Biomedical subjects

D B Light

Publications and source records attributed to D B Light.

10 recordsLinked to original sources

PubMed1998

Protein kinase C and regulatory volume decrease in mudpuppy red blood cells.

This study examined whether protein kinase C (PKC) stimulates K+ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 micrometer), but not with the cyclic nucleotide-dependent kinase antagonists N-(2'-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 micrometer) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 micrometer). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 micrometer). The effect of these three antagonists and H-7 was reversed with gramicidin (5 micrometer in a choline Ringer), indicating PKC was linked to K+ efflux (gramicidin is a cationophore that was used to ensure a high K+ permeability). We also measured cell volume recovery from hypotonic shock (0.5x Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 micrometer), sphingosine (100 nm), chelerythrine (10 micrometer), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 micrometer) nor H-8 (5 micrometer). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 micrometer). Furthermore, using the patch clamp technique, we found H-7 (10 micrometer) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K+ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K+ loss during RVD is regulated, at least in part, by protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

5-Lipoxygenase metabolites of arachidonic acid regulate volume decrease by mudpuppy red blood cells.

We examined whether metabolites of arachidonic acid (AA) regulate K+ efflux during regulatory volume decrease (RVD) by mudpuppy red blood cells (RBCs). Volume regulation was inhibited by the phospholipase A2 antagonists mepacrine (10 microM) and ONO-RS-082 (10 microM); the inhibitory effect of ONO-RS-082 was reversed by gramicidin (5 microM). Eicosatetraynoic acid (ETYA, 100 microM), a general antagonist of AA metabolism, also blocked RVD. In addition, volume regulation was inhibited by the lipoxygenase pathway antagonist nordihydroguaiaretic acid (NDGA, 10 microM), the 5 lipoxygenase antagonists AA-861 (5 microM) and curcumin (20 microM), and by the 5-lipoxygenase activating protein inhibitor L-655,298 (5 microM). Inhibition by all four of these agents was reversed with gramicidin. In contrast, the 12- and 15-lipoxygenase pathway inhibitor ethyl-3,4-dihydroxy-benzylidene-cyanoacetate (EDBCA, 1 microM) and the cytochrome P-450 monooxygenase pathway blocker ketoconazole (20 microM) had no effect. On the other hand, the cyclooxygenase pathway inhibitor aspirin (100 microM) slightly enhanced RVD. Consistent with these findings, a K(+)-selective whole cell conductance responsible for K+ efflux during cell swelling was inhibited by ONO-RS-082 (10 microM), NDGA (10 microM), AA-861 (5 microM), curcumin (20 microM), and L-655,298 (5 microM). In contrast, EDBCA (1 microM), ketoconazole (20 microM), and indomethacin (10 microM) did not block this whole cell conductance. These results indicate that a channel mediating K+ loss during RVD is regulated by a 5-lipoxygenase metabolite of arachidonic acid.

Animals↗

PubMed1996

Potassium conductance activated during regulatory volume decrease by mudpuppy red blood cells.

The cellular basis of regulatory volume decrease (RVD) by mudpuppy (Necturus maculosus) red blood cells (RBCs) was examined. Volume regulation was inhibited by replacing extracellular Na+ with K+. In contrast, addition of gramicidin (5 microM) to the extracellular medium enhanced RVD. The K(+)-channel blocker quinine (1 mM) also inhibited RVD, and this inhibition was reversed by gramicidin (5 microM). In addition, a 0 Ca(2+)-EGTA Ringer blocked RVD, whereas the Ca2+ ionophore A23187 ( microM) enhanced recovery of cell volume. The stretch-activated ion channel antagonist gadolinium (10 microM) inhibited RVD, and this effect was reversed by A23187 (2 microM). Furthermore, the calmodulin inhibitors pimozide (10 microM) and N-(6-aminohexyl)-5-chloro-1-napthalene-sulfonamide (0.1 mM) blocked RVD, and this inhibition was reversed with gramicidin (5 microM). Consistent with these findings, a K(+)-selective membrane conductance was activated by exposing RBCs to a 0.5x Ringer solution (observed with the whole cell patch clamp technique). This conductance was inhibited by quinine (1 mM), gadolinium (10 microM), and pimozide (10 microM). These results indicate that cell swelling activates a K+ conductance by a Ca(2+)-calmodulin-dependent mechanism and that this channel mediates K+ loss during RVD.

Animals↗

PubMed1990

A GTP-binding protein activates chloride channels in a renal epithelium.

Although G proteins have been shown to regulate cation channels, regulation of Cl- channels by G proteins has not been demonstrated directly. Accordingly, the objective of this study was to examine whether a G protein regulates Cl- channels in the apical membrane of rabbit kidney CCD cells grown in culture. Previous studies showed that this channel is activated by adenosine and protein kinase C and has a single channel conductance of 305 picosiemens. The PCl-:PNa+ is 9:1 and the PCl-:PHCO3- is 2:1 (Schwiebert, E.M., Light, D.B., Dietl, P., Fejes-Toth, G., Naray-Fejes-Toth, A., and Stanton, B. (1990) Kidney Int. 37,216). In the present study, Cl- channels in the apical membrane of CCD cells were studied by the patch clamp technique. GTP and guanosine 5'-O(3-thiophosphate) (GTP gamma S), a nonhydrolyzable analog of GTP, increased the single channel open probability (Po). In contrast, guanosine 5'-O-(2-thiophosphate), a nonhydrolyzable analog of GDP, and pertussis toxin (PTX) decreased the Po. GTP gamma S, but not GTP, reversed PTX inhibition of the channel. The alpha i-3-subunit of Gi increased the Po in both untreated and PTX-treated membrane patches. Because GTP gamma S activated the Cl- channel in the presence of H8, a protein kinase inhibitor, we conclude that the G protein does not activate the channel by stimulating a protein kinase. Thus, a PTX-sensitive G protein activates a Cl- channel in the apical membrane of renal CCD cells.

Adenosine↗

PubMed1990

Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.

Atrial natriuretic peptide, acting through its second messenger guanosine 3',5'-cyclic monophosphate (cGMP), suppresses Na+ absorption across the renal inner-medullary collecting duct and increases urinary Na+ excretion. Patch clamp studies show that cGMP reduces Na+ absorption by inhibiting an amiloride-sensitive cation channel in the apical membrane. We have now examined, using the patch clamp technique, the molecular mechanisms of cGMP inhibition. Cyclic GMP directly and specifically reduced the probability of a single channel being open (open probability, Po) by 39% (inhibition constant, Ki = 7.6 x 10(-7) M) by a phosphorylation-independent mechanism. Cyclic GMP also inhibited the channel by activating cGMP-dependent protein kinase (cGMP-kinase). Exogenous cGMP-kinase completely inhibited the channel by a phosphorylation-dependent mechanism. Activation of a pertussis toxin-sensitive G protein by GTP-gamma-S blocked cGMP-kinase inhibition of the channel. By contrast, cGMP-kinase inhibition of Po was completely reversed by GTP-gamma-S. Taken together with the results of a previous study showing that a G protein activates the cation channel, these data indicate that cGMP-kinase and a G protein sequentially regulate the cation channel. Our results show that atrial natriuretic peptide, acting through cGMP, inhibits Na+ absorption across the inner-medullary collecting duct by a dual mechanism, and that cGMP-kinase inhibits the channel by a pathway involving a G protein.

Amiloride↗

PubMed1990

Chloride channels in the apical membrane of cortical collecting duct cells.

Ion channels in the apical membrane of cortical collecting duct (CCD) cells in culture were studied by the patch-clamp technique. CCD cells from rabbit kidney were isolated by solid-phase immunoadsorption with a monoclonal antibody. The majority of CCD cells (93%) had phenotypic characteristics similar to intercalated cells (ICC). Although Cl- channels were present in the apical membrane of the ICC cells, they were rarely active in cell-attached patches; however, channels were activated after patch excision. In inside-out patches, the channels exhibited rapid flickering, substrates, and large unitary currents. The single-channel conductance was 303 pS, the Cl(-)-to-Na+ permeability ratio was 10:1 and the Cl(-)-to-HCO3- permeability ratio was 1.5:1. The channel was inhibited by the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Although a reduction in the cytoplasmic Ca2+ concentration inhibited channel activity in both inside-out patches and cell-attached patches, alterations of Ca2+ within the physiological range did not change the channel open probability. Finally, changing the cytoplasmic pH (6.5 to 8.0) did not alter the open probability. Thus a large conductance anion channel is present in the apical membrane of CCD cells in culture. This channel may be involved in cell volume regulation or in Cl- and HCO3- secretion.

