Effects of putative diuretic factors on intracellular second messenger levels in the Malpighian tubules of Aedes aegypti

Intracellular levels of the second messengers, 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP(3)) were measured in the Malpighian tubules of Aedes aegypti following the in vitro application of 5-hydroxytryptamine (5-HT) and the putative mosquito diuretic peptides, Culex salinarius diuresin and mosquito leucokinins (culekinin depolarizing peptides (CDPs) I, II, III, A. aegypti leucokinin peptides (ALPs) I, II, III). The C. salinarius diuresin significantly (p<0.05) increased tubule intracellular cAMP concentrations. Treatment of tubules with either 5-HT or CDP-II resulted in significant increases in both intracellular cAMP and IP(3) concentrations. All of the mosquito leucokinins, with the exception of CDP-I, significantly stimulated intracellular IP(3) in isolated tubules. These data suggest that the mosquito leucokinins may function on the Malpighian tubules of A. aegypti by increasing the intracellular Ca(2+) levels through the release of IP(3) sensitive Ca(2+) stores. The physiological relevance of these data to the regulation of mosquito Malpighian tubule function is discussed.     

Oscillations of voltage and resistance in Malpighian tubules of Aedes aegypti

The transepithelial voltage (V(t)) of isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti spontaneously oscillates in more than half the tubules. Typically, V(t) decreases and then rises at a frequency of 2 oscillations/min with a duration of 16 s. In 6 isolated perfused tubules studied in detail, V(t) oscillates between 50.5 mV and 15.7 mV in parallel with (1) oscillations of the transepithelial resistance (R(t)) between 7.61 kOmegacm and 3.63 kOmegacm, (2) oscillations of the basolateral membrane voltage of principal cells between -56.7 mV and -72.2 mV, and (3) oscillations of the apical membrane voltage between 107.2 mV and 87.8 mV. The oscillations are dependent on the Cl concentration in the extracellular solutions. As R(t) decreases during the oscillations V(t) goes to the transepithelial equilibrium potential of Cl (E(cl)) indicating transient changes in transepithelial Cl conductance as the mechanism of voltage and resistance oscillations. Since the largest voltage oscillations take place across the whole epithelium and not across cell membranes, oscillating Cl conductances are localized to a single transepithelial Cl diffusion barrier such as the paracellular pathway. This conclusion is supported by the analysis of electrically equivalent circuits that identify the shunt pathway as the site of oscillating Cl conductances.     

Beetle diuretic peptides: the response of mealworm (Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle (Onthophagus gazella)

This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH(31), has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH(37) in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH(46) and Dippu-DH(46) fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH(37) increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.     

Isolation,primary structure,and synthesis of leucokinins V and VI: Myotropic peptides of Lleucophaea maderae

1. Two additional octapeptides which stimulate the contractile activity of the cockroach hindgut have been isolated from head extracts of the cockroach, Leucophaea maderae.2. A series of four high performance liquid-chromatographic (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analysis and primary sequence determinations. The sequence determined for two peptides were: Gly-Ser-Gly-Phe-Ser-Ser-Trp-Gly-NH₂ and pGlu-Ser-Ser-Phe-His-Ser-Trp-Gly-NH₂. These octapeptides were designated leucokinins V and VI (L-V, L-VI), respectively.3. Minimum concentrations of natural L—V and L-VI required to produce a response from the isolated cockroach hindgut were 4.5 × and 4.9 × 10⁻¹¹ M, respectively. These concentrations were quite similar to the threshold concentrations observed for the synthetic products.     

Natriuretic and depolarizing effects of a stable fly (Stomoxys calcitrans) factor on Malpighian tubules

A two-step HPLC purification procedure resulted in a factor from the stable fly that depolarizes the lumen-negative transepithelial voltage (V(t)) of the adult stable fly Malpighian tubule. When applied to tubules of the female mosquito, Aedes aegypti, this factor partially mimics the electrophysiological actions of the mosquito natriuretic factor (MNF). It also selectively increases active transepithelial Na transport by the mosquito Malpighian tubule. The blood meal causes a transient increase in hemolymph Na and Cl contents and hemolymph volume during the course of the 24-h post-feeding period. The level of a factor that is immunologically cross-reactive with the human atrial natriuretic peptide (ANP) increases more than 6-fold within 6h following a blood meal by the stable fly. The temporal pattern of the levels of the ANP-immunoreactive factor closely parallels the blood meal-induced rise and subsequent fall in hemolymph NaCl content and hemolymph volume, suggesting a functional correlation between the ANP-immunoreactive factor and the rate of NaCl and fluid loss from the hemolymph.     

