Cobicistat Boosts the Intestinal Absorption of Transport Substrates, Including HIV Protease Inhibitors and GS-7340,In Vitro

ABSTRACTThe experimental pharmacoenhancer cobicistat (COBI), a potent mechanism-based inhibitor of cytochrome P450 3A enzymes, was found to inhibit the intestinal efflux transporters P-glycoprotein and breast cancer resistance protein. Consistent with its transporter inhibition, COBI significantly increased the absorptive flux of potential candidates for clinical coadministration, including the HIV protease inhibitors atazanavir and darunavir and the lymphoid cell- and tissue-targeted prodrug of the nucleotide analog tenofovir, GS-7340, through monolayers of Caco-2 cellsin vitro.

Active sulfate secretion by the intestine of winter flounder is through exchange for luminal chloride

SO[Formula: see text]transport by winter flounder intestine in Ussing chambers was characterized. With 50 mM SO[Formula: see text] (physiological level) bathing the lumen, net absorption (lumen to blood) dominated. Under short-circuited conditions, 1 mM SO[Formula: see text] on both sides, net active SO[Formula: see text] secretion occurred (8.55 ± 0.96 nmol · cm−2 · h−1). NaCN (10 mM), ouabain (10−4 M), and luminal DIDS (0.2 mM) inhibited net secretion. Removal of luminal Cl− and HCO[Formula: see text] together (Cl−-HCO[Formula: see text]) or Cl− alone blocked net secretion, whereas removal of luminal HCO[Formula: see text] alone increased net secretion. SO[Formula: see text] uptake into foregut brush-border membrane vesicles was stimulated by a trans-Cl− gradient (in > out) and unaffected by a trans-HCO[Formula: see text] gradient (in > out). Short-circuiting with K+ (in = out) and valinomycin had no effect on Cl−-stimulated SO[Formula: see text] uptake, suggesting electroneutral exchange. Satiety (i.e., full stomach) stimulated the unidirectional absorptive flux, eliminating net secretion. It was concluded that the intestine is a site of SO[Formula: see text] absorption in marine teleosts and that active SO[Formula: see text] secretion is in exchange for luminal Cl−.

Intestinal NaCl transport in NHE2 and NHE3 knockout mice

Sodium/proton exchangers [Na+/H+ (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na+ and Cl− flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(−)], and NHE3 knockout [NHE3(−)] mice. Under basal conditions, NHE(+) and NHE2(−) jejuna had similar rates of net Na+ (∼6 μeq/cm2 · h) and Cl− (∼3 μeq/cm2 · h) absorption. In contrast, NHE3(−) jejuna had reduced net Na+ absorption (∼2 μeq/cm2 · h) but absorbed Cl− at rates similar to NHE(+) and NHE2(−) jejuna. Treatment with 100 μM 5-( N-ethyl- N-isopropyl) amiloride (EIPA) completely inhibited net Na+ and Cl−absorption in all genotypes. Studies of the Na+ absorptive flux ( J [Formula: see text]) indicated that J [Formula: see text] in NHE(+) jejunum was not sensitive to 1 μM EIPA, whereas J [Formula: see text] in NHE3(−) jejunum was equally sensitive to 1 and 100 μM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMPi) abolished net NaCl absorption and stimulated electrogenic Cl− secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(−) and NHE3(−) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na+ absorption; 2) an amiloride-sensitive Na+ transporter partially compensates for Na+ absorption in NHE3(−) jejunum; 3) cAMPi stimulation abolishes net Na+ absorption in NHE(+), NHE2(−), and NHE3(−) jejunum; and 4) electroneutral Cl− absorption is not directly dependent on either NHE2 or NHE3.

Increased CO2 stimulates K/Rb reabsorption mediated by H-K-ATPase in CCD of potassium-restricted rabbit

Apical H-K-ATPase in the cortical collecting duct (CCD) plays an important role in urinary acidification and K reabsorption. Our previous studies demonstrated that an H-K-ATPase mediates, in part, Rb reabsorption in rabbit CCD (Zhou X and Wingo CS. Am J Physiol Renal Fluid Electrolyte Physiol 263: F1134–F1141, 1992). The purpose of these experiments was to examine using in vitro microperfused CCD from K-restricted rabbits 1) whether an acute increase in Pco 2 and, presumably, intracellular acidosis stimulate K absorptive flux; and 2) whether this stimulation was dependent on the presence of a functional H-K-ATPase. Rb reabsorption was significantly increased after exposure to 10% CO2 in CCD, and this effect was persistent for the entire 10% CO2 period, whereas 10 μM SCH-28080 in the perfusate totally abolished the stimulation of Rb reabsorption by 10% CO2. After stimulation of Rb reabsorption by 10% CO2, subsequent addition of 0.1 mM methazolamide, an inhibitor of carbonic anhydrase, failed to affect Rb reabsorption. However, simultaneous exposure to 10% CO2 and methazolamide prevented the stimulation of Rb reabsorption. Treatment with the intracellular calcium chelator MAPTAM (0.5 μM) inhibited the stimulation of Rb reabsorption by 10% CO2. Similar inhibition was also observed in the presence of either a calmodulin inhibitor, W-7 (0.5 μM), or colchicine (0.5 mM), an inhibitor of tubulin polymerization. In time control studies, the perfusion time did not significantly affect Rb reabsorption. We conclude the following: 1) stimulation of Rb reabsorption on exposure to 10% CO2 is dependent on the presence of a functional H-K-ATPase and appears to be regulated in part by the insertion of this enzyme into the apical plasma membrane by exocytosis; 2) insertion of H-K-ATPase requires changes in intracellular pH and needs a basal level of intracellular calcium concentration; and 3) H-K-ATPase insertion occurs by a microtubule-dependent process.

