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.     

Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules

Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.     

Immunolocalization of potassium-chloride cotransporter polypeptides in rat exocrine glands

Potassium-chloride cotransporters (KCCs) encoded by at least four homologous genes are believed to contribute to cell volume regulation and transepithelial ion transport. We have studied KCC polypeptide expression and immunolocalization of KCCs in rat salivary glands and pancreas. Immunoblot analysis of submandibular, parotid, and pancreas plasma membrane fractions with immunospecific antibodies raised against mouse KCC1 revealed protein bands at ca 135 kDa and ca 150 kDa. Immunocytochemical analysis of fixed salivary and pancreas tissue revealed basolateral KCC1 distribution in rat parotid and pancreatic acinar cells, as well as in parotid, submandibular, and pancreatic duct cells. KCC1 or the polypeptide product(s) of one or more additional KCC genes was also expressed in the basolateral membranes of submandibular acinar cells. Both immunoblot and immunofluorescence signals were abolished in the presence of the peptide antigen. These results establish the presence in rat exocrine glands of KCC1 and likely other KCC polypeptides, and suggest a contribution of KCC polypeptides to transepithelial Cl(-) transport.     

Expression of K+-Cl- cotransporters in the alpha-cells of rat endocrine pancreas

The expression of K+-Cl- cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in alpha-cells, but not pancreatic beta-cells nor delta-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both alpha-cell and beta-cells. Exposure of isolated alpha-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 microM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in beta-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of alpha-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on beta-cell volume. In conclusion, KCCs are expressed in pancreatic alpha-cells and beta-cells. However, they make a significant contribution to volume homeostasis only in alpha-cells.     

Expression of K-Cl cotransporters in the α-cells of rat endocrine pancreas

The expression of K⁺-Cl⁻ cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in α-cells, but not pancreatic β-cells nor δ-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both α-cell and β-cells. Exposure of isolated α-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 μM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in β-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of α-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on β-cell volume. In conclusion, KCCs are expressed in pancreatic α-cells and β-cells. However, they make a significant contribution to volume homeostasis only in α-cells.     

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.     

Chloride influx provokes lamellipodium formation in microglial cells.

Lamellipodium extension and retraction is the driving force for cell migration. Although several studies document that activation of chloride channels are essential in cell migration, little is known about their contribution in lamellipodium formation. To address this question, we characterized chloride channels and transporters by whole cell recording and RT-PCR, respectively, as well as quantified lamellipodium formation in murine primary microglial cells as well as the microglial cell-line, BV-2, using time-lapse microscopy. The repertoire of chloride conducting pathways in BV-2 cells included, swelling-activated chloride channels as well as the KCl cotransporters, KCC1, KCC2, KCC3, and KCC4. Swelling-activated chloride channels were either activated by a hypoosmotic solution or by a high KCl saline, which promotes K(+) and Cl(-) influx instead of efflux by KCCs. Conductance through swelling-activated chloride channels was completely blocked by flufenamic acid (200 microM), SITS (1 mM) and DIOA (10 microM). By exposing primary microglial cells or BV-2 cells to a high KCl saline, we observed a local swelling, which developed into a prominent lamellipodium. Blockade of chloride influx by flufenamic acid (200 microM) or DIOA (10 microM) as well as incubation of cells in a chloride-free high K(+) saline suppressed formation of a lamellipodium. We assume that local swellings, established by an increase in chloride influx, are a general principle in formation of lamellipodia in eukaryotic cells.     

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.     

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.     

Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells

We studied whether K+-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.     

Use of Rb(+) and Br(-) as tracers for investigating ion transport by X-ray microanalysis in the Malpighian tubules of the black field cricket Teleogryllus oceanicus

Substitution of Rb(+) for K(+) in the incubation saline for in vitro preparations of Malpighian tubules had little effect on tubule function. Secretion rates increased by 10% for whole tubules, 9% for distal segments and 10% for main segments. In the secreted fluids Rb(+) almost completely replaced K(+). Within the cells of the main segment of the tubules Rb replaced the majority of the intracellular K. Treatment by ouabain in Rb saline resulted in a considerable increase in intracellular Na and Cl concentrations but no change in Rb concentration. This suggests that Rb(+) did not enter the cell via Na K ATPase and that the latter was not directly involved in Rb(+) secretion and by inference K(+) secretion. Substitution of Br(-) for Cl(-) in the incubation saline resulted in a 30% reduction in secretion rate from the distal segments but only a 10% reduction for the main segment. Cl(-) was almost completely replaced by Br(-) in fluid from both main and distal segments. In cells of the main segment the intracellular concentration of Br(-) did not exceed 30mmol kg(-1) dry weight and the Cl(-) concentration was unchanged in the apical region of the cell and increased in the basal region. These data suggest that Br(-) was transported across the tubule epithelium by a paracellular route and that the basal cell membrane is relatively impermeable to Cl(-). By inference Cl(-) may also be transported by a paracellular route.     

