Recent experiments towards a model for fluid secretion in Rhodnius Upper Malpighian Tubules (UMT)

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na(+) concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na(+)-K(+)-2Cl(-) cotransporter; of intermediate importance are the Na(+)-K(+)-ATPase and a ouabain-insensitive Na(+)-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H(+)-ATPase; and a K(+) and/or Na(+)-H(+) exchanger.     

A Model for Fluid Secretion in <Emphasis Type="Italic">Rhodnius</Emphasis> Upper Malpighian Tubules (UMT)

We have measured fluid secretion rate in Rhodnius prolixus upper Malpighian tubules (UMT) stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. Thirteen pharmacological agents were applied: ouabain, Bafilomycin A₁, furosemide, bumetanide, DIOA, Probenecid, SITS, acetazolamide, amiloride, DPC, BaCl₂, pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter-transporters and ion and water channels. The basic assumption is that water movement changes reflect changes in ion transport mechanisms, which we localize as follows: (i) At the basolateral cell membrane, fundamental are a Na⁺-K⁺-2Cl^– cotransporter and a Cl^–-HCO₃^– exchanger; of intermediate importance are the Na⁺-K⁺-ATPase, Cl^– channels and Rp-MIP water channels; K⁺ channels play a lesser role: (ii) At the apical cell membrane, most important are a K⁺-Cl^– cotransport that is being located for the first time, a V-H⁺-ATPase; and a Na⁺-H⁺ exchanger; a urate-anion exchanger and K⁺ channels are less important, while Cl^– channels are not important at all. A tentative model for the function of the UMT cell is presented.Symbols and abbreviations:ACTZ, acetazolamide; cAMP, cyclic adenosine-mono-phosphate; DIOA, [(dihydroindenyl)oxy] alkanoic acid; DPC, diphenylamine-2-carboxylate; DTT, dithiothreitol; 5-HT, 5-hydroxy-tryptamine; IR, Insects Ringer; J_v, secretion rate [nl/cm².s]; pCMBS, parachloro-mercuri-benzene-sulphonate; Rp-MIP, Rhodnius prolixus water channels; SITS, 4-acetamido-4-isothiocyanatostilbene -2,2-disulfonic Acid; UMT, upper malpighian tubules.     

Number of Malpighian Tubules in Larvae and Adults of Stingless Bees (Hymenoptera: Apidae) from Amazonia

The number of Malpighian tubules in larvae and adults of bees is variable. Larvae of Apis mellifera L. have four Malpighian tubules, while adults have 100 tubules. In stingless bees, this number varies from four to eight. The objectives of this study were to provide characteristics of the Malpighian tubules as well as to quantify their number in larvae and adults of six species of Meliponinae, Melipona seminigra merrillae Cockerell, Melipona compressipes manaosensis Schwarz, Melipona rufiventris Lepeletier, Scaptotrigona Moure, Frieseomelitta Ihering, and Trigona williana Friese. Malpighian tubules were dissected from larvae and adults, measured, quantified, and maintained in microtubes with Dietrich??s solution. The numbers of Malpighian tubules were constant only for larvae of M. rufiventris (four and eight) and Scaptotrigona sp. (four). The most frequent number of tubules in the Melipona group was seven and eight in larvae, and 70 and 90 in adults. In the Trigona group were four and 20 to 40, for larvae and adults, respectively. The results showed differences in the number of Malpighian tubules among the species analyzed and also between the larvae and adults of the same species. Despite the variation observed, species of the group Melipona always have a larger number and longer Malpighian tubules in both larvae and adults as compared to the Trigona group, which may indicate an evolutionary trend of differentiation between these groups.     

桃小食心虫幼虫的消化道和马氏管的显微结构观察

利用光学显微镜和扫描电子显微镜,在形态学和组织学水平上研究_『桃小食心虫 Carposina sasakii 幼虫消化道和屿氏管的结构.桃小食心虫幼虫消化道由前肠、中肠和后肠组成.前肠细短,肌肉层薄.前肠与中肠交界处有突出的胃盲囊.中肠长且粗大,内有围食膜,肠壁细胞较大,外层为发达的环肌和纵肌.后肠上皮细胞内陷很深.6根念珠状的马氏管位于中、后肠分界处.     

