Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

4-Chloromercuribenzoic acid enhances carbapenem sensitivity among pathogenic Gram negative bacteria by altering blaVIM, adeB and ompC expression.

BACKGROUND: Rapid global dissemination of carbapenem resistant Gram negative bacteria (CRGNB) is supposed to be clinically most alarming. Since, p-chloromercuribenzoic acid (pCMB) is a well known metallo-beta-lactamase inhibitor; evaluation of its bactericidal and carbapenem resistance reversing potential would be important. METHODS: In this study, bactericidal and meropenem resistance reversing potential of pCMB was investigated against CRGNB by MIC determination, checkerboard assay, time-kill assay and cellular viability assay. Effect of pCMB on cellular morphology was visualized by Scanning Electron Microscopy. Further, quantitative Real Time-PCR was performed to evaluate effects of pCMB on clinically relevant metallo-beta-lactamases, major efflux pumps and outer membrane proteins expression. RESULTS: pCMB exhibited at least four fold reduced MIC value (2-256µg/ml) than that of meropenem against CRGNB. Moreover, pCMB exhibited synergism with meropenem against 86.06% of CRGNB. MIC of pCMB (16-32µg/ml) could kill upto 99.96% bacteria within 6-8h of dosing. pCMB exerted bactericidal activity by severely disrupting cell wall integrity. Reversal of carbapenemase property of CRGNB by pCMB might have developed through alteration of blaVIM, acrB, mexB and ompk36 expression. CONCLUSIONS: Hence, the current study identified pCMB as a potential bactericidal agent which enhanced meropenem sensitivity by altering blaVIM, acrB, mexB and ompk36 expression.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin.

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 µM) and PD123319 (10 µM) plus 100 µM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1-20 µM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 µM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

Pharmacological characterization of a novel non-AT1, non-AT2 angiotensin binding site identified as neurolysin.

The discovery of a novel non-AT1, non-AT2 binding site for angiotensins in the rodent brain and testis that is unmasked by the organomercurial compound para-chloromercuribenzoic acid (PCMB) has catalyzed efforts to purify and characterize this protein. We recently reported that this protein is neurolysin and now report upon the specificity of this binding site for various neuropeptides. Competition binding assays in rat brain and testis used (125)I-Sar(1), Ile(8) angiotensin II (Ang II) as the radioligand in the presence of saturating concentrations of AT1 and AT2 receptor antagonists and 100 µM parachloromercuribenzoate. Primary screening of 36 peptides and other compounds at 10 µM concentration revealed seven peptides that inhibited specific binding >50 %: ghrelin, Tyr(1) S36057 (a melanin-concentrating hormone receptor ligand), orphanin FQ and its congeners (Tyr(1) and Tyr(14)), Dynorphin A (1-8), and Ang (1-9). The selective neurolysin inhibitor Proline-Isoleucine dipeptide was inactive at 1 mM. These results suggest that the ability of PCMB to unmask high affinity binding of Ang II to neurolysin is a pharmacological effect and that neurolysin may significantly affect the activity of the renin-angiotensin system.

Reinvestigation of drugs and chemicals as aquaporin-1 inhibitors using pressure-induced hemolysis in human erythrocytes.

Recently, we have found that pressure-induced hemolysis is enhanced by inhibiting water transport via aquaporin-1 (AQP1), as seen in p-chloromercuribenzoate (pCMB)-treated erythrocytes. So, using this method we reinvestigated the functions as AQP1 inhibitors of drugs and chemicals such as acetazolamide, sodium nitroprusside, tetraethylammonium ions (TEA(+)), and dimethylsulfoxide (DMSO). The values of hemolysis at 200 MPa were almost unaffected by acetazolamide or sodium nitroprusside, decreased by TEA(+), and increased significantly by DMSO. Furthermore, the erythrocytes were exposed to pCMB in the presence of TEA(+) or DMSO. The enhancement effect of pCMB on pressure-induced hemolysis was unaffected by TEA(+) but attenuated by DMSO. Taken together, these results suggest that, of drugs and chemicals examined here, DMSO only is an AQP1 inhibitor, but the effect of DMSO is small compared with pCMB.

Purification and characterization of a novel (R)-imine reductase from Streptomyces sp. GF3587.

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.

Association of the novel non-AT1, non-AT2 angiotensin binding site with neuronal cell death.

We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.

Soybean (Glycine max) urease: significance of sulfhydryl groups in urea catalysis.

