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B A Stanton

Publications and source records attributed to B A Stanton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel.

The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.

Amiloride↗
PubMed1998

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, which mediates transepithelial Cl- transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl- secretion in part by increasing the number of CFTR Cl- channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl- secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl- secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl- channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl- secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl- secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Cystic fibrosis transmembrane conductance regulator (CFTR) and renal function.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is expressed in all nephron segments. Although mutations in CFTR are not associated with major changes in renal function, drug excretion by the kidneys is altered in cystic fibrosis (CF) as is the ability of the kidneys to concentrate and dilute the urine and excrete a salt load. It is not clear if these changes in renal function are secondary to decreased extracellular fluid volume caused by excessive losses of NaCl in sweat and feces or if they are related to primary defects in renal function caused by mutations in CFTR. Considerable evidence supports a role for CFTR in mediating Cl- secretion by the distal tubule, principal cells in the cortical collecting duct (CCD) and the inner medullary collecting duct (IMCD). In addition, CFTR is responsible for Cl- secretion into the lumen of cysts in polycystic kidneys and, therefore, contributes to cyst enlargement. Under some conditions--when Na+ absorption across the apical membrane of principal cells in the CCD is stimulated and the apical membrane potential is depolarized--the electrochemical gradient for Cl- will support Cl- absorption via CFTR Cl- channels. In addition to its function as a 3',5'-cAMP-activated Cl- channel, CFTR may play a role in intracellular vesicle acidification, protein processing, protein trafficking, secretion of ATP and the regulation of the epithelial Na channel (ENaC) and the secretory K+ channel (ROMK2) which mediate Na+ and K+ transport, respectively, across the CCD. Thus, CFTR may regulate Na+ and K+ excretion by the kidneys. The most common mutation in CFTR is delta F508, a mutation which causes improper folding of CFTR such that it is retained within the endoplasmic reticulum where it is degraded. Thus, in the majority of cases, CF is a trafficking disease. However, nothing is known about the intracellular trafficking of CFTR in the kidney. In preliminary studies we have developed a living cell model to study the intracellular trafficking of CFTR and delta F508-CFTR in renal epithelial cells in real time. Our ultimate goal is to elucidate the intracellular trafficking of CFTR and to identify therapeutic approaches to restore normal function to renal cells in CF and to block CFTR-mediated Cl- secretion in cysts in polycystic kidneys.

Animals↗
PubMed1997

Cyclic nucleotide-gated cation channels mediate sodium absorption by IMCD (mIMCD-K2) cells.

The inner medullary collecting duct cell line, mIMCD-K2, absorbs Na+ by an amiloride-sensitive, electrogenic mechanism. The goal of the present study was to characterize the amiloride-sensitive, Na+ -conducting channels responsible for electrogenic Na+ absorption. To this end, we measured Na+ currents in single cells with the patch-clamp technique and Na+ currents across monolayers mounted in Ussing-type chambers. In whole cell patch-clamp experiments, amiloride-sensitive, inward Na+ currents were mediated by nonselective cation channels. In single-channel patch-clamp experiments, amiloride- and guanosine 3',5'-cyclic monophosphate (cGMP)-sensitive, 20-pS nonselective cation channels (i.e., CNG channels) were identified in the apical membrane. CNG channels were inhibited by amiloride, diltiazem, ethylisopropylamiloride (EIPA), and 8-bromo-cGMP and were permeable to Ca2+ and Mg2+. Epithelial Na+ channels were never observed in whole cell or single-channel recordings. Na+ absorption across confluent monolayers was inhibited with a rank order potency of benzamil > amiloride > phenamil >> EIPA > diltiazem. Our data are most consistent with the view that CNG channels mediate electrogenic Na+ absorption across mIMCD-K2 cells.

Amiloride↗
PubMed1996

Predicting completeness of symptom relief after major heart surgery.

