Sertraline inhibits pre-synaptic Na⁺ channel-mediated responses in hippocampus-isolated nerve endings

In the present study, a possible sertraline action on cerebral pre-synaptic Na(+) channels was investigated. For this purpose, the effect of sertraline on responses induced by the Na(+) channel opener, veratridine, namely the increase in Na(+) and in neurotransmitter release in hippocampus-isolated nerve endings was investigated. Results show that sertraline in the low μM range (1.5-25?μM) progressively inhibits the rise in Na(+) and the release of pre-loaded [(3) H]Glu as well as the release of endogenous 5-HT, Glu and GABA (detected by HPLC) induced by veratridine depolarization either under external Ca(2+) -free conditions or in the presence of external Ca(2+) . In addition, under non-depolarized conditions, sertraline (25 μM) increased the external concentration of 5-HT at expense of its internal concentration, and unchanged the external and internal concentrations of the amino acid neurotransmitters and of the 5-HT main metabolite, 5-HIAA. This result is consistent with the sertraline inhibitory action of the serotonin transporter. However, sertraline is unlikely to inhibit pre-synaptic Na(+) channels permeability by increasing external 5-HT. Because 5-HT in a wide concentration range (1-1000 μM) did not change the veratridine-induced increase in Na(+) . In summary, present findings demonstrate that besides the inhibition of 5-HT reuptake, sertraline is an effective inhibitor of pre-synaptic Na(+) channels controlling neurotransmitter release.     

Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato

Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato plants that can adapt to saline soil, AVP1, a vacuolar H(+)-pyrophosphatase gene from Arabidopsis thaliana, and PgNHX1, a vacuolar Na(+)/H(+) antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these transgenic plants accumulated a 1.5 times higher Na(+) content in their leaf tissue than the single gene transformants. The toxic effect of Na(+) accumulation in the cytosol is reduced by its sequestration into the vacuole. The physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na(+).     

Gut sensing of dietary K⁺ intake increases renal K⁺excretion

Dietary K(+) intake may increase renal K(+) excretion via increasing plasma [K(+)] and/or activating a mechanism independent of plasma [K(+)]. We evaluated these mechanisms during normal dietary K(+) intake. After an overnight fast, [K(+)] and renal K(+) excretion were measured in rats fed either 0% K(+) or the normal 1% K(+) diet. In a third group, rats were fed with the 0% K(+) diet, and KCl was infused to match plasma [K(+)] profile to that of the 1% K(+) diet group. The 1% K(+) feeding significantly increased renal K(+) excretion, associated with slight increases in plasma [K(+)], whereas the 0% K(+) diet decreased K(+) excretion, associated with decreases in plasma [K(+)]. In the KCl-infused 0% K(+) diet group, renal K(+) excretion was significantly less than that of the 1% K(+) group, despite matched plasma [K(+)] profiles. We also examined whether dietary K(+) alters plasma profiles of gut peptides, such as guanylin, uroguanylin, glucagon-like peptide 1, and glucose-dependent insulinotropic polypeptide, pituitary peptides, such as AVP, α-MSH, and γ-MSH, or aldosterone. Our data do not support a role for these hormones in the stimulation of renal K(+) excretion during normal K(+) intake. In conclusion, postprandial increases in renal K(+) excretion cannot be fully accounted for by changes in plasma [K(+)] and that gut sensing of dietary K(+) is an important component of the regulation of renal K(+) excretion. Our studies on gut and pituitary peptide hormones suggest that there may be previously unknown humoral factors that stimulate renal K(+) excretion during dietary K(+) intake.     

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T_CM) and effector (=T_EM) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL).In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7^?CD45RA^?CD8⁺ T_EM cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T_EM cells was significantly increased. T_EM phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result.Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T_EM cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.     

The immunoregulatory role of CD4⁺ FoxP3⁺ CD25⁻ regulatory T cells in lungs of mice infected with Bordetella pertussis

The identification of regulatory T (Treg) cells was originally based on CD25 expression; however, CD25 is also expressed by activated effector T cells. FoxP3 is a more definitive marker of Treg cells, and CD4(+) FoxP3(+) CD25(+) T cells are considered the dominant natural Treg (nTreg) population. It has been suggested that certain CD4(+) FoxP3(+) Treg cells do not express CD25. In this study, we used a murine model of respiratory infection with Bordetella pertussis to examine the role of Treg cells in protective immunity in the lung. We first demonstrated that CD4(+) FoxP3(+) CD25(-) cells are the dominant Treg population in the lung, gut and liver. Pre-activated lung CD4(+) FoxP3(+) CD25(-) cells suppressed CD4(+) effector T cells in vitro, which was partly mediated by IL-10 and not dependent on cell contact. Furthermore, CD4(+) FoxP3(+) CD25(-) IL-10(+) T cells were found in the lungs of mice at the peak of infection with B. pertussis. The rate of bacterial clearance was not affected by depletion of CD25(+) cells or in IL-10-deficient (IL-10(-/-) ) mice, but was compromised in CD25-depleted IL-10(-/-) mice. Our findings suggest that IL-10-producing CD4(+) FoxP3(+) CD25(-) T cells represent an important regulatory cell in the lung.     

