in response to Drosokinin, demonstrating novel roles for leucokinin. A functional genomic approach has thus yielded the first complete characterization of a leucokinin receptor in an insect. (+info)
Leucokinin activates Ca(2+)-dependent signal pathway in principal cells of Aedes aegypti Malpighian tubules.
(37/268)
The role of Ca(2+) in mediating the diuretic effects of leucokinin-VIII was studied in isolated perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Peritubular leucokinin-VIII (1 microM) decreased the transepithelial resistance from 11.2 to 2.6 kOmega. cm, lowered the transepithelial voltage from 42.8 to 2.7 mV, and increased transepithelial Cl(-) diffusion potentials 5.1-fold. In principal cells of the tubules, leucokinin-VIII decreased the fractional resistance of the basolateral membrane from 0.733 to 0.518. These effects were reversed by the peritubular Ca(2+)-channel blocker nifedipine, suggesting a role of peritubular Ca(2+) and basolateral Ca(2+) channels in signal transduction. In Ca(2+)-free Ringer bath, the effects of leucokinin-VIII were partial and transient but were fully restored after the bath Ca(2+) concentration was restored. Increasing intracellular Ca(2+) with thapsigargin duplicated the effects of leucokinin-VIII, provided that peritubular Ca(2+) was present. The kinetics of the effects of leucokinin-VIII is faster than that of thapsigargin, suggesting the activation of inositol-1,4,5-trisphosphate-receptor channels of intracellular stores. Store depletion may then bring about Ca(2+) entry into principal cells via nifedipine-sensitive Ca(2+) channels in the basolateral membrane. (+info)
Changes in nuclear phenotype frequencies following sequential cold shocks in Triatoma infestans (Hemiptera, Reduviidae).
(38/268)
The nuclear phenotypes of Malpighian tubule cells in fifth instar nymphs of Triatoma infestans, one of the most important vectors of Chagas disease, were studied following sequential shocks at 0 degrees C, separated by intervals of 8 h and 24 h at 30 degrees C, under conditions of moderate fasting and full nourishment. The insects pertained to colonies reared in the laboratory and originated from domestic specimens collected in the Brazilian states of Sao Paulo (north) and Minas Gerais (south). Since nuclear phenotypes in this species are affected by single cold shocks, it was expected that these phenotypes could also be changed by sequential shocks. Nuclear phenotypes indicative of mechanisms of cell survival (nuclear fusion and heterochromatin decondensation) and cell death (apoptosis and necrosis) were observed concomitantly in all the conditions tested. Nuclear fusion and heterochromatin decondensation were not found relevant for the presumed acquisition of the cold-hardening response in T. infestans. The decreased frequency of apoptosis and necrosis following sequential cold shocks including under fasting conditions, indicated that tolerance to sequential cold shocks occurred in T. infestans of the mentioned origin. (+info)
Amino acids modulate ion transport and fluid secretion by insect Malpighian tubules.
(39/268)
Insect haemolymph typically contains very high levels of free amino acids. This study shows that amino acids can modulate the secretion of ions and water by isolated Malpighian tubules of Rhodnius prolixus and Drosophila melanogaster. Secretion rates of Rhodnius tubules in amino-acid-free saline increase after addition of serotonin to a peak value, then slowly decline to a plateau. Addition of glutamine, glutamate or aspartate to such tubules increases secretion rates dramatically relative to the controls in amino-acid-free saline, and these increases are sustained for 1-2 h. Seven other amino acids have more modest stimulatory effects, whereas lysine and arginine are inhibitory. Secreted fluid pH and Na(+) concentration increase and K(+) concentration decreases in response to glutamine. Pre-incubation of unstimulated tubules in saline solutions containing amino acids followed by stimulation with serotonin in amino-acid-free saline shows that the effects of amino acids far outlast the duration of exposure to them. Amino acids do not appear to be important as metabolites in Rhodnius tubules, nor do they act to draw significant amounts of water into the lumen by osmosis. Significant stimulation of fluid secretion can be achieved by physiological levels of particular amino acids, whereas those amino acids that inhibit fluid secretion only do so at concentrations much above those at which they occur naturally in the haemolymph. Secretion rates of unstimulated or stimulated Drosophila tubules are increased by pre-incubation in saline solutions containing glutamine or methionine or by continuous exposure to glutamine, methionine or tyrosine. Cysteine dramatically inhibited fluid secretion by Drosophila tubules, but only at concentrations well above the physiological range. We suggest that the amino acids probably function as compatible intracellular osmolytes that are necessary for sustained secretion at high rates by the Malpighian tubules. (+info)
The dependence of electrical transport pathways in Malpighian tubules on ATP.
(40/268)
The relationship between the intracellular ATP concentration [ATP](i) and the electrical properties of principal cells was investigated in Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Under control conditions, [ATP](i) was 0.91 mmol l(-1), the input resistance of the principal cell (R(pc)) was 334.1 k Omega, and the basolateral membrane was marked by a large K(+)-conductance and a membrane voltage (V(bl)) of -75.8 mV. Peritubular cyanide (CN, 0.3 mmol l(-1)) reduced [ATP](i) to 0.08 mmol l(-1) in less than 2 min; however, V(bl) dropped to -8 mV and R(pc) increased to 3150.8 k Omega in 8 min, while the K(+)-conductance of the basolateral membrane disappeared. Upon washout of CN, V(bl) and R(pc) returned to control values within 2 min, and the basolateral membrane recovered its K(+)-conductance. The recovery of normal [ATP](i) took 15 min. Dose-dependence and EC(50) values for the CN-inhibition of V(bl) and the increase in R(pc) were strikingly similar (184.0 micromol l(-1) and 164.4 micromol l(-1)). Similar effects of metabolic inhibition were observed with dinitrophenol (DNP), but the EC(50) values were 50.3 micromol l(-1) and 71.7 micromol l(-1) for the effects on V(bl) and R(pc), respectively. Barium, a blocker of K(+)-channels, significantly hyperpolarized V(bl) to -89.1 mV and increased R(pc) to 769.4 k Omega under control conditions, but had no effects during metabolic inhibition. These results illustrate a temporal relationship between [ATP](i) and electrogenic and conductive transport pathways in principal cells that is consistent with the role of ATP as an integrator of transport steps at apical and basolateral membranes of the cell. When [ATP](i) drops to levels that are 10% of control, the V-type H(+)-ATPase is inhibited, preventing the extrusion of K(+) to the tubule lumen. At the same time, basolateral membrane K(+)-channels close, preventing the entry of K(+) from the hemolymph. Intracellular K(+) homeostasis is thus protected during metabolic inhibition, allowing the cell to re-establish K(+) transport when ATP is synthesized again. (+info)