The soybean urease (urea amidohydrolase; EC 3.5.1.5) was investigated to elucidate the presence of sulfhydryl (-SH) groups and their significance in urea catalysis with the help of various -SH group specific reagents. The native urease incubated with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) showed exponential increase in the absorbance, thereby revealing the presence of -SH groups. A total of 34 -SH groups per hexamer enzyme molecule were estimated from the absorption studies which represents nearly six -SH groups per subunit. The time-dependent inactivation of urease with DTNB, p-chloromercuribenzoate (p-CMB), N-ethylmaleimide (NEM) and iodoacetamide (IAM) showed biphasic kinetics, where half of the enzyme activity was lost more rapidly than the other half. This study reveals the presence of two categories of "accessible" -SH groups, one category being more reactive than the other. The inactivation of urease by p-CMB was largely reversed on treatment with cysteine, which might be due to unblocking of -SH group by mercaptide exchange reaction. Finally, when NEM inactivated urease was incubated with sodium fluoride, a time-dependent regain of activity was observed with higher concentrations of fluoride ion.

Esterase patterns in species of the triatome bug Rhodnius.

The aim of the present study was to analyse esterase patterns in three triatomine species of Rhodnius genus. Four loci, Est 1, Est 2, Est 3 and Est 4, were found. The corresponding enzymes were characterized as carboxylesterases (E.C. 3.1.1.1) or cholinesterases (E.C. 3.1.1.8) based on inhibitory experiments, using eserine sulphate, malathion, mercury chloride, p-chloromercuribenzoate (pCMB) and iodoacetamide. Low genetic variability was observed: Est 1, Est 2 and Est 3 were monomorphic in Rhodnius domesticus, Rhodnius robustus and Rhodnius neivai, whereas locus Est 4 was polymorphic in the first two species. The UPGMA analysis based on esterase genotypic frequencies indicated greater similarity between R. domesticus and R. robustus when compared with R. neivai. The present study expands our knowledge about genetic variability among triatomines and accords with the hypothesis that R. domesticus is a species derived from R. robustus.

Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries.

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused 'click' chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine K(i) and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (K(i)=K(i)(')=1.5+/-0.2microM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, K(i)=0.41+/-0.03microM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, K(i)=1.4+/-0.10microM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XV. Agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi).

The water diffusional permeability (P(d)) of red blood cells (RBC) from agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi) was monitored using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. The P(d) (cm s(-1)) values of agile wallaby RBCs were 7.5 x 10(-3) at 25 degrees C, 9 x 10(-3) at 30 degrees C, 11 x 10(-3) at 37 degrees C, and 13 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing sulfhydryl (SH)-modifying reagent p-chloromercuribenzoate (PCMB) on agile wallaby RBCs was investigated. The maximal inhibition was reached in 90 min at 37 degrees C with 2 mmol L(-1) PCMB. The value of maximal inhibition was approximately 63% when measured at 25 degrees C, approximately 52% at 37 degrees C and approximately 45% at 42 degrees C. The lowest value of P(d) (corresponding to the basal permeability to water) was approximately 3 x 10(-3) cm s(-1) at 25 degrees C. For the RBCs from red-necked wallaby (M. rufogriseus) the values of P(d) (cm s(-1)) were 7 x 10(-3) at 25 degrees C, 8 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C, and 12 x 10(-3) at 42 degrees C. Higher values of P(d) (cm s(-1)) were found for the RBCs from Goodfellow's tree kangaroo (D. goodfellowi): 8.5 x 10(-3) at 25 degrees C, 10 x 10(-3) at 30 degrees C, 13 x 10(-3) at 37 degrees C, and 15 x 10(-3) at 42 degrees C. The mean values of the activation energy of water diffusion (E(a,d)) were approximately 25 kJ mol(-1) for RBCs from the agile wallaby and tree kangaroo, respectively, and approximately 23 kJ mol(-1) for RBCs from red-necked wallaby. The values of E(a,d) increased after exposure of agile wallaby RBCs to PCMB, reaching a value of approximately 43-46 kJ mol(-1) when the maximal inhibition of P(d) was achieved.

Dietary pectin up-regulates monocaboxylate transporter 1 in the rat gastrointestinal tract.