Diuretic factors and second messengers stimulate secretion of the organic cation TEA by the Malpighian tubules of Drosophila melanogaster

This study showed that four factors which stimulate transepithelial fluid secretion and inorganic ion transport across the main segment of the Malpighian tubules of Drosophila melanogaster also stimulate transepithelial secretion of the prototypical organic cation tetraethylammonium (TEA). TEA fluxes across the Malpighian tubules and gut were measured using a TEA-selective self-referencing (TEA-SeR) microelectrode. TEA flux across isolated Malpighian tubules was also measured using a TEA-selective microelectrode positioned in droplets of fluid secreted by tubules set up in a modified Ramsay assay. TEA flux was stimulated by the intracellular second messengers cAMP and cGMP, which increase the lumen-positive transepithelial potential (TEP), and also by tyramine and leucokinin-I (LK-I), which decrease TEP. The largest increase was measured in response to 1 micromol l-1 LK-I which increased transepithelial TEA flux by 72%. TEA flux in the lower tubule was stimulated slightly (13%) by 1 micromol l-1 tyramine but not by any of the other factors. TEA flux across the midgut was unaffected by cAMP, cGMP or tyramine. This is the first study to demonstrate the effects of insect diuretic factors and second messengers on excretion of organic cations.     

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P-glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.     

Inorganic and organic anion transport by insect renal epithelia

Insect renal organs typically exhibit high rates of transport of inorganic and organic anions, and therefore provide useful models for the study of epithelial anion transport and its control. Isolated Malpighian tubules of some species secrete a volume of iso-osmotic fluid equal to their own volume in 10-15 s, which means that cellular Cl(-) content is exchanged every 3-5 s. Anion transport can also be achieved against extreme thermodynamic gradients. The concentration of K(+) and Cl(-) in the lumen of the Malpighian tubules of some desert beetles approaches or exceeds saturation. A basolateral Na(+):K(+):2Cl(-) cotransporter plays an important role in vectorial ion transport in Malpighian tubules of many species, but there is also evidence for coupling of Cl(-) transport to the movement of a single cationic species (Na(+) or K(+)). Although an apical vacuolar H(+)-ATPase plays a primary role in energizing transepithelial secretion of chloride via channels or cotransporters in the secretory segment of the Malpighian tubule, several different ATPases have been implicated in reabsorption of Cl(-) by the lower Malpighian tubule or hindgut. Chloride transport is known to be controlled by several neuropeptides, amines and intracellular second messengers. Insect renal epithelia are also important in excretion of potentially toxic organic anions, and the transporters involved may play a role in resistance to insecticides of natural or anthropogenic origin.     

Na(+)/H(+) exchange in mosquito Malpighian tubules

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.     

昆虫激肽的生理功能及机制

昆虫激肽是一类高度保守的小分子神经活性物质,自其从马德拉蟑螂脑中分离得到至今,人们在多种无脊椎动物体内均发现了这一激肽家族成员。它们具有促进昆虫后肠收缩、马氏管扭动、原尿分泌,调节血淋巴量和水盐平衡,使马氏管跨膜电位去极化,抑制昆虫体内消化酶释放、幼虫体重增长等功能。然而,天然的昆虫激肽很容易被蛋白酶所降解,因此须对其进行结构改造及构效关系研究,以开发出更有潜力的假肽和非肽模拟抗酶解昆虫激肽类似物,对今后实现环境友好型害虫防治策略具有重要意义。     

Localization and role of inward rectifier K+ channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti

Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K⁺ (Kir) channel subunits: AeKir1, AeKir2B and AeKir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of AeKir1 and AeKir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that AeKir1 and AeKir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; AeKir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K⁺, the peritubular application of VU590 (10 μM), a selective inhibitor of AeKir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (V_bl) of principal cells, but it significantly reduced the cell input conductance (g_in) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 μM), an inhibitor of both AeKir1 and AeKir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ∼35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the V_bl and g_in of principal cells to respectively drop to values 62% and 56% of the control values within only ∼30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that AeKir1 and AeKir2B respectively contribute to 46% and 20% of the transepithelial K⁺ secretion when the tubules are bathed in a Ringer solution containing 34 mM K⁺. Thus, we uncover an important role of AeKir1 and stellate cells in transepithelial K⁺ transport under conditions of peritubular K⁺ challenge. The physiological role of AeKir3 in intracellular membranes of both stellate and principal cells remains to be determined.     

