X-ray microanalysis of the Malpighian tubules of the black field cricket Teleogryllus oceanicus: the roles of Na K ATPase and the Na K 2Cl cotransporter

Both main and distal segments of the Malpighian tubules were sensitive to ouabain and furosemide but in different ways. Oubain had no effect on secretion rate by the main segment but in the secreted fluid Na(+) concentration increased substantially whereas K(+) decreased. Similarly intracellular elemental Na concentration increased and K decreased. Furosemide decreased the secretion rate of the main segment by 80%. The Na(+) concentration in the secreted fluid increased markedly but K(+) was not affected. Intracellular elemental Na concentration also increased but K was unchanged. In the distal segments both ouabain and furosemide decreased secretion rate by 40% but although ouabain had no effect on the composition of the secreted fluid, furosemide caused a substantial reduction in the concentrations of Mg(2+) and Cl(-) and a substantial increase in Na(+) and K(+) concentrations. The evidence suggests that the main segment contains a Na K ATPase and possibly a Na K 2Cl cotransporter whereas the distal segment may contain a Na K ATPase and a furosemide sensitive Mg(2+) transporter. K(+) entry into the cells of the main segment may be partially effected by a Na K 2Cl cotransporter but may be primarily via Na K ATPase in the distal segment.     

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.     

Dopamine-induced epithelial K(+) and Na(+) movements in the salivary ducts of Periplaneta americana

K(+)- and Na(+)-selective double-barrelled microelectrodes were used for intracellular and luminal measurements in salivary ducts of Periplaneta americana. The salivary ducts were stimulated with dopamine (10(-6) mol l(-1)). Dopamine decreased intracellular [K(+)] from 112+/-17 mmol l(-1) to 40+/-13 mmol l(-1) (n=6) and increased intracellular [Na(+)] from 22+/-19 mmol l(-1) to 92+/-4 mmol l(-1) (n=6). Luminal [K(+)] was 15+/-3 mmol l(-1) in the unstimulated salivary ducts and increased to 26+/-11 mmol l(-1) upon stimulation with dopamine (n=10). Luminal [Na(+)] was insignificantly increased from 105+/-25 mmol l(-1) to 116+/-22 mmol l(-1) (n=12) by stimulation with dopamine. The potential difference across the basolateral membrane (PD(b)) was depolarized from -65+/-6 mV to -31+/-13 mV (n=12) and the transepithelial potential difference (PD(t)) was hyperpolarized from -13+/-6 mV to -22+/-7 mV (n=22, lumen negative) upon stimulation with dopamine. The re-establishment of prestimulus values of intracellular [K(+)] and [Na(+)] and PD(b) was inhibited by basolateral addition of ouabain (10(-4) mol l(-1)). Furosemide (10(-4) mol l(-1)) in the bath inhibited the dopamine-induced increase in intracellular [Na(+)], the decrease in intracellular [K(+)] and the depolarization of PD(b). We propose a model for dopamine-stimulated ion transport in the salivary ducts involving basolateral Na(+)-K(+)-2Cl(-) cotransport and active extrusion of K(+) via the apical membrane.     

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.     

The action of inhibitors of protein kinases on fluid and ion secretion by Malpighian tubules of Locusta migratoria,L

Fluid production in Locusta Malpighian tubules was stimulated by corpora cardiaca extract (c. 100%) and dibutyryl cAMP (c. 50%). Chelerythrine and staurosporine (Protein kinase C, PKC inhibitors) inhibited it in the range 0.07-60&mgr;M (IC(50)3&mgr;M), whereas Rp-cAMP (Protein kinase A, PKA inhibitor) caused inhibition over the concentration range 10-1000&mgr;M (IC(50)264&mgr;M). The protein phosphatase inhibitor, okadaic acid, was also inhibitory over the concentration range 0.1-1000nM (IC(50) 91nM). CC extract stimulation increased fluid [Na(+)] from 41 to 59mM and decreased [K(+)] from 127 to 107mM; stimulation with cAMP had no such effect. The PKC inhibitors reduced the [K(+)] in the secreted fluid from 126 to 107mM but had no effect on the [Na(+)]. Subsequent addition of CC extract stimulated fluid production and caused an increase in [Na(+)] from 41 to about 50mM. The addition of Rp-cAMP reduced fluid production but caused a decrease in [Na(+)] from 37 to 28mM and an increase in its [K(+)] from 124 to 148mM. Fluid production by Rp-cAMP inhibited tubules was not stimulated by corpora cardiaca extract or cAMP, but [Na(+)] rose to 36mM. Protein phosphorylation plays a role in the regulation of fluid production probably via the apical and basal membrane cation transporters.     

Ion transport across the epithelium of the rabbit caecum.

The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36Cl and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/Cl- symport in the apical barrier.     

Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation,and effects of ouabain and bumetamide

Isolated salivary glands of Periplaneta americana were used to measure secretion rates and, by quantitative capillary electrophoresis, Na(+), K(+), and Cl(-) concentrations in saliva collected during dopamine (1 micro M) and serotonin (1 micro M) stimulation in the absence and presence of ouabain (100 micro M) or bumetanide (10 micro M). Dopamine stimulated secretion of a NaCl-rich hyposmotic saliva containing (mM): Na(+) 95 +/- 2; K(+) 38 +/- 1; Cl(-) 145 +/- 3. Saliva collected during serotonin stimulation had a similar composition. Bumetanide decreased secretion rates induced by dopamine and serotonin; secreted saliva had lower Na(+), K(+) and Cl(-) concentrations and osmolarity. Ouabain caused increased secretion rates on a serotonin background. Saliva secreted during dopamine but not serotonin stimulation in the presence of ouabain had lower K(+) and higher Na(+) and Cl(-) concentrations, and was isosmotic. We concluded: The Na(+)-K(+)-2Cl(-) cotransporter is of cardinal importance for electrolyte and fluid secretion. The Na(+)/K(+)-ATPase contributes to apical Na(+) outward transport and Na(+) and K(+) cycling across the basolateral membrane in acinar P-cells. The salivary ducts modify the primary saliva by Na(+) reabsorption and K(+) secretion, whereby Na(+) reabsorption is energized by the basolateral Na(+)/K(+)-ATPase which imports also some of the K(+) needed for apical K(+) extrusion.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Electrochemical characteristics of ion secretion in malpighian tubules of the New Zealand alpine weta (Hemideina maori)

Characteristics of ion and fluid secretion were investigated in isolated Malpighian tubules of the New Zealand Alpine Weta (Hemideina maori). Fluid secretion by tubules in iso-osmotic saline (500mOsm) occurred at a rate of 15+/-3nlh(-1) and was enriched in K(+) (approx. 125mmoll(-1)) relative to the saline (10mmoll(-1)). Maximal fluid secretion (112nlh(-1)) during simultaneous exposure to hypo-osmolality and dibutyryl cAMP resulted in an 8.8x increase in the quantity of K(+) secreted, compared to only a 2.4x increase in Na(+) secretion. Measurements of intracellular ion activities and membrane potentials indicated that Na(+) and K(+) were transported against a strong electrochemical gradient across the apical surface, regardless of saline osmolality. On the basolateral surface, there was a large driving force for Na(+) entry, while K(+) was distributed near its equilibrium potential. Neither bumetanide nor ouabain in the bathing saline had a significant effect on fluid secretion, but Ba(2+) and amiloride decreased fluid secretion by 79 and 57%, respectively. The effect of Ba(2+) on fluid secretion was consistent with a high basolateral permeability to K(+), relative to Na(+) and Cl(-). These results indicate that the characteristics of fluid secretion in this primitive insect are largely conserved with characteristics reported for other insects.     

Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.     

Secretion of water and ions by malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity

The effects of changes in the salinity of the rearing medium on Malpighian tubule fluid secretion and ion transport were examined in larvae of the freshwater mosquito Aedes aegypti and the saltwater species Ochlerotatus taeniorhynchus. For unstimulated tubules of both species, the K(+) concentration of secreted fluid was significantly lower when larvae were reared in 30% or 100% seawater (O. taeniorhynchus only), relative to tubules from freshwater-reared larvae. The Na(+) concentration of secreted fluid from unstimulated tubules of O. taeniorhynchus reared in 30% or 100% seawater was higher relative to tubules from freshwater-reared larvae. The results suggest that changes in salinity of the larval rearing medium lead to sustained changes in ion transport mechanisms in unstimulated tubules. Furthermore, alterations of K(+) transport may be utilized to either conserve Na(+) under freshwater (Na(+)-deprived) conditions or eliminate more Na(+) in saline (Na(+)-rich) conditions. The secretagogues cyclic AMP [cAMP], cyclic GMP [cGMP], leucokinin-VIII, and thapsigargin stimulated fluid secretion by tubules of both species. Cyclic AMP increased K(+) concentration and decreased Na(+) concentration in the fluid secreted by tubules isolated from O. taeniorhynchus larvae reared in 100% seawater. Interactions between rearing salinity and cGMP actions were similar to those for cAMP. Leucokinin-VIII and thapsigargin had no effect on secreted fluid Na(+) or K(+) concentrations. Results indicate that changes in rearing medium salinity affect the nature and extent of stimulation of fluid and ion secretion by secretagogues.     

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.     

Na+ and K+ ion imbalances in Alzheimer's disease

Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aβ 1-40, but not by Aβ 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.     

Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the role of organic acid transport

Secretion of primary urine by upper Malpighian tubules of the blood-sucking insect Rhodnius prolixus has recently been shown to be inhibited by cyclic GMP (cGMP). In the present work, we have demonstrated that cGMP has effects antagonistic to those of cAMP in Rhodnius tubules and have further characterized the effects of cGMP on tubular secretion. Cyclic GMP inhibited secretion at all concentrations from 5x10(-6) to 10(-3)M, though this inhibition was partially or wholly reversed by large (2mM) doses of cAMP. While sub-maximal concentrations of cGMP did not significantly alter [K(+)] and [Na(+)] of secreted fluid, high external [cGMP] reduced secretion to minimal levels and caused [K(+)] and [Na(+)] to approach pre-stimulation levels. Cyclic GMP does not appear to affect the permeability of the lower Malpighian tubule to water. Both cAMP and cGMP likely enter tubule cells by way of an organic acid transporter whose activity is induced by feeding. Sensitivity of the tubules to exogenous cGMP and cAMP, which is assumed to be a function of transport activity, reaches a peak approximately 5 days after the blood meal and declines rapidly thereafter. Transport of anions into upper tubules involves at least two different transporters: one for acylamides (e.g., p-aminohippuric acid) and another for sulphonates (e.g., amaranth, phenol red). Amaranth and phenol red blocked the actions of both cGMP and cAMP, whereas p-aminohippuric acid was without effect. This suggests that cyclic nucleotides enter by way of the sulphonate transporter.     

Intracellular ion concentrations in the frog cornea epithelium during stimulation and inhibition of Cl secretion

Summary The intracellular electrolyte concentrations in the isolated cornea of the American bullfrog were determined in thin freeze-dried cryosections using energy-dispersive X-ray microanalysis. Stimulation of Cl secretion by isoproterenol resulted in a significant increase in the intracellular Na concentration but did not change the intracellular Cl concentration. Similar results were obtained when Cl secretion was stimulated by the Ca ionophore A23187. Inhibition of Cl secretion by ouabain produced a large increase in the intracellular Na concentration and an equivalent fall in the K concentration. Again, no increase or decrease in the intracellular Cl concentration was detectable. Clamping of the transepithelial potential to ±50 mV resulted in parallel changes in the transepithelial current and intracellular Na concentration, but, with the exception of the outermost cell layer, in no changes of the Cl concentration. Only when Cl secretion was inhibited by bumetanide or furosemide, together with a decrease in the Na concentration, was a large fall in the Cl concentration observed. Application of loop diuretics also produced significant increases in the P concentration and dry weight, consistent with some shrinkage of the epithelial cells. The results suggest the existence of a potent regulatory mechanism which maintains a constant intracellular Cl concentration and, thereby, a constant epithelial cell volume. Through the operation of this system any variation in the apical Cl efflux is compensated for by an equal change in the rate of Cl uptake across the basolateral membrane. Cl uptake is sensitive to loop diuretics, directly coupled to an uptake of Na, and dependent on the Na and K concentration gradients across the basolateral membrane. Isoproterenol and A23187 seem to increase the Cl permeability of the apical membrane and thus stimulate Cl efflux. Ouabain inhibits Cl secretion by abolishing the driving Na concentration gradient for Cl uptake across the basolateral membrane.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

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.     

Role of ouabain and diuretics on sodium, potassium and chloride retention in perfused rat kidney

A novel in situ kidney perfusion technique is described in Sprague-Dawley rats. The procedure involves retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. Ouabain (15 mM) in perfusate increased Na retention by 92%, decreased K retention by 53% and produced no effect on Cl retention. Ethacrynic acid (1 mM) in perfusate decreased Na retention by 52%, increased K retention by 105% and decreased Cl retention by 27%. Furosemide (1.5 mM) in perfusate decreased Na retention by 52%, increased K retention by 47% and decreased Cl retention by 56%. The Na-K-ATPase pump localized at the peritubular side of the proximal tubule cell is ouabain sensitive and Mg dependent. An Na-K pump responsible for Na influx and K effux exists at the luminal side of the proximal tubule cell and is ethacrynic acid and furosemide sensitive.     

Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes

Both Cs(+) and NH(4)(+) alter neuronal Cl(-) homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K(+)-Cl(-) cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive (86)Rb(+) influx as a function of external Rb(+) concentration at different fixed external cation concentrations (Na(+), Li(+), K(+), Cs(+), and NH(4)(+)). Neither Na(+) nor Li(+) affected furosemide-sensitive (86)Rb(+) influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb(+) and K(+) as alternate substrates, K(+) was a competitive inhibitor of Rb(+) transport by KCC2. Like K(+), both Cs(+) and NH(4)(+) behaved as competitive inhibitors of Rb(+) transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb(+) and Cs(+) influxes, we determined that although KCC2 was capable of transporting Cs(+), it did so with a lower apparent affinity and maximal velocity compared with Rb(+). To assess NH(4)(+) transport by KCC2, we monitored intracellular pH (pH(i)) with a pH-sensitive fluorescent dye after an NH(4)(+)-induced alkaline load. Cells expressing KCC2 protein recovered pH(i) much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH(4)(+) uptake. Consistent with KCC2-mediated NH(4)(+) transport, pH(i) recovery in KCC2-expressing cells could be inhibited by furosemide (200 microM) or removal of external [Cl(-)]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl(-) homeostasis in the presence of Cs(+) and NH(4)(+).