Animals↗

PubMed1989

Atrial natriuretic peptide inhibits a cation channel in renal inner medullary collecting duct cells.

The patch-clamp technique was used to examine the effects of atrial natriuretic peptide (ANP) and its second messenger guanosine 3',5'-monophosphate (cGMP) on an amiloride-sensitive cation channel in the apical membrane of renal inner medullary collecting duct cells. Both ANP (10(-11) M) and dibutyryl guanosine 3',5'-monophosphate (10(-4) M) inhibited the channel in cell-attached patches, and cGMP (10(-5) M) inhibited the channel in inside-out patches. The inner medullary collecting duct is the first tissue in which ANP, via its second messenger cGMP, has been shown to regulate single ion channels. The results suggest that the natriuretic action of ANP is related in part to cGMP-mediated inhibition of electrogenic Na+ absorption by the inner medullary collecting duct.

Aminoquinolines↗

PubMed1989

Guanine nucleotide-binding protein, alpha i-3, directly activates a cation channel in rat renal inner medullary collecting duct cells.

We examined whether GTP binding proteins (G proteins) regulate sodium conducting channels in the apical membrane of renal inner medullary collecting duct (IMCD) cells and thereby modulate sodium absorption. Patch clamp studies were conducted on inside-out patches of the apical membrane of IMCD cells grown in primary culture. Guanosine 5'-triphosphate (GTP) and the nonhydrolyzable GTP analogue, GTP gamma S, which activate G proteins, increased the open probability of the cation channel. In contrast, the nonhydrolyzable GDP analogue, GDP beta S, which decreases G protein activity, inhibited the channel. Pertussis toxin also reduced the open probability of the channel. Addition of the alpha *i-3 subunit of Gi to the solution bathing the cytoplasmic surface of the membrane increased the open probability in a dose-dependent manner (2-200 pM). The threshold concentration for activation by alpha *i-3 was 2 pM. Activation of the cation channel by alpha *i-3 was not mediated via a protein kinase. The IMCD is the first polarized epithelium in which an ion channel has been shown to be directly regulated by a G protein. Thus, G proteins are important elements in regulating sodium absorption by the IMCD.

Animals↗

PubMed1988

Amiloride-sensitive cation channel in apical membrane of inner medullary collecting duct.

Ion channels in the apical membrane of rat inner medullary collecting duct (IMCD) cells in primary culture were studied with the patch-clamp technique. A 27.5 +/- 1.2 pS non-selective cation channel was characterized in inside-out patches. The channel did not discriminate between Na+ and K+ (n = 8) and had a Na permeability-to-Cl permeability (PNa:PCl) ratio of 13:1 (n = 3). Amiloride (5 x 10(-7) M, n = 5) on the extracellular side of the membrane inhibited channel activity 10-fold at negative membrane voltages (voltage refers to cell interior with respect to patch pipette). This diuretic decreased the mean open time and increased the mean closed time without altering single-channel conductance. Voltage-dependent inhibition of this channel by amiloride distinguishes its behavior from other known nonselective cation channels. Neither voltage, Ba2+ (1 mM), tetraethylammonium (5 mM) nor changes in the bath pH (6.1 to 8.0) altered channel activity. Although the channels were active in 41% of the patches in the inside-out configuration, with a percent open time lying between 35 and 60 (-70 to +60 mV), channels were only active in 9% of the cell-attached patches. In preliminary microelectrode studies we have observed an amiloride-inhibited conductance in the apical membrane of isolated and perfused rat IMCD. Therefore, this novel nonselective cation channel identified in IMCD cells in culture may represent the amiloride-sensitive conductance observed in isolated and perfused IMCD and may mediate electrogenic Na+ absorption.

Amiloride↗

PubMed1987

Potassium-selective ion channels in a transformed insulin-secreting cell line.

K+ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K+-selective channels ranging from 15 to 211 pS in symmetrical 140 mM KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140 mM KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H+ ions (pH = 6.8); a 211 pS channel that was Ca2+-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917 +/- 128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4 +/- 8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140 mM KCl solutions. Thus, there appears to be a correlation between the presence of functional K+ channels and insulin secretion.

Adenoma, Islet Cell↗