This work was undertaken to study the effect of pectin feeding on the expression level, cellular localization and functional activity of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of rats. The results indicated that MCT1 protein level was significantly increased along the entire length of the gastrointestinal tract of pectin-fed rats in comparison with control animals. Immunohistochemical analysis revealed an increase in MCT1 in the stratified squamous epithelia of the forestomach as well as in the basolateral membranes of the cells lining the gastric pit of the glandular stomach of pectin-fed rats when compared with control animals. The parietal cells, which showed barely any or no detectable MCT1 in the control group, exhibited a strong intensity of MCT1 on the basolateral membranes in pectin-fed rats. In the small intestine of pectin-fed rats, strong immunopositivity for MCT1 was detected in the brush border and basolateral membranes of the absorptive enterocytes lining the entire villi, while in control rats, weak reactivity was detected on the brush border membrane in a few absorptive enterocytes in the villus tip. In the large intestine of control animals, MCT1 was detected on the basolateral membranes of the epithelia lining the caecum and colon. This staining intensity was markedly increased in pectin-fed rats, along with the appearance of strong reactivity for MCT1 on the apical membranes of the surface and crypt epithelia of caecum and colon. Our results also showed that MCT1 co-localizes with its chaperone, basigin (CD147), in the rat gastrointestinal tract, and that the pectin feeding increased the expression of CD147. In vivo functional studies revealed an enhanced acetate absorption in the colon of pectin-fed in comparison with control animals. We conclude that MCT1 is up-regulated along the gastrointestinal tract of pectin-fed rats, which might represent an adaptive response to the increased availability of its substrates.

Cultured ruminal epithelial cells express a large-conductance channel permeable to chloride, bicarbonate, and acetate.

The absorption of short-chain fatty acids (SCFA) from the rumen requires efficient mechanisms for both apical uptake and basolateral extrusion. Previous studies suggest that the rumen expresses a basolateral chloride conductance that might be permeable to SCFA. In order to characterize this conductance in more detail, isolated cultured ruminal epithelial cells were studied with the patch-clamp technique, revealing a whole-cell conductance with p(Cl(-)) approximately p(NO(3) (-)) > p(HCO(3) (-)) > p(acetate(-)) > p(gluconate(-)). Currents could be blocked by diisothiocyanato-stilbene-2,2'-disulfonic acid (1 mmol l(-1) > 100 micromol l(-1)), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (50 micromol l(-1)), niflumic acid (100 micromol l(-1)), and p-chloromercuribenzoate (1 mmol l(-1)). Single-channel conductance was 350 +/- 7 pS for chloride and 142 +/- 7 pS for acetate. Open probability could be fitted with a three-state gating model. We propose a role for this channel in mediating the permeation of chloride, bicarbonate, and acetate across the basolateral membrane of the ruminal epithelium.

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides.

AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.

Characterization of the brain-specific non-AT(1), non-AT(2) angiotensin binding site in the mouse.

In the present study the existence of a non-AT(1), non-AT(2) angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT(1), non-AT(2) angiotensin binding site was similar to the rat brain: Sar(1)-Ile(8)-Ang II > Ang III >or= Ang II > Ang I> p-aminophenylalanine(6) Ang II> CGP42112 >> Ang IV > Ang 1-7 congruent with shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K(i) values >10 microM, while substance P and VIP had K(i) values of approximately 2 microM. The non-AT(1), non-AT(2) angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [(125)I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT(1a), AT(1b), AT(2), Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT(1), AT(2), AT(4) and Ang 1-7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study.

The role of monocarboxylate transporter 2 and 4 in the transport of gamma-hydroxybutyric acid in mammalian cells.

Monocarboxylate transporter 1 (MCT1) is an important determinant of the renal transport of the drug of abuse, gamma-hydroxybutyric acid (GHB). The objective of this study was to investigate the role of MCT2 and MCT4, present in tissues including intestine, kidney, skeletal muscle, and brain, in the membrane transport of GHB and the MCT substrate l-lactate. mRNA and protein of MCT2 and MCT4 were expressed in MDA-MB231 cells, as detected by reverse transcription-polymerase chain reaction and Western blot analysis; MCT1 and MCT3 were not detected. The uptake of GHB or l-lactate by MDA-MB231 cells was pH-dependent but not sodium-dependent. The concentration-dependent uptake of GHB was best fitted to a single-transporter model with a diffusional clearance component (K(m) of 17.6 +/- 1.5 mM, V(max) of 50.6 +/- 9.0 nmol x mg(-1) min(-1) and diffusional clearance of 0.20 +/- 0.07 microl x mg(-1) min(-1)). On the other hand, the concentration-dependent uptake of l-lactate was best fitted to a two-transporter model (K(m) of 21 +/- 2.5 and 3.0 +/- 1.5 mM, and V(max) of 268 +/- 72 and 62.9 +/- 42.2 nmol x mg(-1)min(-1), respectively). The uptake of GHB and l-lactate was inhibited by MCT inhibitors alpha-cyano-4-hydroxycinnamate (CHC), phloretin, and p-chloromercuribenzoic acid; CHC inhibited GHB and l-lactate uptake with IC(50) values of 1.71 +/- 0.39 and 0.71 +/- 0.11 mM, respectively. Small interfering RNA treatment to silence MCT2 or MCT4 significantly decreased their protein expression and the uptake of l-lactate and GHB; however, the decrease in GHB uptake with MCT2 inhibition was smaller than that for MCT4. This investigation demonstrated that GHB is a substrate for both MCT2 and MCT4; these transporters may be important in the nonlinear disposition of GHB, as well as influencing its tissue distribution.

