Alkaline Phosphatase Activity Is a Key Determinant of Vascular Responsiveness to Norepinephrine.

Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode's-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, Ki=56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α1-adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α1-adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α1-adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A1-adenosine receptors, which are known to participate in coincident signaling with α1-adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α1-adrenoceptor signaling via a mechanism involving A1 receptor signaling.

Catalytic Site-Selective Acylation of Carbohydrates Directed by Cation-n Interaction.

Site-selective functionalization of hydroxyl groups in carbohydrates is one of the long-standing challenges in chemistry. Using a pair of chiral catalysts, we now can differentiate the most prevalent trans-1,2-diols in pyranoses systematically and predictably. Density functional theory (DFT) calculations indicate that the key determining factor for the selectivity is the presence or absence of a cation-n interaction between the cation in the acylated catalyst and an appropriate lone pair in the substrate. DFT calculations also provided a predictive model for site-selectivity and this model is validated by various substrates.

Kinetic resolution of racemic α-hydroxyphosphonates by asymmetric esterification using achiral carboxylic acids with pivalic anhydride and a chiral acyl-transfer catalyst.

A practical protocol is developed to directly provide chiral α-acyloxyphosphonates and α-hydroxyphosphonates from (±)-α-hydroxyphosphonates utilizing the transacylation process to generate the mixed anhydrides from acid components and pivalic anhydride in the presence of organocatalysts (s-value = 33-518).

Kinetic resolution of N-acyl-thiolactams via catalytic enantioselective deacylation.

Methanolysis of N-acyl-thiazolidin-2-thiones and -oxazolidin-2-thiones in the presence of acyl transfer catalyst benzotetramisole (BTM) proceeds in a highly enantioselective fashion thus enabling kinetic resolution of these substrates.

Determination of absolute configuration of secondary alcohols using thin-layer chromatography.

A new implementation of the competing enantioselective conversion (CEC) method was developed to qualitatively determine the absolute configuration of enantioenriched secondary alcohols using thin-layer chromatography. The entire process for the method requires approximately 60 min and utilizes micromole quantities of the secondary alcohol being tested. A number of synthetically relevant secondary alcohols are presented. Additionally, (1)H NMR spectroscopy was conducted on all samples to provide evidence of reaction conversion that supports the qualitative method presented herein.

Origin of enantioselectivity in benzotetramisole-catalyzed dynamic kinetic resolution of azlactones.

Density functional theory (DFT) calculations were performed to investigate the origins of enantioselectivity in benzotetramisole (BTM)-catalyzed dynamic kinetic resolution of azlactones. The transition states of the fast-reacting enantiomer are stabilized by electrostatic interactions between the amide carbonyl group and the acetate anion bound to the nucleophile. The chiral BTM catalyst confines the conformation of the α-carbon and the facial selectivity of the nucleophilic attack to promote such electrostatic attractions.

Kinetic resolution of α-substituted alkanoic acids promoted by homobenzotetramisole.

A new method for catalytic nonenzymatic kinetic resolution of α-substituted alkanoic acids has been developed, which relies on their activation with DCC followed by enantioselective alcoholysis of the intermediate symm-anhydrides in the presence of the amidine-based catalyst homobenzotetramisole (HBTM). Moderate to excellent selectivity factors (s=5-96) have been obtained in the case of several classes of substrates, namely, α-aryl-, α-aryloxy/alkoxy-, α-halo-, α-azido-, and α-phthalimido-alkanoic acids. Under similar conditions, α-(arylthio/alkylthio)-alkanoic acids undergo dynamic kinetic resolution providing corresponding esters in up to 92% ee and up to 93% yield.

Kinetic resolution of N-acyl-ß-lactams via benzotetramisole-catalyzed enantioselective alcoholysis.

The first nonenzymatic kinetic resolution of ß-lactams has been achieved. Alcoholysis of their N-aroyl derivatives in the presence of a simple chiral acyl transfer catalyst, benzotetramisole, produces ß-amino acid derivatives with excellent enantioselectivity.

Kinetic resolution of racemic alpha-arylalkanoic acids with achiral alcohols via the asymmetric esterification using carboxylic anhydrides and acyl-transfer catalysts.

