Francisco Alvarado

Direct Inhibitory Effect of Rotavirus NSP4(114-135) Peptide on the Na+-d-Glucose Symporter of Rabbit Intestinal Brush Border Membrane

ABSTRACT The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport ofd-glucose and of l-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na+-d-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na+-l-leucine symport about equally and partially via a different transport mechanism, in that Na+behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

Characterization of the d-glucose/Na+ cotransport system in the intestinal brush-border membrane by using the specific substrate, methyl α-d-glucopyranoside

By using isolated membrane vesicles, we have investigated the tenet that D-glucose transport across the intestinal brush-border membrane involves at least two distinct, Na+-activated agencies (D-glucose transport systems S-1 and S-2), only one of which (S-1) can use methyl alpha-D-glucopyranoside (methyl alpha-glucoside) as a substrate. Our results with this glucose analogue show that: (a) As a function of time, methyl alpha-glucoside uptake exhibits a typical overshoot, similar to but smaller than that given by D-glucose with the same vesicle batch. (b) Nonlinear regression analysis of substrate-saturation curves reveals that, contrary to D-glucose, methyl alpha-glucoside transport involves a single transport system which we have identified as S-1. (c) Methyl alpha-glucoside exhibits an apparent affinity (defined as the reciprocal of Km) 4-times smaller than that of D-glucose for S-1 (Km(Dglucose) = 0.5 mM; Km(methyl alpha-glucoside) = 2 mM). However, methyl alpha-glucoside has a Vmax (230 pmol/mg protein per s) identical to that characterizing D-glucose transport by this system. (d) In the absence of Na+, methyl alpha-glucoside uptake is indistinguishable from simple diffusion, confirming that Na+ is an obligatory activator of S-1. (e) Phlorizin behaves as a fully competitive inhibitor of methyl alpha-glucoside transport (Ki = 18 microM), again indicating that S-1 is involved. (f) Neither phloretin nor cytochalasin B affects methyl alpha-glucoside uptake. We conclude that methyl alpha-glucoside is a substrate specific for S-1, which permits study of the properties of this system without interference by substrate fluxes taking place through any other channel.

Virtual elimination of the interference of unstirred water layers on intestinal sugar transport kinetics by use of the tissue accumulation method at appropriate shaking rates.

The intestinal transport of sugars and amino acids seems to follow Michaelis-Menten kinetics, but the presence of unstirred water layers at the outer face of the brush border membrane may distort kinetic measurements. According to current theory, the capacity parameter,Jmcmax would not be affected, but theKt would be increased to a higher value,Kt′, in proportion to the thickness of the unstirred water layer,d.We reasoned that by increasing the shaking rate in the tissue accumulation method,d might drop to such small values thatKt′ would fall to a constant level practically equal to the “true”Kt.We measuredd-galactose influx into rings of everted hamster intestine as a function of both the substrate concentration and the shaking rate. Our results show that as the circular stirring rate increases from 0.38–6.2 Hz,Jmcmax remains constant, as expected, butKt′ first drops, then levels off to reach a plateau between 2 and 6.2 Hz. We conclude that the averageKt values in this frequency range (Kt=7.4 mM) represent the true transportKt. Furthermore, all previous kinetic work performed in our laboratory has been carried out under identical conditions, including shaking rates of 4 Hz. The validity of our preceding results is thus upheld.

The effect of harmaline on intestinal sodium transport and on sodium-dependentd-glucose transport in brush-border membrane vesicles from rabbit jejunum

Harmaline inhibition of sodium uptake and of sodium-dependentd-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, “initial”d-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrationsd-glucose uptake was stimulated by 10–15%. When a similar potassium gradient was used, harmaline had no effect. At concentrations upt to 2 mM, harmaline did not alter the equilibrium uptake ofd-glucose ord-mannitol. After pre-equlibration with sodium (25 mM),d-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2 mM. Sodium (10 mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependentd-glucose uptake. Similar to phlorizin, harmaline (1 mM) was able to prevent glucose-induced sodium influx across the brush-border membrane.Sodium uptake into brush-border membrane vesicles seems to be inhibited at lower harmaline concentrations than sodium-dependentd-glucose uptake. At high (2 mM) inhibitor concentrations, however, sodium-dependent glucose uptake is more strongly inhibited than sodium uptake. These results suggest that harmaline inhibits both sodium and sodium-dependent transport across intestinal brush-border membranes by interacting with specific sodium-binding sites.

Sodium-dependent activation of intestinal brush-border sucrase: correlation with activation by deprotonation from pH 5 to 7.

Abstract The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n -butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (p K 1 of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (p K 2 of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, V m and K m . The V m parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent p K (p K 1y , about 5.6) was little affected by sodium. On the contrary, the K m parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent p K (p K 1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent ( F. Alvarado and A. Mahmood, 1979 , J. Biol. Chem. 254 , 9534–9541).

pH-Dependent Inhibitory Effects of Tris and Lithium Ion on Intestinal Brush-Border Sucrase

Tris and two of its hydroxylated amine analogs were examined in a metal-free, universal n-butylamine buffer, for their interaction with intestinal brush border sucrase. Our recent three-proton-families model (Vasseur, van Melle, Frangne and Alvarado (1988) Biochem. J., 251, 667-675) has provided the sucrase pK values necessary to interpret the present work. At pH 5.2, 2-amino-2-methyl-l-propanol (PM) causes activation whereas Tris has a concentration-dependent biphasic effect, first causing activation, then fully competitive inhibition. The amine species causing activation is the protonated, cationic form. The difference between the two amines is related to the fact that Tris has a much lower pKa value than PM (respectively, 8.2 and 9.8). Even at pH 5.2, Tris (but not PM) exists as a significant proportion of the free base which, by inhibiting the enzyme fully competitively, overshadows the activating effect of the cationic, protonated amine. Above pH 6.8, both Tris and PM act as fully competitive inhibitors. These inhibitions increase monotonically between pH 6.5 and 8.0 but, above pH 8, inhibition by 2.5 mM Tris tends to diminish whereas inhibition by 40 mM PM increases abruptly to be essentially complete at pH 9.3 and above. As pH increases from 7.6 to 9.0, the apparent affinity of the free amine bases decreases whereas that of the cationic, protonated amines, increases. In this way, the protonated amines replace their corresponding free bases as the most potent inhibitors at high pH. The pH-dependent inhibition by 300 mM Li+ is essentially complete at pH 8, independent of the presence or absence of either 2.5 mM Tris or 40 mM PM. Even at pH 7.6, an excess (300 mM) of Li+ causes significant increases in the apparent Ki value of each Tris, PD (2-amino-2-methyl-1-3-propanediol) and PM, suggesting the possibility of a relation between the effects of Li+ and those of the hydroxylated amines which in fact are mutually exclusive inhibitors. The inhibitory results are interpreted in terms of a mechanistic model in which the free bases bind at two distinct sites in the enzymes active center. Binding at the glucosyl sub-site occurs through the amines free hydroxyl groups. This positioning facilitates the interaction between the lone electron pair of the deprotonated amino group with a proton donor in the enzymes active center, characterized by a pK0 around 8.1. When this same group deprotonates, then the protonated amines acting as proton donors replace the free bases as the species giving fully competitive inhibition of sucrase.

Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles.

To investigate the inhibitory effect of trans potassium on the Cl-/H+ symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H+ and K+ gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K+ was demonstrated. This inhibition was independent of the presence or absence of any K+ gradient. Electrical effects cannot be involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect of intravesicular K+ involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K+ for cis H+, catalyzed by the K+/H+ antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K+ and H+. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pHout/pHin = 5.0/7.5), charged or not with K+. In the absence of intravesicular K+, proton influx exhibited monoexponential kinetics with a time constant k = 11 s-1. Presence of 100 mM K+ within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s-1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K+ for cis H+, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/H+ symporter and K+/H+ antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.

Quantitative analysis of the mixed activating effects of the alkali metal ions on intestinal brush-border sucrase at pH 5.2.

The activation of rabbit brush-border sucrase by the alkali metal ions, Li+, Na+ and K+, was analyzed using the equations of the random-order allosteric model previously proposed for sucrase (Mahmood, A. and Alvarado, F. (1975) Arch. Biochem. Biophys. 168, 585). The alkali metals have mixed activating effects in tert-butylamine buffers at pH 5.2, including: 1. Affinity-type activation, where the apparent Km decreases as a hyperbolic function of the metal concentration. 2. Capacity-type activation, where the apparent V increases with the metal concentration. These two effects were analyzed quantitatively: firstly, by using linear transformations that allowed us to solve each partial equation separately and secondly, by iteration of the general equation, which permits treating the mixed effects as a whole. Results are consistent with the interpretation that a single metal-binding (activator) site suffices to explain the simultaneous occurrence of the two types of kinetic effect. Nevertheless, complicating factors exist that may require the postulation of additional sites for monovalent cations. In particular, the tert-butylammonium ion appears to interface with the effects of the alkali metals, especially Li+.

