Basolateral Membrane Potential Research Articles

Transport of tetraethylammonium by the Malpighian tubules of Trichoplusia ni: Regional specialization and the influence of diet

Insect Malpighian tubules (MTs) play a major role in elimination of many potentially toxic compounds, including the organic cation tetraethylammonium (TEA). This paper examines transport of TEA by different segments of the MTs of the cabbage looper, Trichoplusia ni. The results show that the proximal ileac plexus (PIP) region of the MTs plays a dominant role in secretion of the organic cation TEA and that the rate of secretion is altered by feeding; principal cells of the proximal ileac plexus in tubules from larvae with full guts secreted TEA at higher rates than did the same cells in tubules of larvae in which the gut was empty. Michaelis-Menten analysis revealed that TEA secretion by the PIP was saturable and was blocked in a concentration-dependent manner by the organic cation cimetidine. For larvae reared from eggs on TEA-rich diet, higher concentrations of TEA in fluid secreted by the ileac plexus of tubules, and lower concentrations of TEA in the hemolymph, relative to larvae reared on control diet, is consistent with an upregulation of TEA transport in response to higher levels of dietary intake of an exogenous organic cation. The distal and proximal regions of the ileac plexus were also differentiated on the basis of transepithelial and basolateral membrane potentials and the influence of these electrical potentials on organic cation transport are discussed.

The genetic spectrum of Gitelman(-like) syndromes.

Purpose of reviewGitelman syndrome is a recessive salt-wasting disorder characterized by hypomagnesemia, hypokalemia, metabolic alkalosis and hypocalciuria. The majority of patients are explained by mutations and deletions in the SLC12A3 gene, encoding the Na+-Cl−-co-transporter (NCC). Recently, additional genetic causes of Gitelman-like syndromes have been identified that should be considered in genetic screening. This review aims to provide a comprehensive overview of the clinical, genetic and mechanistic aspects of Gitelman(-like) syndromes.Recent findingsDisturbed Na+ reabsorption in the distal convoluted tubule (DCT) is associated with hypomagnesemia and hypokalemic alkalosis. In Gitelman syndrome, loss-of-function mutations in SLC12A3 cause impaired NCC-mediated Na+ reabsorption. In addition, patients with mutations in CLCKNB, KCNJ10, FXYD2 or HNF1B may present with a similar phenotype, as these mutations indirectly reduce NCC activity. Furthermore, genetic investigations of patients with Na+-wasting tubulopathy have resulted in the identification of pathogenic variants in MT-TI, MT-TF, KCNJ16 and ATP1A1. These novel findings highlight the importance of cell metabolism and basolateral membrane potential for Na+ reabsorption in the DCT.SummaryAltogether, these findings extend the genetic spectrum of Gitelman-like electrolyte alterations. Genetic testing of patients with hypomagnesemia and hypokalemia should cover a panel of genes involved in Gitelman-like syndromes, including the mitochondrial genome.

Biallelic loss-of-function variants in KCNJ16 presenting with hypokalemic metabolic acidosis

KCNJ16 encodes Kir5.1 and acts in combination with Kir4.1, encoded by KCNJ10, to form an inwardly rectifying K+ channel expressed at the basolateral membrane of epithelial cells in the distal nephron. This Kir4.1/Kir5.1 channel is critical for controlling basolateral membrane potential and K+ recycling, the latter coupled to Na-K-ATPase activity, which determines renal Na+ handling. Previous work has shown that Kcnj16−/− mice and SSKcnj16−/− rats demonstrate hypokalemic, hyperchloremic metabolic acidosis. Here, we present the first report of a patient identified to have biallelic loss-of-function variants in KCNJ16 by whole exome sequencing who presented with chronic metabolic acidosis with exacerbations triggered by minor infections.

