Homer Smith Award recipient--Peter Aronson.
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Awards and Prizes↗
Biomedical subjects
G Giebisch
Publications and source records attributed to G Giebisch.
Effects of a novel KATP channel blocker on renal tubule function and K channel activity.
ATP-sensitive potassium channels (KATP) modulate Na, K and Cl reabsorption in the thick ascending limb of the loop of Henle and K secretion in the cortical collecting tubule. Inhibition of potassium recycling in the apical membrane of the thick ascending limb of the loop of Henle interferes with sodium reabsorption and suppression of the apical potassium conductance in principal cells of the cortical collecting tubule and reduces potassium secretion. Both potassium recycling and potassium secretion depend critically on the activity of KATP, and recent studies indicate that the sulfonylurea compound glyburide, a known KATP channel blocker, is diuretic but does not produce kaliuresis.
Adamantane↗
Regulation of ROMK1 K+ channel activity involves phosphorylation processes.
An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.
Adenosine Triphosphate↗
Acidification in mammalian cortical distal tubule.
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Acid-Base Equilibrium↗
Potassium and sodium transport along the loop of Henle: effects of altered dietary potassium intake.
We assessed the effects of changes in potassium (K+) balance on the function of the loop of Henle by a combination of renal clearance and microperfusion experiments. Rat superficial cortical nephrons were perfused in vivo at 20 nl.min-1 from late proximal to early distal tubule with an artificial end-proximal solution containing either 3.8 or 1.8 mM potassium. Rats were fed a control diet, a low-potassium diet for at least three weeks, or a high-potassium diet for 10 to 14 days. When compared with the appropriate end-proximal potassium concentration in the perfusion fluid, potassium absorption along the loop of Henle (JK) increased in potassium-depletion whereas sodium (JNa) and fluid (Jv) absorption decreased. In rats fed a high-potassium diet, absorption of potassium, sodium and fluid was depressed. We propose that changes of external potassium balance affect the transport of electrolytes and fluid along the loop of Henle in vivo by modulating the transport of potassium and sodium primarily in the thick ascending limb. Changes in potassium reabsorption may also be affected by alterations of potassium-recycling.
Animals↗
A novel approach allows identification of K channels in the lateral membrane of rat CCD.
We have developed a novel approach to study K channels in the lateral membrane of principal cells (PC) in rat cortical collecting ducts (CCD). The technique consists of 1) exposing the CCD apical membrane, 2) removing the intercalated cells adjoining a PC by gentle suction through a pipette, and 3) applying patch-clamp technique to the lateral membrane of PC. Functional viability of the PC was confirmed by three indexes: 1) maintenance of physiological cell membrane potentials (-85 +/- 3 mV); 2) depolarization of the cell membrane potential with 1 mM Ba2+; and 3) hyperpolarization of the cell potential with 0.1 mM amiloride. Two types of K channels were identified: a low-conductance K channel and an intermediate-conductance K channel. In cell-attached patches the slope conductance of the low-conductance K channel was 27 pS and that of the intermediate-conductance K channel was 45 pS. The open probability (Po) of the 27-pS K channel was 0.81 +/- 0.02 and was not voltage dependent. In contrast, the Po of the 45-pS K channel was 0.23 +/- 0.01 at the spontaneous cell membrane potential and was increased by hyperpolarization. In addition, decrease of the bath pH from 7.4 to 6.7 reduced the 27-pS K channel current amplitude in a voltage-dependent manner, but the Po was not affected. Finally, two time constants were required to fit open- and closed-time histograms of both populations of K channels. Application of 1 mM Ba2+ completely blocked these K channels. We conclude that two types of K channel are present in the basolateral membrane of PC.
Amiloride↗
Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct.
We have employed the patch-clamp technique to investigate the role of the actin cytoskeleton in the modulation of the low-conductance K+ channel in the apical membrane of the rat cortical collecting duct (CCD). This K+ channel is inactivated by application of cytochalasin B or D, both compounds known to disrupt actin filaments. The effect of both cytochalasins, B and D, was fully reversible in cell-attached patches, but channel activity could not be fully restored in excised membrane patches. The effect of cytochalasins on channel activity was specific and resulted from depolymerization of the actin cytoskeleton, since application of 10 microM chaetoglobosin C, a cytochalasin analogue that does not depolymerize the actin filaments, had no effect on channel activity in inside-out patches. Addition of either actin monomers or of the polymerizing actin filaments in inside-out patches to the cytosolic medium had no effect on channel activity. This suggests that cytochalasin B- or D-induced inactivation of apical K+ channels is not caused by obstruction of the channel pore by actin. We also observed that channel inhibition by cytochalasin B or D could be blocked by pretreatment with 5 microM phalloidin, a compound that stabilizes actin filaments. We conclude that apical K+ channel activity depends critically on the integrity of the actin cytoskeleton.