PH GRADIENT EFFECTS ON CHLORIDE TRANSPORT ACROSS BASOLATERAL MEMBRANE VESICLES FROM GUINEA-PIG JEJUNUM

1. The effects of alkaline‐inside pH gradients on 36Cl‐ uptake were quantified by using brush‐border membrane (BBM) and basolateral membrane (BLM) vesicles from guinea‐pig jejunum. 2. With BBM vesicles, a pHo/pHi gradient of 5.0/7.5 yielded fast overshoots involving a random, non‐obligatory Cl(‐)‐H+ symport, strongly inhibited by CCCP. In contrast, BLM vesicles responded to similar pH gradients with much smaller, delayed overshoots, unaffected by CCCP. 3. The initial Cl‐ entry rates into BLM vesicles were a function of each pHo, pHi and delta pH value. They were stimulated by valinomycin in the presence of inward‐directed K+ gradients. Short‐circuiting the membrane potential with equilibrated K+ and valinomycin inhibited pH gradient‐dependent Cl‐ uptake, but only partially. 4. Taken together, these results indicate that guinea‐pig jejunal BLM vesicles possess both Cl‐ conductance and Cl(‐)‐H+ symport activities. 5. Even when different, the BBM and the BLM symporters are mechanistically similar. Neither of them involves a Cl(‐)‐OH‐ antiport, nor a simultaneous Cl(‐)‐anion exchange mechanism. Rather, for each membrane, all of these activities (symport, anion exchange) can be explained in terms of a single mobile carrier acting as a random, non‐obligatory Cl(‐)‐H+ symporter where exchange occurs simply by counterflow. Net Cl‐ translocation via either the ternary (Cl(‐)‐C‐H+) or the binary (Cl(‐)‐C) complexes accounts, respectively, for the existence of two, operationally distinct, electroneutral and rheogenic components. 6. The BBM symporter appears to involve an AE2 protein, but the molecular identity of the BLM one remains to be established.

Different Temperature Sensitivity and Cation Specificity of Two Distinct d‐Glucose/Na+ Cotransport Systems in the Intestinal Brush‐Border Membranea

Recently, we proposed’ a general model for organic solute and N a + cotransport based on two key premises: (1) N a + is an obligatory (essential) activator and (2) because net transfer of a positive charge occurs, V,,, should be a function of A+. To test this working hypothesis, we studied D-glucose uptake in the presence and in the absence of Na+, using isolated brush-border membrane vesicles, which permit controlling the composition of the medium a t each side of the membrane. The experimental setup (see the illustration legends for details) included either an alkali-metal chloride salt giving an (out)/(in) = 100/0 m M concentration gradient, or no metal ion at all. In the absence of Na+, D-glucose uptake rates decreased but nevertheless indicated the existence of stereospecific transport. The activating sequence was: Na+ >> Li+ > K+ = sorbitol. However, these results required further clarification because of recent evidence showing that there are two distinct sodiumdependent glucose transport systems in guinea pig jejunum.* Frequently, transport is classified into “sodium-dependent’’ and “sodium-independent,” the implication being that separate transport systems are involved under either condition. However, this distinction seems gratuitous in the absence of a solid understanding of the mechanism(s) of glucose uptake under sodium-free conditions. Rather, the correct question is: To what extent does a sodium-dependent system remain operational when the ‘‘main,” but not necessarily “obligatory” activator, Na+, is absent? To answer this question, we carried out glucose saturation curves at either 25OC or at 35OC. When Na+ is present, a t 35O, our results demonstrate the existence of two distinct (saturable) glucose transport systems, apart from the diffusion component, Kd [S]. In contrast, a t 25O one of these two systems was not readily manifest. We noticed, however, that the apparent value of Kd (given by the slope of the saturation curve a t high [S]; see FIG. 1) is more than twice as large at 2 5 O as a t 35O, strongly suggesting that it hides a very low-affinity second transport system. In effect, if for that system K,,, >> [S], then the corresponding Michaelian term would simplify to: v = (V,,JK,) [S] = k [S]. The experimentally observed diffusion constant would in that case be the sum of two factors: pd = Kd + k. When we fixed Kd to its “true” value (see TABLE l ) , the second system at 25” became patent, showing a negligibly small affinity