Identification of a novel non-AT1, non-AT2 angiotensin binding site in the rat brain.

Efforts to protect radiolabeled angiotensins from metabolism during receptor binding assays date back more than 30 years. However, this continues to be a problem. This study focused on the effects of a protease inhibitor, p-chloromercuribenzoate (PCMB), on the binding of (125)I-Ang II to rat brain membranes. Addition of PCMB to the incubation medium revealed a high affinity binding site for (125)I-Ang II in brain membranes (K(d)=1-4 nM) with a greater amount of binding than revealed in previous studies of brain Ang II receptors. Further characterization of this binding, revealed it to be insensitive to inhibition by losartan (an AT(1) receptor antagonist) and PD123319 (an AT(2) receptor antagonist). This non-AT1, non-AT2 binding site was not present in liver or adrenal membranes. It was activated by a limited range of concentrations of PCMB, with maximal activation at 0.3-1 mM. This binding site was equally abundant in cerebral cortex (a brain region with few Ang II receptors) and the hypothalamus (a brain region with abundant Ang II receptors). The binding site was also present in mouse brain, but not mouse liver. The binding site shows high affinity for Ang I, Ang II and Ang III (K(i) approximately 40-100 nM), but lesser affinity for smaller angiotensin fragments and other neuropeptides. This binding site shares some characteristics with the liver cytosolic Ang II binding proteins, later identified as endopeptidases EC 3.4.24.15 and/or EC 3.4.24.16. However, some unique characteristics of this non-AT1, non-AT2 binding site suggest that it may be a novel angiotensin binding substance.

The role of monocarboxylate transporters in uptake of lactic acid in HeLa cells.

This study was aimed to identify the monocarboxylate transporters (MCTs) in HeLa cells and to delineate their role in transportation of L-lactic acid. The functional role of MCTs in lactic acid transport was evaluated at various mucosal pHs (4.5-7.4) or in the presence of various loading doses (0.2-2mM) of lactic acid, MCT substrates (nicotinic acid, n-butyric acid, etc.) and inhibitors (alpha-cyano-4-hydroxycinnamate and para-chloromercuribenzoic acid). The molecular properties of MCTs were characterized using reverse transcription-polymerase chain reaction (RT-PCR). The uptake rate of lactic acid by HeLa cells significantly increased from 0.353+/-0.052 to 1.103+/-0.196 micromol/mg protein as the extra-cellular pH changed from 7.4 to 4.5, indicating that activities of MCT were mediated through H(+)-linked mechanism. The uptake profile of lactic acid followed the saturable process with the K(m) value of 0.53 mM. The uptake rate of lactic acid is concentration dependent and is reduced in the presence of MCT inhibitors. MCT isoforms 1, 5 and 6 in HeLa cells were identified by RT-PCR. HeLa cell line can be used as an effective screening tool for intravaginally administered drugs targeted toward MCT.

Monocarboxylate transporter 1 (MCT1) mediates transport of short-chain fatty acids in bovine caecum.

The present study was undertaken to investigate the functional role of monocarboxylate transporter 1 (MCT1) in the ruminant large intestine. Messenger RNA encoding for MCT1 was verified by reverse transcriptase-polymerase chain reaction in caecum, proximal colon and distal colon of adult cattle. Both immunohistochemistry and confocal laser microscopy verified that the MCT1 protein was abundant in the surface epithelium of the large intestine, and the amount decreased from the opening of the crypt to its base. In the immunopositive cells, MCT1 was primarily localized in the basolateral membranes of epithelium lining the large intestine. Western blotting indicated that the levels of MCT1 protein were highest in the caecum, followed by proximal colon and then distal colon. In vitro studies were conducted to elucidate the possible involvement of MCT1 in the transport of short-chain fatty acids (SCFA) across the isolated mucosal sheets of cattle caecum using the Ussing chamber technique. Acetate absorption was found to be pH dependent, and the rate of acetate absorption increased as pH decreased. The serosal application of the MCT1 inhibitor 'p-chloromercuribenzoic acid (pCMB)' significantly reduced the transport of acetate across the caecal epithelium of cows. In addition, the transport of acetate was significantly reduced in the presence of its analogue, propionate, indicating that acetate and propionate compete for binding to the same transporter. The results show that MCT1 is a major route for SCFA efflux across the basolateral membrane of bovine large intestine and that it could play a role in the regulation of intracellular pH.

Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors.

The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC(50) values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB (p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC(50) values of PCMB and bestatin for VACV and GVACV were found to be 3.81+/-0.94 and 0.34+/-0.08muM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea.