Coupling Reactions of Anhydro-Aldose Tosylhydrazones with Boronic Acids.

A catalyst-free coupling reaction between O-peracetylated, O-perbenzoylated, O-permethylated, and O-permethoxymethylated 2,6-anhydro-aldose tosylhydrazones (C-(ß-d-glycopyranosyl)formaldehyde tosylhydrazones) and aromatic boronic acids is reported. The base-promoted reaction is operationally simple and exhibits a broad substrate scope. The main products in most of the transformations were open-chain 1-C-aryl-hept-1-enitol type compounds while the expected ß-d-glycopyranosylmethyl arenes (benzyl C-glycosides) were formed in subordinate yields only. A mechanistic rationale is provided to explain how a complex substrate may change the well-established course of the reaction.

Bicarbonate Inhibits Bacterial Growth and Biofilm Formation of Prevalent Cystic Fibrosis Pathogens.

We investigated the effects of bicarbonate on the growth of several different bacteria as well as its effects on biofilm formation and intracellular cAMP concentration in Pseudomonas aeruginosa. Biofilm formation was examined in 96-well plates, with or without bicarbonate. The cAMP production of bacteria was measured by a commercial assay kit. We found that NaHCO3 (100 mmol l-1) significantly inhibited, whereas NaCl (100 mmol l-1) did not influence the growth of planktonic bacteria. MIC and MBC measurements indicated that the effect of HCO 3 - is bacteriostatic rather than bactericidal. Moreover, NaHCO3 prevented biofilm formation as a function of concentration. Bicarbonate and alkalinization of external pH induced a significant increase in intracellular cAMP levels. In conclusion, HCO 3 - impedes the planktonic growth of different bacteria and impedes biofilm formation by P. aeruginosa that is associated with increased intracellular cAMP production. These findings suggest that aerosol inhalation therapy with HCO 3 - solutions may help improve respiratory hygiene in patients with cystic fibrosis and possibly other chronically infected lung diseases.

Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice.

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration.

Depressed calcium cycling contributes to lower ischemia tolerance in hearts of estrogen-deficient rats.

OBJECTIVE: Estrogens enhance ischemia tolerance (IT) in the myocardium, the mechanism of which remains unclear. We investigated the effects of long-term estrogen deprivation on the intracellular calcium (Ca(2+)(i)) transient of the heart and its possible influence on IT. METHODS: Hearts of ovariectomized (OVX) and sham-operated (control) adult female rats (some receiving estrogen therapy) were studied 10 weeks after surgical operation: control (n = 8), OVX (n = 10), sham-operated estrogen-substituted (n = 7), and ovariectomized estrogen-substituted (n = 9). In vivo heart function was assessed by echocardiography, whereas Ca(2+)(i) transients were recorded, concomitantly with left ventricular pressure and coronary flow, by Indo-1 surface fluorometry in isolated Langendorff-perfused hearts. Isolated hearts were subjected to a 30-minute global ischemia-30-minute reperfusion protocol. Left ventricular expression of myocardial sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), and Ser16-phosphorylated PLB was measured. RESULTS: Ovariectomy did not influence resting cardiac function in vivo or ex vivo. However, Ca(2+) removal was slower. During ischemia, Ca(2+)(i) elevation and ischemic contracture were more pronounced after ovariectomy. Postischemic restitution of inotropic function (developed pressure; +dP/dt(max)) and lusitropic function (-dP/dt(max)) and Ca(2+)(i) transient recovery (amplitude; ±dCa(2+)(i)/dt(max)) were decreased in OVX hearts. Sarcoendoplasmic reticulum Ca(2+)-ATPase expression was unaltered, whereas PLB and Ser16-phosphorylated PLB levels were higher after ovariectomy. All effects of ovariectomy were restored by estrogen therapy. CONCLUSIONS: Ovariectomy impairs myocardial Ca(2+) removal by increasing the expression of the SERCA2a inhibitor PLB. Defective Ca(2+) transport causes ischemic Ca(2+)(i) overload and insufficient postischemic recovery of Ca(2+)(i) transients, which entail depressed hemodynamic restitution. Protection of intact Ca(2+) cycling in the myocardium by estrogens plays a major role in enhancing IT.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods.

BACKGROUND/AIMS: ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS: We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS: Our data show that ATP (< 1 µM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS: Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.

Inhibition of the human epithelial calcium channel TRPV6 by 2-aminoethoxydiphenyl borate (2-APB).

TRPV6, a highly calcium-selective member of the transient receptor potential (TRP) channel superfamily, is a major pathway for calcium absorption in the fetal and adult body. It is expressed abundantly in the duodenum, the placenta and exocrine tissues. TRVP6 was postulated to contribute to store-operated calcium channel (SOC) activity in certain cell types such as exocrine cells. In this study, we tested 2-APB, a widely used SOC inhibitor on human TRPV6 (hTRPV6) activity using fluorescence imaging, patch clamp and radioactive tracer techniques in transiently and stably transfected HEK293 cells. We found that the basal calcium and cadmium influx was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells. 2-APB inhibited hTRPV6 activity in both transient and stably transfected cells. This effect was slightly sensitive toward extracellular calcium. The extracellular sodium concentration did not affect the inhibition of hTRPV6 by 2-APB. However, N-methyl-d-glucamine significantly diminished the inhibitory effect of 2-APB presumably through direct interaction with this compound. Furthermore, 2-APB inhibited the activity of TRPV6 orthologs but not human TRPV5. 2-APB may serve as a parental compound for the development of therapeutic strategies specifically targeting the hTRPV6 calcium channel.

Age-dependent changes in ion channel mRNA expression in canine cardiac tissues.

The expression pattern of cardiac ion channels displays marked changes during ontogeny. This study was designed to follow the developmental changes in the expression of major ventricular and atrial ion channel proteins (including both pore forming and regulatory subunits) in canine cardiac tissues at the mRNA level using competitive reverse transcription polymerase chain reaction. Therefore, the corresponding mRNA levels were compared in myocardial tissues excised from embryonic (25-60 days of gestation) and adult (2-3 years old) canine hearts. Expression level of Kv4.3, Kv1.4, KChIP2, KvLQT1, and Cav3.2 mRNAs were higher in the adult than in the embryonic hearts, while expression of Nav1.5 and minK mRNAs were higher in the embryonic than in the adult myocardium. No change in Kir2.1, HERG, Kv1.5, and Cav1.2 mRNA was observed during ontogeny. Direction of the developmental change in the mRNA level, determined for any specific channel protein, was identical in the atrial and ventricular samples. The age-dependent increase observed in the expression of Kv4.3, Kv1.4, KChIP2, and KvLQT1 is congruent with the greater repolarization reserve of the adult myocardium, associated with higher densities of Ito and IKs. The results indicate that age-dependent changes in the expression pattern of many ion channels are similar in canine and healthy human myocardium, therefore, canine cardiac muscle can be considered as a good model of studying developmental changes in the human heart.

Heavy metal cations permeate the TRPV6 epithelial cation channel.

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.