Voltage-gated calcium channels regulate K+ transport in the Malpighian tubules of the larval cabbage looper, Trichoplusia ni

Transporting epithelia are tissues that specialize in the directional movements of ions and water and are typically either secretory or reabsorptive. Recent work on the Malpighian tubule of larval lepidopterans (caterpillars) demonstrated that the distal ileac plexus segment of this epithelium is capable of rapidly switching between ion secretion and reabsorption. Subsequent transcriptomic studies suggested expression of voltage-gated ion channels in the lepidopteran MTs (which are not contractile and not innervated). The present study shows that isolated MTs of larval Trichoplusia ni express α1, β2, and α2δ4 subunits of voltage-gated Ca2+ channel CaV1 and that pan-CaVα immunoreactivity is present in the apical and basolateral membranes of the principal cells. Basolateral membrane potential (Vbl) in isolated MTs of larval Trichoplusia ni was influenced by CaV1 functioning; pharmacological inhibition of CaV1 reversed Vbl from inside-negative to inside-positive, and also reduced transepithelial potential (Vte), lowered [Ca2+]i and reversed the direction of K+ transport from secretion to reabsorption. Thus, our findings indicate that a functional CaV1 channel is necessary for constitutive K+ secretion observed in isolated preparations of lepidopteran MTs. Lastly, Vte and Vbl of isolated MTs were influenced by changes in bathing saline [K+]. Our findings suggest that epithelia may rely on CaV channels to enable robust ion secretion and downregulation of CaV channels, together with other transcriptional changes, enables ion reabsorption.

Mechanisms coupling sodium and magnesium reabsorption in the distal convoluted tubule of the kidney.

Hypomagnesaemia is a common feature of renal Na+ wasting disorders such as Gitelman and EAST/SeSAME syndrome. These genetic defects specifically affect Na+ reabsorption in the distal convoluted tubule, where Mg2+ reabsorption is tightly regulated. Apical uptake via TRPM6 Mg2+ channels and basolateral Mg2+ extrusion via a putative Na+‐Mg2+ exchanger determines Mg2+ reabsorption in the distal convoluted tubule. However, the mechanisms that explain the high incidence of hypomagnesaemia in patients with Na+ wasting disorders of the distal convoluted tubule are largely unknown. In this review, we describe three potential mechanisms by which Mg2+ reabsorption in the distal convoluted tubule is linked to Na+ reabsorption. First, decreased activity of the thiazide‐sensitive Na+/Cl− cotransporter (NCC) results in shortening of the segment, reducing the Mg2+ reabsorption capacity. Second, the activity of TRPM6 and NCC are determined by common regulatory pathways. Secondary effects of NCC dysregulation such as hormonal imbalance, therefore, might disturb TRPM6 expression. Third, the basolateral membrane potential, maintained by the K+ permeability and Na+‐K+‐ATPase activity, provides the driving force for Na+ and Mg2+ extrusion. Depolarisation of the basolateral membrane potential in Na+ wasting disorders of the distal convoluted tubule may therefore lead to reduced activity of the putative Na+‐Mg2+ exchanger SLC41A1. Elucidating the interconnections between Mg2+ and Na+ transport in the distal convoluted tubule is hampered by the currently available models. Our analysis indicates that the coupling of Na+ and Mg2+ reabsorption may be multifactorial and that advanced experimental models are required to study the molecular mechanisms.

Contribution of Kir4.1/Kir5.1 Channels to the Control of ENaC‐Mediated Apical Sodium Transport in the Cortical Collecting Duct

Recent studies using genetically modified animal models reveal that inwardly rectifying potassium channels Kir4.1 and Kir5.1, encoded by Kcnj10 and Kcnj16 genes, play an essential role in determining plasma potassium level and regulating blood pressure. Moreover, we have revealed recently that the deletion of Kcnj16 gene in Dahl salt‐sensitive (SS) rats has the profound effect on the blood pressure and renal damage. These data uncover a new potential target for the treatment of salt‐induced hypertension. Protein products of Kcnj10 and Kcnj16 are localized to the basolateral membrane of distal nephron segments forming functional homomeric (Kir4.1) and heteromeric (Kir4.1/Kir5.1) potassium channels that play an essential role in establishing a negative basolateral membrane potential and recycling K+ ions moved into the cytosol by the Na+/K+‐ATPase. In the present study we examined the effect of pharmacological inhibition of Kir4.1 and Kir4.1/Kir5.1 channels on sodium transport through the epithelial Na+ channel (ENaC) in the cortical collecting duct (CCD) cells. We have reported recently that Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM) inhibited whole‐cell potassium currents recorded from native principal CCD cells. First we examined whether fluoxetine and nortriptyline have different selectivity in targeting Kir4.1 and Kir4.1/Kir5.1 expressed in Chinese hamster ovary (CHO) cells. Single channel analysis revealed that application of 100 μM of amitriptyline produces a significant decrease in NPo of Kir4.1/Kir5.1 but does not affect Kir4.1 current activity, whereas fluoxetine selectively inhibits Kir4.1. Next we examined whether selective inhibition of Kir4.1 and Kir4.1/Kir5.1 affects an apical sodium transport via ENaC. We showed that amitriptyline, but not fluoxetine reduced (IC50=15.0 μM) the amiloride‐sensitive short‐circuit current (ISC) measured in polarized epithelial mCCDcl1 cells. In control experiments, application of amitriptyline did not directly affect ENaC activity overexpressed in CHO cells. Using different concentrations of recently developed Kir4.1 inhibitor, VU0134992, and patch‐clamp studies of single‐channel activity on mCCD cell line and native rodent and human CCD tubules, we confirmed that inhibition of Kir4.1/Kir5.1 channel but not Kir4.1 produces suppressive effect on ENaC activity. In summary, inhibition of Kir4.1/Kir5.1 channels in CCD cells indirectly reduces apical sodium conductance through ENaC, demonstrating the importance of basolateral potassium channels in the control of sodium reabsorption in distal nephron.Support or Funding InformationNational Institutes of Health grants R01 DK120821 (to J.D. and A.S.), R56 DK121750 (to O.P.), and R35 HL135749 (to A.S.)