Morphometric analysis of Rhodnius prolixus Stal (Hemiptera:Reduviidae) midgut cells during blood digestion

Post-feeding ultrastructural modifications to the midgut cells of Rhodnius prolixus are quantified using morphometry. Changes in relative and absolute volumes and/or surface areas are demonstrated for the whole cells, nuclei, mitochondria, rough endoplasmic reticulum, lysosomes, Golgi apparatus, storage vesicles, glycogen, microvilli, and basal labyrinth, before and during blood digestion. These parameters are separately determined for cells from each of the three midgut regions, and are correlated against previously published cycles of digestive enzyme activities. The results support the proposed division of the midgut of R. prolixus into three functional regions: the anterior midgut or crop is the site of water transport immediately after feeding, and of lipid and glycogen storage. No protein digestion occurs in this region. The anterior intestine is the site of most proteinase synthesis and secretion, although limited absorption and nutrient storage also occurs. The posterior intestine is responsible for some secretory activity, but is also implicated as the most important region for absorption of digested nutrients and for carbohydrate absorption and storage.     

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.     

中国角蝉总科部分昆虫马氏管分泌物“网粒体”的超微结构研究(半翅目:蝉亚目)

运用光学显微镜、扫描电镜和透射电镜分别对中国角蝉总科18种昆虫(叶蝉17种,角蝉1种)的成虫“网粒体”进行了超微结构及合成部位研究.研究结果证实体表网粒体均合成于马氏管第三区( MT3)管壁细胞的高尔基体;这些网粒体可被分为4种类型:小球形网粒体(SB)、棒状网粒体(RB)、多室大球形网粒体( LMB)及少室大球形网粒...     

Transepithelial potential in Malpighian tubules of Rhodnius prolixus: lumen-negative voltages and the triphasic response to serotonin

Previous studies of the Malpighian tubules of Rhodnius reported lumen-negative values of transepithelial potential (TEP), and a characteristic triphasic change in TEP in response to stimulation of tubule fluid secretion by serotonin. TEP was measured using the Ramsay technique, in which electrodes are positioned in bathing and secreted fluid droplets for tubules isolated under paraffin oil. The validity of this method of TEP measurement has been questioned on the grounds that, in tubules of some species, it may permit shunting of current from lumen to bath through the cells or through the thin layer of fluid adherent to the surface of that portion of the tubule in the oil. The triphasic response of TEP to serotonin has been confirmed in this study of tubules of fifth instar Rhodnius prolixus using two different techniques that eliminate the possibility of shunting artefacts. From an initially negative value in unstimulated tubules ( approximately -25 mV, lumen-negative), TEP shifted to approximately -33 mV in phase 1, approximately +30 mV in phase 2 and approximately -32 mV in phase 3. TEP during each phase was similar irrespective of the measurement technique. Ion substitution experiments and the effects of specific pharmacological reagents support the proposal that the three phases of the response of TEP to serotonin correspond to sequential activation of an apical Cl(-) channel, an apical V-type H(+) ATPase and a basolateral Na(+):K(+):2Cl(-) cotransporter.     

Osmosis in Cortical Collecting Tubules : A Theoretical and Experimental Analysis of the Osmotic Transient Phenomenon

This paper reports a theoretical analysis of osmotic transients and an experimental evaluation both of rapid time resolution of lumen to bath osmosis and of bidirectional steady-state osmosis in isolated rabbit cortical collecting tubules exposed to antidiuretic hormone (ADH). For the case of a membrane in series with unstirred layers, there may be considerable differences between initial and steady-state osmotic flows (i.e., the osmotic transient phenomenon), because the solute concentrations at the interfaces between membrane and unstirred layers may vary with time. A numerical solution of the equation of continuity provided a means for computing these time-dependent values, and, accordingly, the variation of osmotic flow with time for a given set of parameters including: P_f (cm s^–1), the osmotic water permeability coefficient, the bulk phase solute concentrations, the unstirred layer thickness on either side of the membrane, and the fractional areas available for volume flow in the unstirred layers. The analyses provide a quantitative frame of reference for evaluating osmotic transients observed in epithelia in series with asymmetrical unstirred layers and indicate that, for such epithelia, P_f determinations from steady-state osmotic flows may result in gross underestimates of osmotic water permeability. In earlier studies, we suggested that the discrepancy between the ADH-dependent values of P_f and P_DDw (cm s^–1, diffusional water permeability coefficient) was the consequence of cellular constraints to diffusion. In the present experiments, no transients were detectable 20–30 s after initiating ADH-dependent lumen to bath osmosis; and steady-state ADH-dependent osmotic flows from bath to lumen and lumen to bath were linear and symmetrical. An evaluation of these data in terms of the analytical model indicates: First, cellular constraints to diffusion in cortical collecting tubules could be rationalized in terms of a 25-fold reduction in the area of the cell layer available for water transport, possibly due in part to transcellular shunting of osmotic flow; and second, such cellular constraints resulted in relatively small, approximately 15%, underestimates of P_f.     