Leucokinin and the modulation of the shunt pathway in Malpighian tubules

Transepithelial secretion in Malpighian tubules of the yellow fever mosquito (Aedes aegypti) is mediated by active transport of Na(+) and K(+) through principal cells and passive Cl(-) transport through the shunt. Permeation through the shunt was assessed by measuring transepithelial halide diffusion potentials in isolated perfused Malpighian tubules, after first inhibiting active transport with dinitrophenol. Diffusion potentials were small under control conditions, revealing Eisenman selectivity sequence I (I(-)>Br(-)>Cl(-)>F(-)) which is the halide mobility sequence in free solution. Accordingly, electrical field strengths of the shunt are small, selecting halides for passage on the basis of hydrated size. Leucokinin-VIII (LK-VIII) significantly increased the shunt conductance from 57.1 μS/cm to 250.0 μS/cm. In parallel, the shunt selectivity sequence shifted to Eisenman sequence III (Br(-)>Cl(-)>I(-)>F(-)), revealing increased electrical field strengths in the shunt, now capable of selecting small, dehydrated halides for passage. High concentrations of peritubular F(-) (142.5 mM) duplicated the effects of LK-VIII on shunt conductance and selectivity, suggesting a role for G-protein. In the presence of LK-VIII (or F(-)), coulombic interactions between the shunt and I(-) and F(-) may be strong enough to cause binding, thereby blocking the passage of Cl(-). Thus, LK-VIII increases both shunt conductance and selectivity, presumably via G-protein.     

Intracellular and luminal pH measurements of Malpighian tubules of the mosquito Aedes aegypti: the effects of cAMP

In order to understand the critical role that hydrogen ions play in fluid secretion in Malpighian tubules, intracellular and luminal pH and K+ measurements were performed in isolated Malpighian tubules of the yellow fever mosquito (Aedes aegypti). The intracellular pH was 7.03+/-0.05 (n=15 Malpighian tubules (MT)) and the luminal pH was 7.19+/-0.09 (n=99 MT) when bathed in saline at a pH of 7.0. The lumen potential is positive, thus net proton secretion into the lumen is active. The intracellular and the luminal K+ concentrations were 75+/- 9 mM (n=15) and 102+/-13 mM (n=9 MT) respectively. Cyclic AMP analogues accelerated fluid secretion and at the same time acidified the cell without affecting the luminal pH. Both effects were abolished by an isomer of adenosine-3',5' cyclic monophosphothioate (cAMPS), the Rp-cAMPS, known to inhibit protein kinase A. The results suggest that in the presence of cAMP the properties of the cation/H+ exchanger are affected and that this may be a result of phosphorylation of a Na+/2H+ antiporter located on the apical membrane.     

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

Calcium homeostasis in larval and adult Drosophila melanogaster

Calcium homeostasis in Drosophila melanogaster was examined in response to the challenges imposed by growth, reproduction and variations in dietary calcium content. Turnover time for calcium, calculated as the time for (45)Ca(2+)to accumulate to half the steady state value of 3.46 nmol/fly, was 3.3 days. Although larvae weighed 2x as much as adults, they contained 3-4x as much calcium. Anterior Malpighian tubules (Mts) contain much more calcium than posterior Mts, accounting for 25-30% of the calcium content of the whole fly. In response to a 6.2-fold increase in dietary calcium level, calcium content of whole flies increased only 10%. Hemolymph calcium concentration ( approximately 0.5 mM) was similar in males and females and in animals raised on diets differing in calcium content. Fluid secretion rate, secreted fluid calcium concentration, and transepithelial calcium flux in tubules isolated from flies raised on high and low calcium diets did not differ significantly. Malpighian tubules secrete calcium at rates sufficient to eliminate whole body calcium content in 0.5 and 3 days for tubules secreting fluid at basal and maximal rates, respectively. It is suggested that flies absorb high quantities of calcium from the diet and maintain homeostasis through the combined effects of elimination of calcium in fluid secreted by the Malpighian tubules and the sequestration of calcium in granules, especially within the distal segment of the anterior pair of Malpighian tubules.     

The biological activity of diuretic factors in Rhodnius prolixus

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones.In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards.Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.