Actins↗
Living renal epithelial cells imaged by atomic force microscopy.
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Animals↗
Bicarbonate transport along the loop of Henle. II. Effects of acid-base, dietary, and neurohumoral determinants.
The loop of Henle contributes to renal acidification by reabsorbing about 15% of filtered bicarbonate. To study the effects on loop of Henle bicarbonate transport (JHCO3) of acid-base disturbances and of several factors known to modulate sodium transport, these in vivo microperfusion studies were carried out in rats during: (a) acute and chronic metabolic acidosis, (b) acute and chronic (hypokalemic) metabolic alkalosis, (c) a control sodium diet, (d) a high-sodium diet, (e) angiotensin II (AII) intravenous infusion, (f) simultaneously intravenous infusion of both AII and the AT1 receptor antagonist DuP 753, (g) acute ipsilateral mechanicochemical renal denervation. Acute and chronic metabolic acidosis increased JHCO3; acute metabolic alkalosis significantly reduced JHCO3, whereas chronic hypokalemic alkalosis did not alter JHCO3. Bicarbonate transport increased in animals on a high-sodium intake and following AII administration, and the latter was inhibited by the AII (AT1) receptor antagonist DuP 753; acute renal denervation lowered bicarbonate transport. These data indicate that bicarbonate reabsorption along the loop of Henle in vivo is closely linked to systemic acid-base status and to several factors known to modulate sodium transport.
Acid-Base Imbalance↗
Aldosterone-mediated Na/K-ATPase expression is alpha 1 isoform specific in the renal cortical collecting duct.
In the renal cortical collecting duct (CCD), mineralocorticoid hormones, like aldosterone, augment the abundance of Na/K-ATPase molecules. It has been postulated that this response involves an isoform switch of the Na/K-ATPase catalytic subunit, alpha, as the molecular basis for the differential regulation of mineralo-corticoid-induced and constitutively expressed Na/K-ATPase pools. In opposition to this attractive hypothesis, three lines of independent evidence are presented which demonstrate that the CCD exclusively expresses the alpha 1 form despite mineralocorticoid-mediated changes in functional Na/K pump density. First, aldosterone increased [3H]ouabain binding in CCD 2.5-fold without changing the ouabain dissociation constant. Second, an electrophysiological assay for pump activity revealed that aldosterone increased maximum Na/K pump current in parallel with the change in ouabain binding without altering the apparent sodium affinity. Third, Western blot analysis with alpha isoform-specific, antipeptide antibodies demonstrated that aldosterone exclusively increased the total chemical pool of the alpha 1 form of the pump without inducing other alpha subunit isoforms. In summary, aldosterone increases the abundance of Na/K-ATPase molecules in the CCD which are pharmacologically, physiologically, and chemically indistinguishable from those that are normally expressed.
Aldosterone↗
Mechanism of activation of K+ channels by minoxidil-sulfate in Madin-Darby canine kidney cells.
We studied the mechanism of K+ channel activation by minoxidil-sulfate (MxSO4) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca(2+)-dependent inward-rectifying K+ channel with slope conductances of 53 +/- 3 (negative potential range) and 20 +/- 3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO4 initiated both transient channel activation and an increase of intracellular Ca2+ (from 94.2 +/- 9.1 to 475 +/- 12.6 nmol/liter). The observation that K+ channel activity of excised inside-out patches was detected only at Ca2+ concentrations in excess of 10 mumol/liter suggests the involvement of additional mechanisms during channel activation by MxSO4. Transient K+ channel activity was also induced in cell-attached patches by 10 mumol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mumol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca2+ sensitivity of the K+ channel in inside-out patches significantly by lowering the Km for Ca2+ from 100 mumol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO4 on K+ channel activation. We conclude that MxSO4-induced K+ channel activity is mediated by the synergistic effects of an increase in intracellular Ca2+ and a PKC-mediated enhancement of the K+ channel's sensitivity to Ca2+.
Alkaloids↗
Imaging the lamellipodium of migrating epithelial cells in vivo by atomic force microscopy.