The Aplysia californica Cl- pump is a P-type ATPase: evidence through inhibition studies

Utilizing a proteoliposomal preparation containing Cl–-ATPase from Aplysia californica foregut, it was shown that orthovanodate inhibited Cl–-ATPase activity, ATP-dependent Cl– transport, ATP-dependent membrane potential change and ATP-dependent phosphorylation. N-ethylmalemide and p-chloromercurobenzoate also inhibited the Cl– pump biochemical and physiological transport characteristics. However, bafilomycin, azide, N, N'-dicyclohexylcarboiimide (DCCD), and efrapeptin had no effect on the Cl– pump biochemical or physiological characteristics, suggesting that this Cl– pump was a P-type ATPase. It was concluded that this P-type ATPase Cl– pump is the mechanism that is responsible for the net absorptive flux of Cl– in the A. californica foregut.Key words: Cl– pump, P-type ATPase, orthovanadate.

Diurnal rhythmicity in intestinal SGLT-1 function, V max, and mRNA expression topography

Mechanisms underlying the circadian rhythmicity in intestinal sugar absorption remain unclear. To test whether this rhythmicity is caused by changes in Na+-glucose cotransporter 1 (SGLT-1) function, we measured phloridzin-inhibitable sugar fluxes as an index of SGLT-1 activity. Jejunum obtained from rats killed at 6-h intervals during a 12-h light-dark cycle (CT0 is circadian time 0 h, time of light onset) were mounted in Ussing chambers, and 3- O-methylglucose (3-OMG) fluxes were calculated before and after addition of phloridzin. 3-OMG-induced change in short-circuit current and absorptive flux were significantly greater at CT9 than at CT3. This increase was phloridzin inhibitable. Kinetic studies indicated a significant increase in SGLT-1 maximal velocity ( V max) at CT9. Food intake between CT3 and CT9 was <10% of the daily total, indicating that the increased SGLT-1 activity was anticipatory. Diurnicity of SGLT-1 mRNA was confirmed by Northern blotting. Expression topography analyzed by in situ hybridization revealed more intense labeling along the entire villus axis at CT9 and CT15 compared with CT3 and CT21. We conclude that diurnicity in intestinal sugar absorption is caused by periodicity in SGLT-1 V max.

HCO 3 − absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase

Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H+ secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H+ secretion, we measured net [Formula: see text] transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg. Chem. 24: 262–272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net[Formula: see text] flux were performed for each drug. Basal [Formula: see text] absorptive flux was 12 pmol ⋅ min−1 ⋅ mm−1in control segments and significantly increased to 16 pmol ⋅ min−1 ⋅ mm−1in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited[Formula: see text] absorption by 50% were ∼0.1 and ∼5 μM, similar to the K i for CA IV inhibition by these agents (0.2 and 4.0 μM, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98–104, 1997). Adding exogenous CA to the inhibitor in the perfusate nearly restored basal [Formula: see text] transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited[Formula: see text] absorption by 50% were 50 and 500 μM, respectively, >100 times the K i for CA IV inhibition and for inhibition of [Formula: see text]transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited ∼50% of CA IV activity also comparably inhibited [Formula: see text] transport, indicating that H+ secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of [Formula: see text]transport to CA inhibition.

Do Thiazides Reduce Intestinal Oxalate Absorption?: A Study In Vitro Using Rabbit Colon

1. The purpose of the present study was to examine the effects of various diuretics on intestinal oxalate transport. Transmural oxalate fluxes were measured across isolated, short-circuited tissue segments removed from rabbits and placed in Ussing chambers. 2. The net absorptive flux of oxalate across the distal colon was significantly reduced in the presence of trichlormethiazide at 10−4 mol/l. In contrast, this diuretic had no effect on oxalate transport in the other intestinal segments examined. Several of the thiazide diuretics tested had some inhibitory effect on colonic oxalate absorption, but at higher concentrations of 10−3 mol/l or 10−2 mol/l. 3. We conclude that the previously reported hypooxaluric effects of hydrochlorothiazide and chlorthalidone are most likely not the result of an exclusive or primary effect on intestinal oxalate transport. It is suggested that the reduction in colonic oxalate absorption that was observed with the thiazides probably involves the transport system responsible for oxalate efflux across the basolateral membrane of the colonocyte.