Secretion of Na+, K+ and fluid by the Malpighian (renal) tubule of the larval cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae)

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na⁺ and K⁺ in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na⁺, K⁺, H⁺) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K⁺ and Na⁺ were secreted by the distal iliac plexus (DIP) and K⁺ was reabsorbed in downstream regions. The fluid secretion rate decreased > 50% after 25 μM bafilomycin A1, 500 μM amiloride or 50 μM bumetanide was added to the bath. The concentration of K⁺ in the secreted fluid did not change, whereas the concentration of Na⁺ in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500 μM cAMP or 1 μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K⁺ concentration in the secreted fluid. An increase in Na⁺ concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K⁺, whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K⁺/Na⁺ ratio.     

The ultrastructure of Malpighian tubules and hindgut of Frankliniella occidentalis (Pergande) (Thysanoptera : Thripidae)

The ultrastructure of the western flower thrips, Frankliniella occidentalis (Pergande) (Order : Thysanoptera), has 4 Malpighian tubules that are free of the intestine as they leave their junction at the pyloric region. The tubules consist of an epithelium with a single type of microvillated cells; proximally, the cells are lined by a thin cuticle. Numerous mitochondria, basal infoldings of the plasma membrane and vesicles with varying densities suggest active transit of fluid in the cell for osmoregulation. Two of the Malpighian tubules are bent posteriorly and closely adhere to the hindgut in the region of the rectal pads where the 2 epithelia are separated only by a basal lamina. The ultrastructure of this region suggests possible fluid reabsorption from the gut lumen.     

Feeding and defecation of Rhodnius (hemiptera: Reduviidae) fed human blood

Feeding and defecation behavior of Rhodnius prolixus Stal, 1859, R. robustus Larrousse, 1927, R. neivai Lent, 1953 and R. pictipes Stal, 1872, artificially fed on human blood, were studied under laboratory conditions. In all species, first instar nymphs did not defecate in the first 30 minutes after feeding. R. pictipes did not accept artificial feeding but fed directly on humans. Nymph and adult R. prolixus had a higher defecation index (DI) than other species; third instar nymphs had the highest DI = 1.62. In all instars, most individuals accepted the food in 3 Pounds minutes and finished feeding in less than 15 minutes.     

Studies on the morphology and ultrastructure of the Malpighian tubules of Halobiotus crispae Kristensen, 1982 (Eutardigrada)

The excretory and osmoregulatory system of Halobiotus crispae consists of two lateral and one smaller dorsal Malpighian tubules, which empty into the digestive tract in the transition zone of the midgut and rectum. The tubules are identical at the ultrastructural level, and consist of an initial segment with three large cells, a thin transitional distal part lacking a nucleus, and a proximal part with 9–12 nuclei. The initial segment possesses deep basal infoldings and interdigitating, finger-shaped processes of the plasma membrane, large mitochondria and giant nuclei. The distal part is a short section which supports the initial segment. Cellular offshoots from the succeeding proximal part constitute the distal part. The distal and proximal parts contain intercellular canals with concretions of variable size. The exit of the proximal part into the digestive tract is characterized by the presence of microvilli. Correlated with the different stages in the cyclomorphosis of H. crispae , we observed size variation of the Malpighian tubules; thus, pseudosimplex stages have the largest tubules. We present suggestions concerning the physiology of the tubules and compare the Malpighian tubules of Tardigrada with the Malpighian papillae of Protura.     

Osmotic Adjustment to Water Stress in Pearl Millet (Pennisetum americanum (L. ) Leeke) in a Controlled Environment

A pressure-volume (P-V) and an expressed sap (cryoscopic) techniquewere compared for assessing osmotic adjustment to water stressby pearl millet (Pennisetum americanum (L. ) Leeke) plants grownin a controlled environment cabinet. For leaf water potentials( ) above the point of zero turgor, there was good agreementbetween estimates of solute potential ( _s)and turgor ( _p) obtainedby the two methods. Reductions in pre-dawn leaf to –1.8 MPa over 5–6d resulted in net solute accumulation as indicated by a fallin s at full hydration of about 0.3 MPa. The degree of osmoticadjustment increased linearly with the decrease in pre-dawn. Adjustment in cv. BJ 104 was significantly (P < 0.05) lessduring a second drought than during a first, and cv. Serere39 was significantly (P < 0.05) less able to adjust osmoticallythan BJ 104. Adjustment was greater in leaves which were undergoing extensiongrowth during the drought than in leaves already fully extendedbefore drought started. Much of the adjustment was lost within24 h following rewatering, the loss being most complete in theolder, fully extended leaves.     

Morphometric and Quantitative Immunohistochemical Analysis of Disease-Related Changes in the Upper (Suburothelial) Lamina Propria of the Human Bladder Dome

The upper (suburothelial) lamina propria (ULP) is a distinct region in the human bladder with dense populations of interstitial cells (IC), fine vascular networks and variable development of muscularis mucosae (MM). It is more and more obvious that the ULP plays an important role in bladder physiology and bladder disease, and in the present study we have quantified changes in the cellular key players of the ULP in bladders from patients with carcinoma in situ (CIS), multiple sclerosis (MS) and bladder pain syndrome (BPS). Tissue samples for the different patient groups were obtained from radical cystectomy-specimens. Standardized immunohistochemistry with a panel of specific cell markers was used to characterise the ULP cellular structures, followed by digitalised morphometry and quantitative staining analysis. Alterations in the ULP area were most pronounced in MS bladders, but also present in BPS and CIS bladders. We observed an increased thickness and increased variability in thickness of the ULP IC area in MS and BPS bladders; a significantly increased development of MM in MS bladders; a changed organization of vascular plexuses in the lamina propria in most pathologic bladders and a changed phenotype of ULP IC: a significantly decreased expression of progesterone receptor in MS bladders and a trend towards decreased expression of alpha-smooth muscle actin in BPS bladders. We show here for the first time the presence of disease-specific changes in organisation and/or phenotype of the different key players of the ULP area in human bladder. The present findings further support the hypothesis that the ULP area is involved and altered in different bladder diseases.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> Heat Stable (STa) Enterotoxin and the Upper Small Intestine: Lack of Evidence in Vivo for Net Fluid Secretion

Heat stable (STa) enterotoxin from E. coli reduced fluid absorption in vivo in the perfused jejunum of the anaesthetized rat in Krebs-phosphate buffer containing lactate and glucose (nutrient buffer), in glucose saline and in glucose free saline. Bicarbonate ion enhanced fluid absorption of 98 ± 7 (6) μl/cm/h was very significantly (P < 0.0001) reduced by STa to 19 ± 4 (6) μl/cm/h, but net secretion was not found. When impermeant MES substituted for bicarbonate ion, net fluid absorption of 29 ± 3 (6) μl/cm/h was less (P < 0.01) than the values for phosphate buffer and bicarbonate buffer. With STa in MES buffer, fluid absorption of 3 ± 2 (6) μl/cm/h was less than (P < 0.001) that in the absence of STa and not significantly different from zero net fluid absorption. E. coli STa did not cause net fluid secretion in vivo under any of the above circumstances. Neither bumetanide nor NPPB when co-perfused with STa restored the rate of fluid absorption. In experiments with zero sodium ion-containing perfusates, STa further reduced fluid absorption modestly by 20 μl/cm/h. Perfusion of ethyl-isopropyl-amiloride (EIPA) with STa in zero sodium ion buffers prevented the small increment in fluid entry into the lumen caused by STa, indicating that the STa effect was attributable to residual sodium ion and fluid uptake that zero sodium-ion perfusates did not eradicate. These experiments, using a technique that directly measures mass transport of fluid into and out of the in vivo proximal jejunum, do not support the concept that E. coli STa acts by stimulating a secretory response.