Anthracene-9-carboxylic acid inhibits an apical membrane,chloride conductance in canine tracheal epithelium

Summary Canine tracheal epithelium secretes Cl from the submucosal to the mucosal surface via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. Cl exit across the apical membrane is thought to be a passive, electrically conductive process. To examine the cellular mechanism of Cl secretion we studied the effect of anthracene-9-carboxylic acid (9-AC), an agent known to inhibit the Cl conductance of muscle membrane. When added to the mucosal solution, 9-AC rapidly and reversibly decreases short-circuit current and transepithelial conductance, reflecting a reduction in electrogenic Cl secretion. The inhibition is concentration-dependent and 9-AC does not appear to compete with Cl for the transport process. The decrease in current and conductance results from a decrease in the net and both unidirectional transepithelial Cl fluxes without substantial alterations of Na fluxes. Furthermore, 9-AC specifically inhibits a Cl conductance: tissues bathed in Cl-free solutions showed no response to 9-AC. Likewise, when the rate of secretion and Cl conductance were minimized with indomethacin, addition of 9-AC did not alter transepithelial conductance. In contrast, neither removal of Na from the media nor blockade of the apical Na conductance with amiloride prevented a 9-AC-induced decrease in transepithelial conductance. We also found that the effect of 9-AC is independent of transepithelial transport: 9-AC decreases transepithelial conductance despite inhibition of Cl secretion with ouabain or furosemide. Intracellular electrophysiologic techniques were used to localize the effect of 9-AC to a reduction of the electrical conductance of the apical cell membrane: 9-AC hyperpolarizes the electrical potential difference across the apical membrane and decreases its relative conductance. 9-AC also prevents the characteristic changes in the cellular electrical potential profile, transepithelial conductance, and the ratio of membrane conductances produced by a reduction in mucosal bathing solution Cl concentration. These results indicate that 9-AC inhibits Cl secretion in tracheal epithelium by blocking an electrically conductive Cl exit step in the apical cell membrane. Thus, they support a cellular model of Cl secretion in which Cl leaves the cell across a Cl permeable apical membrane driven by its electrochemical gradient.     

Phosphoregulation of the Na–K–2Cl and K–Cl cotransporters by the WNK kinases

Precise regulation of the intracellular concentration of chloride [Cl?]i is necessary for proper cell volume regulation, transepithelial transport, and GABA neurotransmission. The Na–K–2Cl (NKCCs) and K–Cl (KCCs) cotransporters, related SLC12A transporters mediating cellular chloride influx and efflux, respectively, are key determinants of [Cl?]i in numerous cell types, including red blood cells, epithelial cells, and neurons. A common “chloride/volume-sensitive kinase”, or related system of kinases, has long been hypothesized to mediate the reciprocal but coordinated phosphoregulation of the NKCCs and the KCCs, but the identity of these kinase(s) has remained unknown. Recent evidence suggests that the WNK (with no lysine = K) serine–threonine kinases directly or indirectly via the downstream Ste20-type kinases SPAK/OSR1, are critical components of this signaling pathway. Hypertonic stress (cell shrinkage), and possibly decreased [Cl?]i, triggers the phosphorylation and activation of specific WNKs, promoting NKCC activation and KCC inhibition via net transporter phosphorylation. Silencing WNK kinase activity can promote NKCC inhibition and KCC activation via net transporter dephosphorylation, revealing a dynamic ability of the WNKs to modulate [Cl?]. This pathway is essential for the defense of cell volume during osmotic perturbation, coordination of epithelial transport, and gating of sensory information in the peripheral system. Commiserate with their importance in serving these critical roles in humans, mutations in WNKs underlie two different Mendelian diseases, pseudohypoaldosteronism type II (an inherited form of salt-sensitive hypertension), and hereditary sensory and autonomic neuropathy type 2. WNKs also regulate ion transport in lower multicellular organisms, including Caenorhabditis elegans, suggesting that their functions are evolutionarily-conserved. An increased understanding of how the WNKs regulate the Na–K–2Cl and K–Cl cotransporters may provide novel opportunities for the selective modulation of these transporters, with ramifications for common human diseases like hypertension, sickle cell disease, neuropathic pain, and epilepsy.     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.     

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.     

K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells

Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl(-) cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase alpha1-subunit (alpha1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous alpha1NaK, and it recruited alpha1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of alpha1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with alpha1NaK. In conclusion, KCC3a forms a functional complex with alpha1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates alpha1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.     

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.     

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.