The authors hypothesized that the same biopsychosocial factors that raise the risk of illness incidence would influence the speed and completeness of relief from physical symptoms during recovery following cardiac surgery. This multicenter prospective study involved 463 patients aged 35 to 69 years who underwent coronary artery bypass graft or cardiac valve surgery. Predictor data were gathered 1 to 3 days before surgery, and outcome measures were collected 6 months postoperatively. The following predictors were associated with postoperative freedom from cardiac symptoms: fewer preoperative cardiac hospitalizations; low levels of angina, dyspnea, fatigue, and sleep problems; low levels of anxiety, depression, hostility, and life-change events; and high levels of psychosocial well-being, hopefulness, overall satisfaction, and social support. The predictors of not requiring daytime bed rest because of cardiac symptoms during the 6th postoperative month were male gender, higher education, less cardiac disability preoperatively, low levels of angina, dyspnea, sleep problems, depression and fatigue, and absence of Type A behavior and of intraoperative hypotension. By using separate multiple logistic regression equations, the authors identified the variables printed in bold face as independent significant contributors to prediction. Many of the predictors are strong enough and simple enough to be used in clinical practice. Many of them may also predict recovery after acute illnesses and injuries involving other organ systems.

Activities of Daily Living↗
PubMed1996

Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney.

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

Amiloride↗
PubMed1996

Both the wild type and a functional isoform of CFTR are expressed in kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

Animals↗
PubMed1996

Hyperosmolality inhibits sodium absorption and chloride secretion in mIMCD-K2 cells.

Previously we demonstrated that a cell line derived from mouse inner medullary collecting duct (mIMCD-K2) absorbs Na+ and secretes Cl- by electrogenic mechanisms and that arginine vasopressin (AVP) stimulates Cl- secretion. The objective of the present study was to determine whether hyperosmolality, both acute (minutes) and chronic (weeks), affects electrogenic Na+ absorption IscNa and electrogenic Cl- secretion IscCl across the IMCD. To this end, we measured IscNa and IscCl across monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. Osmolality was increased from 290 to 590 mosmol/kgH2O by adding 200 mosmol/kgH2O of NaCl and 100 mosmol/kgH2O of urea or 300 mosmol/kgH2O of sucrose to the bathing solutions. Acute and chronic hyperosmolality reduced basal IscNa and IscCl and the AVP-stimulated rise in IscCl. These findings indicate that osmolality is an important determinant of IscNa and IscCl across IMCD cells and that the osmolality of the interstitial fluid should be considered when evaluating the effects of hormones and other factors on Na+ and Cl- transport by the IMCD.

Animals↗
PubMed1995

Cloning of a cGMP-gated cation channel from mouse kidney inner medullary collecting duct.

The cDNA sequence coding for the cGMP-gated cation channel expressed in the mouse kidney inner medullary collecting duct has been determined. The kidney cGMP-gated cation channel cDNA has an open reading frame of 2055 nucleotides and encodes a 685 amino acid protein. One cDNA clone is alternatively spliced thereby producing a deletion of 107 bp. Two differentially spliced 5' untranslated regions were determined by 5' RACE.

Amino Acid Sequence↗
PubMed1995

The renal cGMP-gated cation channel: its molecular structure and physiological role.

Cyclic nucleotide-gated cation channels, which are permeable to monovalent and divalent cations, are expressed in a number of tissues. cDNAs encoding cGMP-gated cation channel subunits have been cloned in retinal rods, cones, olfactory neuroepithelium, pineal gland, aorta, testis, heart, and most recently kidney. Patch clamp studies have identified and characterized cGMP-gated cation channels in the cortical collecting duct (CCD) and inner medullary collecting duct (IMCD). cGMP-gated cation channels in kidney share many biophysical and molecular properties with the retinal rod cGMP-gated channel. However, unlike the retinal rod channel, the cGMP-gated cation channel in kidney is inhibited by cGMP and stimulated by increased calcium levels. In the IMCD the cGMP-gated cation channel mediates electrogenic sodium absorption which is inhibited by ANP via cGMP. Recently, cGMP-gated cation channel poly(A+) RNA has been identified in other nephron segments by RT-PCR and in situ PCR hybridization. Furthermore, cGMP-gated cation channel protein has also been identified in all nephron segments by Western blot analysis. These observations suggest that cGMP-gated cation channels, or closely related gene products, may play an important physiological role in all nephron segments. Hormones that increase intracellular cGMP may regulate sodium, and perhaps calcium, uptake in nephron segments proximal to the IMCD. Increases in cell sodium and calcium may regulate other transport and signaling pathways.