Cell locomotion originates at a specific region of the cell surface, the leading edge of a migrating cell. Various factors have been proposed to contribute to the propulsion of a cell over the substratum. Rapid turnover processes of cytoskeletal elements inside the cell and insertion of new plasma membrane at the leading edge of the cell permit the extension of a cell in a given direction. Our goal was to image in vivo plasma membrane turnover by means of atomic force microscopy (AFM) and to resolve dynamic processes at the nanometer level. As an experimental model we used migrating kidney cells derived from the Madin-Darby canine kidney (MDCK) cell line that was transformed by alkaline stress. These so-called MDCK-F cells exhibit spontaneous calcium-dependent oscillatory activity of plasma membrane potential associated with cell locomotion. We imaged cells during migration and observed dynamic invagination processes in the cell surface close to the leading edge, indicating internalization of plasma membrane. Invaginations were prevented by removal of calcium from the perfusate. During calcium reduction plasma membrane uncoupled from the underlying cytoskeleton and lipidic pores with diameters of about 30 nm could be disclosed and imaged. This study demonstrates that the AFM can readily trace dynamic physiological processes in vivo, emphasizing the potential role of calcium in maintaining plasma membrane integrity and function.
Animals↗
Renal and extrarenal sites of action of diuretics.
This paper provides (a) a survey of the basic tubule transport mechanisms of sodium and potassium ions along the nephron, (b) a comparison of the overall renal effects of two diuretic agents (torasemide and furosemide), as assessed by renal clearance techniques, (c) an analysis of the tubule sites of action of torasemide evaluated by free-flow micropuncture and microperfusion techniques, and (d) a brief evaluation of some extrarenal transport effects of loop diuretics.
Animals↗
Diuretic action of potassium channel blockers.
The availability of specific potassium channel blockers has made it possible to examine their effects along the nephron on fluid and electrolyte excretion. Recent experiments indicate that they modulate transepithelial sodium and potassium transport. In the thick ascending limb, potassium channel blockers, by interfering with potassium recycling across the apical cell membrane, reduce the activity of the sodium-2chloride-potassium cotransporter. In the initial and cortical collecting duct their inhibitory action on ATP-sensitive potassium channels reduces potassium secretion. Accordingly, tissue-specific potassium channel blockers are promising diuretic agents that may induce diuresis by inhibiting tubular sodium transport with only minimal loss of potassium.
Animals↗
Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase.
The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)-dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)-dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.
Alkaloids↗
Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells.
M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl-Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M-1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.
Amiloride↗
Recent studies on the characterization of loop diuretics.
This article provides a survey of transport mechanisms in the loop of Henle and of secondary effects on electrolyte excretion of loop diuretics. Results are presented of experiments that define ion transport in the thick ascending limb. New data on ion channels and transport regulation in this tubule segment are summarized. In addition, the mechanisms underlying enhanced sodium chloride and water transport after diuretics in tubule segments that are located downstream of the loop of Henle are surveyed. Finally, the effects of K depletion and chronic metabolic acidosis on diuretic potency of torasemide as well as possible antialdosterone effects of torasemide are evaluated.
Animals↗
Endothelin increases [Ca2+]i in M-1 mouse cortical collecting duct cells by a dual mechanism.
We tested the effects of endothelin-1 (ET-1) on intracellular calcium concentration ([Ca2+]i) of cultured M-1 mouse cortical collecting duct cells. [Ca2+]i was measured using fura 2 and a fluorescent imaging system. At a concentration of extracellular calcium ([Ca2+]o) of 1 mM, ET-1 (10(-12) to 10(-7) M) increased [Ca2+]i. A second application of ET-1 had no effect on Ca2+. In contrast, application of arginine vasopressin after an initial exposure to ET-1 induced a second Ca2+ response. In the absence of extracellular Ca2+ (1 mM EGTA) ET-1 also elicited a Ca2+ peak, indicating participation of Ca2+ release from intracellular stores in the initial Ca2+ peak. At [Ca2+]o of 10 mM, ET-1 also induced an intracellular Ca2+ peak but [Ca2+]i remained significantly elevated. The Ca2+ plateau phase was abolished by nickel (10 or 100 microM) and nifedipine (0.1 or 1 microM). We conclude that ET-1 mediates an increase in [Ca2+]i by Ca2+ release from intracellular stores and activation of a nickel- and nifedipine-sensitive Ca2+ entry mechanism.
Animals↗