A variety of optically active carboxylic esters are produced by the kinetic resolution of racemic alpha-substituted carboxylic acids using achiral alcohols, aromatic or aliphatic carboxylic anhydrides, and chiral acyl-transfer catalysts. The combination of 4-methoxybenzoic anhydride (PMBA) or pivalic anhydride with the modified benzotetramisole-type catalyst ((S)-beta-Np-BTM) is the most effective for promotion of the enantioselective coupling reaction between racemic carboxylic acids and a novel nucleophile, bis(alpha-naphthyl)methanol, to give the corresponding esters with high ee's. This protocol was successfully applied to the production of nonracemic nonsteroidal anti-inflammatory drugs from racemic compounds utilizing the transacylation process to generate the mixed anhydrides from the acid components with the suitable carboxylic anhydrides.

Benzotetramisole-catalyzed dynamic kinetic resolution of azlactones.

Enantioselective acyl transfer catalyst benzotetramisole (BTM) has been found to promote dynamic kinetic resolution of azlactones providing di(1-naphthyl)methyl esters of alpha-amino acids with up to 96% ee.

Homobenzotetramisole: an effective catalyst for kinetic resolution of aryl-cycloalkanols.

Homobenzotetramisole (HBTM), a ring-expanded analogue of the previously reported catalyst BTM, displays higher catalytic activity and a different structure-selectivity profile. It displays good enantioselectivities in kinetic resolution of secondary benzylic alcohols but is particularly effective for 2-aryl-substituted cycloalkanols.

Kinetic resolution of propargylic alcohols catalyzed by benzotetramisole.

[reaction: see text] Kinetic resolution of variously substituted secondary propargylic alcohols catalyzed by benzotetramisole (BTM) proceeds with selectivity factors up to 32, the highest ever achieved with nonenzymatic catalysts for this class of substrates.

Benzotetramisole: a remarkably enantioselective acyl transfer catalyst.

[reaction: see text] A commercially available pharmaceutical, tetramisole, was found to be a competent enantioselective acylation catalyst. Its benzannellated analogue, benzotetramisole (BTM), produced outstanding enantioselectivities in kinetic resolution of secondary benzylic alcohols.

Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

BACKGROUND: In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. RESULTS: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. CONCLUSION: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

Inhibitors of tyrosine phosphatases block angiotensin II inhibition of Na(+) pump.

To determine how angiotensin II inhibits the Na(+) pump (Na(+), K(+)-ATPase) in rat zona glomerulosa, we selectively blocked signaling proteins that could be activated by the angiotensin AT(1) receptor and known to affect Na(+) pump activity. Inhibitors of protein kinase C [calphostin C (1 microM); staurosporine (1 microM)], phospholipase A(2) [arachidonyl triflouromethyl ketone (25 microM); quinacrine (75 microM)], diacylgycerol lipase [RHC-80267 (5 microM)], and tyrosine phosphorylation [tyrphostin 47 (100 microM)] had no effect on angiotensin II inhibition of the Na(+) pump. On the other hand, inhibitors of tyrosine phosphatases [phenylarsine oxide (5 microM) and 4-bromotetramisole oxalate (100 microM)] blocked angiotensin II inhibition, where as inhibitors of serine/threonine phosphatases [okadaic acid (1 microM) and microcystin (1.5 microM)] did not. Thus, angiotensin II inhibition of the Na(+) pump may in part be mediated by a tyrosine phosphatase.

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole.

Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [(32)P]PO(4) release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate.

Enhancement of Ca2+-induced noradrenaline release by vanadate in PC12 cells: possible involvement of tyrosine phosphorylation.

Tyrosine phosphorylation has been shown to participate in the signal cascade after receptor stimulation with neurotransmitters and neurotrophins. However, the role of tyrosine phosphorylation in the process(es) of neurotransmitter release has not been well established. The effects of orthovanadate (Na3VO4), an inhibitor of protein-tyrosine phosphatases, on cytosolic free Ca2+ concentrations ([Ca2+]i), phosphotyrosine accumulation and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. Addition of Na3VO4 enhanced ionomycin-stimulated [3H]NA release in a concentration-dependent manner, although Na3VO4 alone had no effect. Na3VO4 also enhanced [3H]NA release induced by P2 receptor stimulation with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or by depolarization with 50 mM KCl, which stimulated a [Ca2+]i increase. A cell permeable inhibitor of protein-tyrosine phosphatases, L-p-bromotetramisole oxalate, at 0.3 mM enhanced ionomycin-stimulated [3H]NA release, although pervanadate had no effect. Addition of 5 mM Na3VO4 stimulated phosphotyrosine accumulation in several protein bands such as p130cas, but did not increase [Ca2+]i in PC12 cells. These findings suggest that the tyrosine phosphorylation pathway regulates Ca2+-stimulated NA release without changes of [Ca2+]i in PC12 cells.