Amiloride↗
PubMed1995

Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells.

A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.

Base Sequence↗
PubMed1995

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells.

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

Adenosine Triphosphate↗
PubMed1995

CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells.

Previously we demonstrated that the inner medullary collecting duct cell line mIMCD-K2 secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The goal of the present study was to characterize the Cl- channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- secretion. To this end, using the patch-clamp technique, we measured Cl- currents. In whole cell patch-clamp experiments, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activated Cl- currents that were time and voltage independent, inhibited by diphenylamine 2-carboxylate (DPC), and had a linear current-voltage (I-V) relation. In cell-attached patches of the apical membrane, we identified 7-pS Cl- channels that were stimulated by CPT-cAMP. In inside-out patches with Cl- in the pipette and bath solutions, Cl- currents had a linear I-V relation. The halide permeability sequence was PCl = PBr > PI. The Cl- channel inhibitors DPC, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide blocked the 7-pS Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was ineffective. By reverse transcriptase polymerase chain reaction, we isolated a partial cDNA clone encoding the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells. We conclude that cAMP stimulates electrogenic Cl- secretion in inner medullary collecting duct cells by activating cystic fibrosis transmembrane conductance regulator Cl- channels.

Animals↗
PubMed1995

Electrogenic sodium absorption and chloride secretion by an inner medullary collecting duct cell line (mIMCD-K2).

The initial segment of the inner medullary collecting duct (IMCDi) absorbs Na+ by an electrogenic mechanism and plays an important role in regulating the composition and volume of the urine. The purpose of the present study was to establish a permanent cell line derived from the IMCDi, which has the ion transport properties of the IMCDi in vivo. To this end, we isolated IMCD cells from the IMCDi of a mouse, Tg(SV40E) Bri 7, transgenic for the early region of SV40 (large T antigen) and established a permanent cell line, mIMCD-K2, by clonal dilution. mIMCD-K2 cells retain many differentiated characteristics of the IMCDi, including amiloride-sensitive electrogenic Na+ absorption stimulated by nanomolar concentrations of aldosterone. Aldosterone (1.5 x 10(-6) M) increased Na+ absorption from 0.2 +/- 0.1 to 4.6 +/- 1.7 microA/cm2. In addition, the cells secrete Cl- by an electrogenic mechanism at a rate of 0.5 +/- 0.1 microA/cm2. We propose that IMCDi cells either absorb or secrete NaCl depending on NaCl homeostasis. The mIMCD-K2 cell line should be useful for studying the cellular mechanisms responsible for electrogenic Na+ and Cl- transport in the IMCDi.

Absorption↗
PubMed1995

Vasopressin and cAMP stimulate electrogenic chloride secretion in an IMCD cell line.

