Hubert Wiener

Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca2+-activated K+ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca2+-activated K+ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca2+ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K0.5 for Ca2+ was raised from 20nm at a potential of 0 mV to 300nm at −40 mV. A decrease in pH at the cytoplasmic face of the K+ channel reduced the Ca2+ sensitivity dramatically. A loss of the high sensitivity to Ca2+ was also observed after incubation with MgCl2, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca2+ sensitivity between studies on K+ channels from the same tissue. High affinity inhibition (K0.5=10nm) by charybdotoxin of the Ca2+-activated K+ channel from the extracellular face could be lifted by an outward flux of K+ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K+ channels. The high sensitivity for Ca2+ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K+ channel in the epithelial cells.

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells.

SummaryA method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na+, K+)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 2∶1∶1. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-14C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 μm/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na+, K+) pump operating in inside-out vesicles.

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

SummaryLoop diuretic-sensitive (Na+,K+,Cl−)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na+,K+) gradien-driven36Cl fluxes weree half-maximally inhibited by 0.14 μm bumetanide and 44 μm furosimide. While86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl− concentration, Hill coefficient 1.8, indicating a 1 Na+: 1 K+:2 Cl− stoichiometry of ion transport.The interaction of putative (Na+, K+, Cl−)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simulataneous presence of Na+,K+, and Cl−, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+, K+, Cl−)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (KD1=0.13 μm;Bmax1 =6.4 pmol/mg of protein) and low-affinity (KD2=34 μm;Bmax2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl− (K1=60mm), whereas low-affinity sites seem to be unaffected by Cl−. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.

Regulation of Ca2+-activated K+ channel from rabbit distal colon epithelium by phosphorylation and dephosphorylation.

Abstract. The Ca2+-activated maxi K+ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na+ reabsorption from the intestinal lumen. Previous measurements of these basolateral K+ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca2+ with a K0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca2+. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K+ channels lost their high sensitivity to Ca2+, yet they could still be activated by high concentrations of Ca2+ (10 μm). Furthermore, the high sensitivity to Ca2+ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca2+ of the maxi K+ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca2+ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca2+ sensitivities for maxi K+ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems.

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

SummaryIn the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven86Rb+ fluxes through K+ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba2+-sensitive, Ca2+-activated K+ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10−7m free Ca2+. This suggests an important role of cytoplasmic Ca2+ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K+ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca2+-activated K+ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H+ and tetraethylammonium show that both high-and low-sensitive86Rb+ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba2+-sensitive, Ca2+-activated K+ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.

Induction of drug metabolism in the rat by taglutimide, a sedative-hypnotic glutarimide derivative

SummaryPretreatment with taglutimide significantly decreased the plasma dicoumarol level and shortened the duration of hexobarbital-induced narcosis in rats. Furthermore, taglutimide pretreatment accelerated thein vitro metabolism of dicoumarol, hexobarbital, o-nitrophenyl acetate and procaine, but not of 3,4-benzpyrene, as assayed in the 10,000xg supernatant fraction of rat liver homogenate. No definite increase was observed in liver wet weight, nor in the amount of microsomal and total liver protein in comparison with the control values. No marked differences were found between the effects of short-(4-day) and long-term (17-day) pretreatment on any of the studied parameters. The changes in drug metabolism and liver protein observed after taglutimide pretreatment differed from those observed after pretreatment with either phenobarbital or 3,4-benzpyrene. Taglutimide, like other inducing agents, is lipophilic, but differs from them in not being a substrate of monooxygenases.

Vanadium-induced Cl−-secretion in rabbit descending colon is mediated by prostaglandins

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl- secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E2 from the tissue. Inhibitors of phospholipase A2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E2 and Cl- secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl- secretion. Although vanadate is known to inhibit Na+,K(+)-ATPase activity, no inhibition of active Na+ transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na+,K(+)-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl- secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E2.

Active Ca2+ transport systems in basolateral membranes from rabbit distal colon.

Abstract. Active Na+ absorption by tight epithelia such as frog skin and distal colon share common features like feedback inhibition of cellular [Na+] on Na+ influx through amiloride‐sensitive Na+ channels. It is postulated that the negative feedback of increasing cell [Na+] is mediated via a rise in cell [Ca2+]. In this model, cell [Na+] is coupled to cell [Ca2+] via a Na+/Ca2+ exchange system in the basolateral membrane. In the present study, the Ca2+ transporting systems in rabbit distal colon basolateral membranes were characterized. ATP‐dependent Ca2+ uptake could be demonstrated in membrane vesicles from surface cells with the following kinetic parameters: Km = 0.09 μM Ca2+ and Vm = 3.8 nmol Ca2+ mg−1 protein min−1. The ATP‐dependent Ca2+ transport was not responsive to ruthenium red and oxalate, suggesting a plasmalemmal origin. The addition of 75 mM Na+ to the uptake medium, 10 min after addition of ATP, did not release Ca2+ from the vesicles in significant amounts. In the absence of ATP, outwardly directed Na+ gradients were incapable of stimulating Ca2+ uptake. This study demonstrates that rabbit distal colon epithelium lacks a well‐defined Na+/Ca2+ exchange system, and (Ca2+, Mg2+)‐ATPase appears to be the sole Ca2+ extrusion system. Alternatives for the coupling of cell [Na+] to cell [Ca2+] are discussed.

Wasser-, Säure-Basen- und Elektrolytgleichgewicht

Verlust, Uberschus oder Umverteilung von Wasser, Protonen und Elektrolyten konnen zu Storungen des Wasser-, Saure-Basen- und Elektrolytgleichgewichts fuhren. Ziel einer Infusionstherapie ist die Wiederherstellung des Gleichgewichts durch Infusion verschiedener, auf Erhaltungs- und Korrekturbedarf abgestimmter Infusionslosungen.

Renin-Angiotensin-Aldosteron

Das Renin-Angiotensin-Aldosteron-System spielt eine wichtige Rolle bei der Regulation des Blutdrucks und der Aufrechterhaltung des Wasser- und Elektrolytgleichgewichts.