Glibenclamide induces apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and intracellular Ca(2+) release in HepG2 human hepatoblastoma cells.

Glibenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoblastoma cells. Glibenclamide increased intracellular Ca(2+) concentration, which was significantly inhibited by Ca(2+) release blockers dantrolene and TMB-8. BAPTA/AM, an intracellular Ca(2+) chelator, and the Ca(2+) release blockers significantly inhibited glibenclamide-induced apoptosis. Glibanclamide also increased intracellular Cl(-) concentration, which was significantly blocked by CFTR Cl(-) channel activators levamisole and bromotetramisole. These activators also significantly inhibited both intracellular Ca(2+) release and apoptosis induced by glibenclamide. The expression of CFTR protein in the cells was confirmed by Western blot analysis. These results suggest that glibenclamide induced apoptosis through inhibition of CFTR Cl(-) channels and intracellular Ca(2+) release and that this protein may be a good target for treatment of human hepatomas.

Transformation is associated with an increase in sensitivity to TNF-mediated lysis as a result of an increase in TNF-induced protein tyrosine phosphatase activity.

Using the tumor necrosis factor (TNF)-resistant cell line B/CN and an anchorage-independent variant, 10ME, we have shown a relationship between transformation and sensitivity to TNF. Here, we report a role for protein tyrosine phosphorylation in expression of these phenotypes. Several studies have demonstrated the involvement of protein phosphorylation in the TNF signaling pathways that leads to cell death. We show that TNF treatment of the TNF-sensitive, transformed cells results in a marked increase in protein tyrosine phosphatase (PTP) activity and a decrease in protein tyrosine kinase (PTK) activity. In contrast, TNF treatment of the TNF-resistant, non-transformed parental cells results in a marked increase in PTK activity. Also, the PTP inhibitors vanadate and bromotetramisole decrease TNF lytic activity, indicating that the PTP activity observed is an integral part of the lytic process. Treatment of targets with vanadate prior to TNF exposure had no effect on TNF-mediated lysis. In contrast, the addition of vanadate up to 4 hr after TNF-treatment resulted in a decrease in TNF-mediated lysis. Our findings indicate that the phosphatase activity is induced after TNF binds its receptor. Our data also indicate that the decrease in TNF-mediated lysis caused by PTP inhibitors is not due to the inhibition of the TNF lytic mechanism. Instead, vanadate increases a TNF resistance mechanism; it does so by blocking the PTP-mediated inhibition of the TNF resistance mechanism. Further, the lineage relationship of these cell lines suggests that there is a biochemical relationship between anchorage-independence, tumorigenicity and the protein tyrosine phosphorylation that governs sensitivity to TNF.

Levamisole inhibits intestinal Cl- secretion via basolateral K+ channel blockade.

BACKGROUND & AIMS: Phenylimidazothiazoles have recently been shown to activate wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in transfected cells and were proposed as therapy for cystic fibrosis. The aim of this study was to investigate the effects of phenylimidazothiazoles on regulated transepithelial Cl- transport in intact epithelia. METHODS: T84 intestinal epithelial cells grown on permeable supports and stripped human colonic mucosal sheets were studied by conventional current-voltage clamping. Selective permeabilization of apical or basolateral membranes with the monovalent ionophore nystatin was used to isolate basolateral K+ and apical Cl- channel activity, respectively. 86Rb+ uptake was assessed for Na/K/2Cl cotransporter and Na+,K(+)-adenosine triphosphatase activity. RESULTS: In T84 monolayers and human colon, levamisole and its brominated derivative bromotetramisole failed to activate transepithelial secretion. In fact, these compounds dose-dependently inhibited secretory responses to the cyclic adenosine monophosphate agonist forskolin and the Ca2+ agonist carbachol. In permeabilized T84 monolayers, phenylimidazothiazoles weakly activated apical Cl- currents (consistent with their reported action on CFTR) and did not affect bumetanide-sensitive or bumetanide-insensitive 86+Rb+ uptake. Instead, they profoundly inhibited the basolateral Ba(2+)-sensitive and Ba(2+)-insensitive K+ currents. CONCLUSIONS: Phenylimidazothiazoles block K+ channels required for Cl(-)-secretory responses elicited by diverse pathways in model epithelia and native colon, an effect that outweighs their ability to activate apical Cl- channels.