Mechanisms of transport of H+, Na+ and K+, across the distal gastric caecum of larval Aedes aegypti

Measured changes in ion fluxes, transepithelial potential (TEP) and basolateral membrane potential (Vb) in response to ion transporter inhibitors were used to assess the mechanisms of transport of H+, Na+ and K+, across the distal gastric caecum of larval Aedes aegypti, a vector of yellow fever. Preparations were stimulated with 5-hydroxytryptamine (5-HT, 10−6 M) in order to maintain stable rates of H+, Na+, and K+ transport across the distal caecum. Transepithelial potential (TEP), basolateral membrane potential (Vb), and H+, Na+ and K+ fluxes all declined after the addition of a vacuolar-type H+-ATPase (VA) inhibitor, n-ethlymaleimide (NEM), consistent with a primary role for VA in energizing ion transport across the distal gastric caecum. Amiloride also inhibited H+, Na+, and K+ fluxes, consistent with an apically expressed VA that is coupled to a cation:H+ antiporter (AeNHE8), analogous to the coupling of apical VA and cation:nH+ antiporter in Malpighian tubules. A working model of transport of H+, Na+ and K+ across the distal gastric caecum proposes that coupling of VA and AeNHE8 in the apical membrane leads to the removal of intracellular Na+ or K+, thus creating favourable ion gradients to promote the activity of two transporters in the basal membrane, cation:H+ antiporter (AeNHE3) and a bumetanide-sensitive cation chloride cotransporter (CCC).

Deletion of Kir5.1 Impairs Renal Ability to Excrete Potassium during Increased Dietary Potassium Intake.

The basolateral potassium channel in the distal convoluted tubule (DCT), comprising the inwardly rectifying potassium channel Kir4.1/Kir5.1 heterotetramer, plays a key role in mediating the effect of dietary potassium intake on the thiazide-sensitive NaCl cotransporter (NCC). The role of Kir5.1 (encoded by Kcnj16) in mediating effects of dietary potassium intake on the NCC and renal potassium excretion is unknown. We used electrophysiology, renal clearance, and immunoblotting to study Kir4.1 in the DCT and NCC in Kir5.1 knockout (Kcnj16-/- ) and wild-type (Kcnj16+/+ ) mice fed with normal, high, or low potassium diets. We detected a 40-pS and 20-pS potassium channel in the basolateral membrane of the DCT in wild-type and knockout mice, respectively. Compared with wild-type, Kcnj16-/- mice fed a normal potassium diet had higher basolateral potassium conductance, a more negative DCT membrane potential, higher expression of phosphorylated NCC (pNCC) and total NCC (tNCC), and augmented thiazide-induced natriuresis. Neither high- nor low-potassium diets affected the basolateral DCT's potassium conductance and membrane potential in Kcnj16-/- mice. Although high potassium reduced and low potassium increased the expression of pNCC and tNCC in wild-type mice, these effects were absent in Kcnj16-/- mice. High potassium intake inhibited and low intake augmented thiazide-induced natriuresis in wild-type but not in Kcnj16-/- mice. Compared with wild-type, Kcnj16-/- mice with normal potassium intake had slightly lower plasma potassium but were more hyperkalemic with prolonged high potassium intake and more hypokalemic during potassium restriction. Kir5.1 is essential for dietary potassium's effect on NCC and for maintaining potassium homeostasis.

Potassium intake modulates the thiazide-sensitive sodium-chloride cotransporter (NCC) activity via the Kir4.1 potassium channel

Kir4.1 in the distal convoluted tubule plays a key role in sensing plasma potassium and in modulating the thiazide-sensitive sodium-chloride cotransporter (NCC). Here we tested whether dietary potassium intake modulates Kir4.1 and whether this is essential for mediating the effect of potassium diet on NCC. High potassium intake inhibited the basolateral 40 pS potassium channel (a Kir4.1/5.1 heterotetramer) in the distal convoluted tubule, decreased basolateral potassium conductance, and depolarized the distal convoluted tubule membrane in Kcnj10flox/flox mice, herein referred to as control mice. In contrast, low potassium intake activated Kir4.1, increased potassium currents, and hyperpolarized the distal convoluted tubule membrane. These effects of dietary potassium intake on the basolateral potassium conductance and membrane potential in the distal convoluted tubule were completely absent in inducible kidney-specific Kir4.1 knockout mice. Furthermore, high potassium intake decreased, whereas low potassium intake increased the abundance of NCC expression only in the control but not in kidney-specific Kir4.1 knockout mice. Renal clearance studies demonstrated that low potassium augmented, while high potassium diminished, hydrochlorothiazide-induced natriuresis in control mice. Disruption of Kir4.1 significantly increased basal urinary sodium excretion but it abolished the natriuretic effect of hydrochlorothiazide. Finally, hypokalemia and metabolic alkalosis in kidney-specific Kir4.1 knockout mice were exacerbated by potassium restriction and only partially corrected by a high-potassium diet. Thus, Kir4.1 plays an essential role in mediating the effect of dietary potassium intake on NCC activity and potassium homeostasis.

Insight into the Mode of Action of Celangulin V on the Transmembrane Potential of Midgut Cells in Lepidopteran Larvae.

Celangulin V (CV) is the main insecticidal constituent of Celastrus angulatus. The V-ATPase H subunit of the midgut cells of lepidopteran larvae is the putative target protein of CV. Here, we compared the effects of CV on the midgut membrane potentials of Mythimna separata and Agrotis ipsilon larvae with those of the Cry1Ab toxin from Bacillus thuringiensis and with those of inactive CV-MIA, a synthetic derivative of CV. We investigated the changes in the apical membrane potentials (Vam) and basolateral membrane potentials (Vbm) of the midguts of sixth-instar larvae force-fed with the test toxins. We also measured the Vam and Vbm of larval midguts that were directly incubated with the test toxins. Similar to the effect of Cry1Ab, the Vam of CV-treated midguts rapidly decayed over time in a dose-dependent manner. By contrast, CV-MIA did not influence Vam. Meanwhile, the Vam of A. ipsilon larval midguts directly incubated with CV decayed less than that of M. separata larval midguts, whereas that of larvae force-fed with CV did not significantly change. Similar to Cry1Ab, CV did not affect the Vbm of isolated midguts. CV significantly inhibited V-ATPase activity in a dose-dependent manner. Therefore, CV initially inhibits V-ATPase in the apical membrane and affects intracellular pH, homeostasis, and nutrient transport mechanisms in lepidopteran midgut cells.

Dietary K+ and Cl- independently regulate basolateral conductance in principal and intercalated cells of the collecting duct.

The renal collecting duct contains two distinct cell types, principal and intercalated cells, expressing potassium Kir4.1/5.1 (KCNJ10/16) and chloride ClC-K2 (ClC-Kb in humans) channels on their basolateral membrane, respectively. Both channels are thought to play important roles in controlling systemic water-electrolyte balance and blood pressure. However, little is known about mechanisms regulating activity of Kir4.1/5.1 and ClC-K2/b. Here, we employed patch clamp analysis at the single channel and whole cell levels in freshly isolated mouse collecting ducts to investigate regulation of Kir4.1/5.1 and ClC-K2/b by dietary K+ and Cl- intake. Treatment of mice with high K+ and high Cl- diet (6% K+, 5% Cl-) for 1week significantly increased basolateral K+-selective current, single channel Kir4.1/5.1 activity and induced hyperpolarization of basolateral membrane potential in principal cells when compared to values in mice on a regular diet (0.9% K+, 0.5% Cl-). In contrast, basolateral Cl--selective current and single channel ClC-K2/b activity was markedly decreased in intercalated cells under this condition. Substitution of dietary K+ to Na+ in the presence of high Cl- exerted a similar inhibiting action of ClC-K2/b suggesting that the channel is sensitive to variations in dietary Cl- per se. Cl--sensitive with-no-lysine kinase (WNK) cascade has been recently proposed to orchestrate electrolyte transport in the distal tubule during variations of dietary K+. However, co-expression of WNK1 or its major downstream effector Ste20-related proline-alanine-rich kinase (SPAK) had no effect on ClC-Kb over-expressed in Chinese hamster ovary (CHO) cells. Treatment of mice with high K+ diet without concomitant elevations in dietary Cl- (6% K+, 0.5% Cl-) elicited a comparable increase in basolateral K+-selective current, single channel Kir4.1/5.1 activity in principal cells, but had no significant effect on ClC-K2/b activity in intercalated cells. Furthermore, stimulation of aldosterone signaling by Deoxycorticosterone acetate (DOCA) recapitulated the stimulatory actions of high K+ intake on Kir4.1/5.1 channels in principal cells but was ineffective to alter ClC-K2/b activity and basolateral Cl- conductance in intercalated cells. In summary, we report that variations of dietary K+ and Cl- independently regulate basolateral potassium and chloride conductance in principal and intercalated cells. We propose that such discrete mechanism might contribute to fine-tuning of urinary excretion of electrolytes depending on dietary intake.

The gastric caecum of larval Aedes aegypti: stimulation of epithelial ion transport by 5-hydroxytryptamine and cAMP.

We report measurements of ion transport across the gastric caecum of larvae of Aedes aegypti, a vector of yellow fever that inhabits a variety of aquatic habitats ranging from freshwater to brackish water. We provide the first measurements of the effect of 5-hydroxytryptamine (5-HT) on transepithelial potential (TEP), luminal ion concentrations and electrochemical potentials, as well as basolateral membrane potential and H+, Na+ and K+ fluxes. TEP, basolateral membrane potential, and H+, K+ and Na+ fluxes across the gastric caeca declined within 3-6 min after isolation of the entire midgut from the larva. 5-HT restored both the TEP and active accumulation of H+, K+ and Na+ in the lumen. Additionally, 5-HT restored H+, K+ and Na+ fluxes across the distal caecum of freshwater larvae, and restored H+ fluxes across the distal caecum of brackish water larvae. There was no effect of 5-HT on ion fluxes across the proximal caecum. We have also shown that 5-HT restores the basolateral membrane potential in cells of the distal, but not proximal, caecum. Effects of 5-HT on TEP and basolateral membrane potential were mimicked by application of cAMP but not by a phorbol ester. We provide a working model which proposes that 5-HT and cAMP stimulate the vacuolar H+-ATPase of the distal caecum. Our results provide evidence that the gastric caecum is functionally distinct from the adjacent anterior midgut and we discuss possible roles of the gastric caecum in osmoregulation. We also describe similarities in the arrangement of ion transporters in the caecum with those of the Malpighian tubules.

Role of Kir5.1 (Kcnj16) Channel in the Control of Potassium Homeostasis in the Kidney

The inwardly‐rectifying potassium (K+) channels (Kir), Kir4.1/Kir5.1 (Kcnj10/Kcnj16) are critical channels responsible for the recycling of K+ in the distal nephron as well as regulation of basolateral membrane potential. Human mutations in KCNJ10 and KCNJ16 are associated with renal pathologies characterized by salt‐wasting, renal tubulopathy and hypokalemia (EAST/SeSAME and Gitelman syndromes) as well as cardiac phenotypes such as Brugada syndrome). The Kir4.1/Kir5.1 heteromeric channel is expressed on the basolateral side of distal convoluted tubule (DCT) cells and the principal cells of the cortical collecting duct (CCDs). Immunohistochemistry analysis of Dahl salt‐sensitive (SS) rats showed increased Kir5.1 abundance in the CCD compared to the DCT. Moreover, expression of Kir5.1 in both segments was significantly upregulated after 3 weeks of high salt diet (4% NaCl), showing a direct correlation between the development of salt sensitive hypertension and basolateral K+ channel function. To further explore the role of this channel in salt‐sensitive hypertension, we have created a loss‐of‐function mutation in Kir5.1 in the Dahl SS rat (SS‐Kcnj16em1Mcwi) using zinc‐finger nucleases. Overall, Kir5.1 loss‐of‐function mutation in the SS rat induced a renal phenotype characterized by hypokalemia (2.1±0.2 vs 4.3±0.1 mmol/L (mutant vs SS)), low aldosterone level (245.4±26.5 vs 1050±100 pg/ml), salt wasting (FEK+ 30.3±3.1 vs 5.8±0.6; FENa+ 0.42±0.06 vs 0.19±0.03; FEMg2+ 15.8±1.3 vs 6.1±0.9 %) and decreased mean arterial blood pressure (91.3±1.8 vs 104.7±5.5 mmHg). Furthermore, the absence of the Kir5.1 from distal nephron resulted in a compensatory increase of Kir4.1 protein abundance. IHC analysis of Kir4.1 localization revealed a rerouting from its expected location in the basolateral membrane of CCD and DCT tubules to the cytosol instead. We also found that loss‐of‐function mutation of Kir5.1 triggered upregulation of both total and phosphorylated forms of the NCC and NKCC2. In contrast, we were not able to identify any changes in the expression of ENaC subunits. Increasing the salt content of the diet from 0.4 to 4% triggered sodium and potassium excretion in Kir5.1 mutants, which resulted in a rapid reduction of blood K+ to critically low values (1.36±0.04 vs 2.1±0.1mmo/L (HS vs LS)). Na+ blood concentrations (141.6±0.5 vs 139.2±1.5 mmo/L) were unchanged between these two groups. This severe hypokalemia resulted in high mortality rate with most animals dying within 24 hours. These data demonstrate the critical role of renal basolateral Kir4.1/Kir5.1 channels in potassium handling, and identify them as a potential pharmacological target for the treatment of the imbalanced potassium homeostasis.Support or Funding InformationSupported by Advancing a Healthier Wisconsin Research and Education Program #9520217 and HL122662

Low basolateral Na+ pump activity in macula densa cells may be necessary to generate tubuloglomerular feedback responses (892.32)

Macula densa (MD) cells sense changes in tubular fluid [Cl–] and this initiates a chain of events within the juxtaglomerular apparatus (i.e., the tubuloglomerular feedback (TGF) response) that regulates GFR. It is known that MD cells have much lower basolateral Na+/K+ATPase activity than neighboring cortical thick ascending limb (cTAL) cells. Also, an increase in luminal [NaCl] leads to the depolarization of the MD cell basolateral membrane, an event thought to be an essential step in the TGF response. To study the role of the Na+ pump in MD cells, we created a mathematical model that includes kinetic and thermodynamic models of the transport proteins and channels in MD cells. We examined the influence of luminal [NaCl] on basolateral membrane potential difference (PD). For comparison, similar computations were made with a model of a cTAL cell. When the luminal NaCl was varied from 20 to 150 mM, the basolateral membrane of the MD cell depolarized from –71 to –47 mV. For the cTAL cell, the basolateral membrane potential remained near –96 mV. These results suggest that the low expression of the basolateral Na+ pump may be appropriate in MD cells because, in these sensory cells, cytosolic [Na+] tracks luminal [Na+]. In a reabsorbing cTAL cell, the high basolateral Na+ pump expression stabilizes cytosolic [Na+] and basolateral PD.Grant Funding Source: Supported in part by MBRS‐RISE grant 2R25GM061151‐12 and by NIH‐NIGMS grant SC1GM084744.

Direct inhibition of basolateral Kir4.1/5.1 and Kir4.1 channels in the cortical collecting duct by dopamine

It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about the molecular targets of dopamine actions on water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine, which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K(+) channels in the distal renal tubule are critical for K(+) recycling and controlling basolateral membrane potential to establish the driving force for Na(+) reabsorption. Here, we demonstrate that Kir4.1 and Kir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCD. Using patch-clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40-pS and scarce 20-pS single channel conductances, most likely representing Kir4.1/5.1 and Kir4.1 channels, respectively. Dopamine reversibly decreased the open probability of both channels, with a relatively greater action on the Kir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. PKC blockade abolished the inhibition of basolateral K(+) channels by dopamine. Importantly, dopamine significantly decreased the amplitude of Kir4.1/5.1 and Kir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential, as directly monitored using current-clamp mode in isolated CCDs. Therefore, we demonstrate that dopamine inhibits basolateral Kir4.1/5.1 and Kir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of the basolateral membrane and a decreased driving force for Na(+) reabsorption in the distal renal tubule.

Dopamine inhibits basolateral potassium channels in the murine distal nephron

It is recognized that dopamine promotes natriuresis by inhibiting NHE3 and Na+‐K+‐ATPase in the proximal tubule. In contrast, less is known about putative dopamine actions on the water‐electrolyte transport in the distal nephron. Basolateral side of the epithelial cells in the distal nephron is exposed to influence of dopamine secreted from sympathetic nerve endings. Furthermore, dopamine receptors are abundantly expressed at the basolateral membrane of the connecting tubule and the collecting duct cells. Basolateral K+ channels in the distal nephron are critical for potassium recycling and controlling basolateral membrane potential to promote electrogenic Na+ reabsorption. Using patch clamp electrophysiology in freshly isolated distal nephrons from mice, we detected ~20 pS and 40 pS basolateral K+ channels most likely representing Kir4.1, Kir4.1/5.1, respectively. Dopamine reversibly inhibited both channels when applied in a low micromolar range. Importantly, dopamine also decreased the amplitude of unitary currents approximately by 30% indicating depolarization of basolateral membrane and reduction of transepithelial voltage. We propose that consistently with its actions in the proximal tubule, dopamine reduces Na+ transport in the distal nephron, in part, by inhibiting basolateral K+ conductance and decreasing driving force for sodium reabsorption.

Chemosensory function of amphibian skin: integrating epithelial transport, capillary blood flow and behaviour

Terrestrial anuran amphibians absorb water across specialized regions of skin on the posterioventral region of their bodies. Rapid water absorption is mediated by the insertion of aquaporins into the apical membrane of the outermost cell layer. Water moves out of the epithelium via aquaglyceroporins in the basolateral membrane and into the circulation in conjunction with increased capillary blood flow to the skin and aquaporins in the capillary endothelial cells. These physiological responses are activated by intrinsic stimuli relating to the animals' hydration status and extrinsic stimuli relating to the detection of osmotically available water. The integration of these processes has been studied using behavioural observations in conjunction with neurophysiological recordings and studies of epithelial transport. These studies have identified plasma volume and urinary bladder stores as intrinsic stimuli that activate the formation of angiotensin II (AII) to stimulate water absorption behaviour. The coordinated increase in water permeability and capillary blood flow appears to be mediated primarily by sympathetic stimulation of beta adrenergic receptors, although the neurohypopyseal hormone arginine vasotocin (AVT) may also play a role. Extrinsic stimuli relate primarily to the ionic and osmotic properties of hydration sources. Toads avoid NaCl solutions that have been shown to be harmful in acute exposure, approx. 200-250 mm. The avoidance is partially attenuated by amiloride raising the hypothesis that the mechanism for salt detection by toads resembles that for salt taste in mammals that take in water by mouth. In this model, depolarization of the basolateral membrane of taste cells is coupled to afferent neural stimulation. In toad skin we have identified innervation of skin epithelial cells by branches of spinal nerves and measured neural responses to NaCl solutions that elicit behavioural avoidance. These same concentrations produce depolarization of the basolateral membrane in isolated epithelial preparations. As with salt taste in mammals, the neural responses and depolarization of basolateral membrane potential are partially inhibited by amiloride. In addition, toads are more tolerant of sodium gluconate solution which is consistent with the phenomenon in mammalian taste physiology termed the anion paradox in which sodium salts with larger molecular weight anions produce a reduced intensity of salt taste. Finally, toads also avoid concentrated solutions of a non-electrolyte, mannitol, which differs from NaCl solutions in not affecting transepithelial conductance and requires a longer time to depolarize the basolateral membrane. Osmotic stimuli may mediate sensory processes for longer term detection of conditions with low water potential while ionic stimuli are more important for shorter term analysis of rehydration sources.

Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands

Cockroach salivary glands are innervated by dopaminergic and serotonergic neurons. Both transmitters elicit saliva secretion. We studied the distribution pattern of neurons containing gamma-aminobutyric acid (GABA) and their physiological role. Immunofluorescence revealed a GABA-immunoreactive axon that originates within the subesophageal ganglion at the salivary neuron 2 (SN2) and this extends within the salivary duct nerve towards the salivary gland. GABA-positive fibers form a network on most acinar lobules and a dense plexus in the interior of a minor fraction of acinar lobules. Co-staining with anti-synapsin revealed that some putative GABAergic terminals seem to make pre-synaptic contacts with GABA-negative release sites. Many putative GABAergic release sites are at some distance from other synapses and at distance from the acinar tissue. Intracellular recordings from isolated salivary glands have revealed that GABA does not affect the basolateral membrane potential of the acinar cells directly. When applied during salivary duct nerve stimulation, GABA enhances the electrical response of the acinar cells and increases the rates of fluid and protein secretion. The effect on electrical cell responses is mimicked by the GABA(B) receptor agonists baclofen and SKF97541, and blocked by the GABA(B) receptor antagonists CGP52432 and CGP54626. These findings indicate that GABA has a modulatory role in the control of salivation, acting presynaptically on serotonergic and/or dopaminergic neurotransmission.

Oscillating Cortical Thick Ascending Limb Cells at the Juxtaglomerular Apparatus

While studying the intracellular calcium dynamics in cells of the macula densa, the observation was made that tubular epithelial cells located near the macula densa and associated with the renal arterioles exhibit spontaneous Ca2+ oscillations. In this study, the cortical thick ascending limb-distal tubule, with attached glomerulus, was isolated and perfused. At a low luminal sodium chloride concentration, Ca2+ oscillations at a frequency of 63 mHz were observed in tubular cells that were within 100 microm of the macula densa plaque using four-dimensional multiphoton microscopy and wide-field fluorescence microscopy with fura-2. The Ca2+ oscillations were absent in the macula densa cells. Spontaneous oscillations in basolateral membrane potential suggested that Ca2+ oscillations occurred, at least in part, through depolarization-induced increases in Ca2+ entry. The amplitude of these Ca2+ oscillations was significantly enhanced by the activation of the Ca2+-sensing receptor. Increasing the luminal sodium chloride concentration or luminal flow resulted in a significant increase in both the amplitude of Ca2+ oscillations and the intracellular Ca2+ concentration in perimacular cortical thick ascending limb cells. In addition, luminal furosemide attenuated the [NaCl]L-dependent changes in intracellular Ca2+ concentration, but hydrochlorothiazide had no effect. These findings demonstrate that tubular epithelial cells at the perimeter of the macula densa exhibit spontaneous oscillations in intracellular Ca2+ concentration, enhanced by tubular flow and luminal sodium chloride. These oscillatory patterns may play a role in juxtaglomerular signaling.

Chemosensory function of amphibian skin epithelium: Amiloride inhibition of basolateral membrane depolarization in response to increased bath osmolality correlates with afferent neural activity and behavior.

In dehydrated toads cutaneous water absorption is associated with initiation a behavior, the water absorption response (WR), in which the toads press the skin to a rehydration surface. WR initiation is suppressed in toads given a hyperosmotic (250 mM) NaCl solution. Associated with WR suppression the activity of spinal nerve #6 increased when the skin was exposed to this solution and the basolateral membrane potential (Vb) of principal cells in the isolated skin depolarized. These results support our hypothesis that the chemosensory properties of the skin are analogous to taste cells of the mammalian tongue where depolarization of Vb also stimulates neural activity. Here we show the frequency of WR initiation increased from 21% to 50% of trials in toads given 250 mM NaCl with 10 μM amiloride, that the time toads allow ventral skin contact is greater in the presence of amiloride, and that the depolarization of Vb is greatly reduced, relative to NaCl alone. The frequency of WR initiation was also greater with 250 mM Na gluconate as the rehydration solution (40% vs 21% with NaCl) and was increased to 80% of trials when 10 μM amiloride was added to the Na gluconate solution. Inhibition of the transcellular pathway with amiloride and the paracellular pathway with gluconate have similar behavioral responses, and variability in the behavioral responses correspond with variability in Vb and paracellular conductance.