Previously, we demonstrated that the mIMCD-K2 cell line, derived from the inner medullary collecting duct (IMCD) of a transgenic mouse, secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton, Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995]. The objective of the present study was to characterize the cellular mechanisms of electrogenic Cl- secretion (IscCl) and to determine whether arginine vasopressin (AVP) and adenosine 3',5'-cyclic monophosphate (cAMP) stimulate IscCl. To this end, we measured IscCl across monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. AVP increased IscCl with a Michaelis constant (Km) of 2.1 +/- 0.7 x 10(-12) M. 1-Desamino-8-D-AVP, a specific V2 receptor agonist, increased IscCl from 3.3 +/- 0.4 to 17.4 +/- 1.3 microA/cm2, 8-(4-Chlorophenylthio)-cAMP, a cell-permanent analogue of cAMP, a second messenger of AVP, increased IscCl from 1.4 +/- 0.3 to 15.2 +/- 1.2 microA/cm2. Furosemide and bumetanide, inhibitors of Na(+)-2Cl(-)-K+ cotransport, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- exchange, reduced IscCl when added to the basolateral solution. Our data suggest that AVP, via V2 receptors, and the second messenger cAMP stimulate IscCl and that Cl- secretion by mIMCD-K2 cells involves uptake of Cl- across the basolateral membrane by Na(+)-2Cl(-)-K+ cotransport and Cl-/HCO3- exchange and diffusion out of the cells across the apical membrane by cystic fibrosis transmembrane conductance regulator Cl- channels.

Absorption↗
PubMed1995

Adenosine inhibits arginine vasopressin-stimulated chloride secretion in a mouse IMCD cell line (mIMCD-K2).

Previously, we demonstrated that a mouse inner medullary collecting duct cell line (mIMCD-K2) secretes Cl- by an electrogenic mechanism via cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995; D. Vandorpe, N. Kizer, F. Ciampolillo-Bates, B. Moyer, K. Karlson, W. B. Guggino, and B. A. Stanton. Am. J. Physiol. 269 (Cell Physiol. 38): C683-C689, 1995]. The objective of the present study was to determine whether adenosine, and adenosine A1 receptors (A1AR) specifically, regulate electrogenic Cl- secretion (IscCl) in mIMCD-K2 cells. Neither N6-cyclohexyladenosine (CHA), a specific A1AR agonist, nor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1AR antagonist, altered basal, unstimulated IscCl in monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. In contrast, DPCPX increased arginine vasopressin (AVP)-stimulated IscCl, an effect that was reversed by CHA. Adenosine deaminase (ADA), which oxidatively deaminates adenosine to inosine, increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of ADA on AVP-stimulated IscCl. These results suggest that adenosine, via A1AR, inhibits AVP-stimulated IscCl. To identify the source(s) of extracellular adenosine, we examined the effects of dipyridamole, an inhibitor of nucleoside transport, and alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP), an inhibitor of ecto-5'-nucleotidase, on AVP-stimulated IscCl. Both compounds increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of dipyridamole and AOPCP on IscCl. Neither ADA nor CHA had an effect on 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated IscCl. Moreover, U-73122, an inhibitor of phospholipase C, failed to attenuate the increase in AVP-stimulated IscCl elicited by dipyridamole and AOPCP or the decrease in AVP-stimulated IscCl elicited by CHA. We conclude that adenosine, released by a nucleoside transporter and formed extracellularly by the breakdown of AMP, binds to A1AR, and decreases AVP-stimulated IscCl in mIMCD-K2 cells by reducing intracellular cAMP levels.

Adenosine↗
PubMed1994

Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line.

The regulatory volume decrease (RVD) of a renal cortical collecting duct cell line (RCCT-28A) exposed to a hypotonic solution was studied using electronic cell sizing to measure cell volume and the patch clamp technique to measure Cl- channel activity. Results demonstrate that RVD was mediated in part by KCl loss through separate K+ and Cl- channels. The Cl- channel had a conductance of 305 pS and was activated by cell swelling, membrane stretch, and disruption of F-actin by dihydrocytochalasins. In contrast, stabilizing F-actin with phalloidin prevented swelling and stretch activation of the Cl- channel and inhibited the RVD. Thus, the state of actin polymerization regulates the probability of the 305 pS Cl- channel being open. Short actin filaments activate whereas long actin filaments inactivate the channel. Taken together, our studies suggest that RVD in this renal collecting duct cell line cell is mediated in part by a 305 pS Cl- channel, which is activated, during cell swelling, by a signaling pathway that includes disruption of F-actin.

Actins↗