The evolution of cardenolide-resistant forms of Na⁺,K⁺ -ATPase in Danainae butterflies

Cardenolides are a class of plant secondary compounds that inhibit the proper functioning of the Na⁺, K⁺‐ATPase enzyme in susceptible animals. Nonetheless, many insect species are able to sequester cardenolides for their own defence. These include butterflies in the subfamily Danainae (Family: Nymphalidae) such as the monarch (Danaus plexippus). Previous studies demonstrated that monarchs harbour an asparagine (N) to histidine (H) substitution (N122H) in the α subunit of Na⁺, K⁺‐ATPase (ATPα) that reduces this enzyme’s sensitivity to cardenolides. More recently, it has been suggested that at ATPα position 111, monarchs may also harbour a leucine (L)/glutamine (Q) polymorphism. This later amino acid could also contribute to cardenolide insensitivity. However, here we find that incorrect annotation of the initially reported DNA sequence for ATPα has led to several erroneous conclusions. Using a population genetic and phylogenetic analysis of monarchs and their close relatives, we show that an ancient Q111L substitution occurred prior to the radiation of all Danainae, followed by a second substitution at the same site to valine (V), which arose before the diversification of the Danaus genus. In contrast, N122H appears to be a recent substitution specific to monarchs. Surprisingly, examination of a broader insect phylogeny reveals that the same progression of amino acid substitutions (Q111L → L111V + N122H) has also occurred in Chyrsochus beetles (Family: Chrysomelidae, Subfamily: Eumolpinae) that feed on cardenolide‐containing host plants. The parallel pattern of amino acid substitution in these two distantly related lineages is consistent with an adaptive role for these substitutions in reducing cardenolide sensitivity and suggests that their temporal order may be limited by epistatic interactions.     

Human CD8⁺ and CD4⁺ T cell memory to lymphocytic choriomeningitis virus infection

Although cellular immunity to acute lymphocytic choriomeningitis virus (LCMV) infection has been well characterized in experimental studies in mice, the T cell response to this virus in humans is incompletely understood. Thus, we analyzed the breadths, magnitudes, and differentiation phenotypes of memory LCMV-specific CD8(+) and CD4(+) T cells in three human donors displaying a variety of disease outcomes after accidental needle stick injury or exposure to LCMV. Although only a small cohort of donors was analyzed at a single time point postinfection, several interesting observations were made. First, we were able to detect LCMV-specific CD8(+) and CD4(+) T cell responses directly ex vivo at 4 to 8 years after exposure, demonstrating the longevity of T cell memory in humans. Second, unlike in murine models of LCMV infection, we found that the breadths of memory CD8(+) and CD4(+) T cell responses were not significantly different from one another. Third, it seemed that the overall CD8(+) T cell response was augmented with increasing severity of disease, while the LCMV-specific CD4(+) T cell response magnitude was highly variable between the three different donors. Next, we found that LCMV-specific CD8(+) T cells in the three donors analyzed seemed to undergo an effector memory differentiation program distinct from that of CD4(+) T cells. Finally, the levels of expression of memory, costimulatory, and inhibitory receptors on CD8(+) and CD4(+) T cell subsets, in some instances, correlated with disease outcome. These data demonstrate for the first time LCMV-specific CD8(+) and CD4(+) T cells in infected humans and begin to provide new insights into memory T cell responses following an acute virus infection.     

Ammonia excretion via Rhcg1 facilitates Na⁺ uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ～ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.     

Cyclic AMP-induced K⁺ secretion occurs independently of Cl⁻ secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.     

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei

The epithelial Na? channels (ENaCs) are present in kidney and contribute to Na? and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.     

Extracellular proton depression of peak and late Na⁺ current in the canine left ventricle

Cardiac ischemia reduces excitability in ventricular tissue. Acidosis (one component of ischemia) affects a number of ion currents. We examined the effects of extracellular acidosis (pH 6.6) on peak and late Na(+) current (I(Na)) in canine ventricular cells. Epicardial and endocardial myocytes were isolated, and patch-clamp techniques were used to record I(Na). Action potential recordings from left ventricular wedges exposed to acidic Tyrode solution showed a widening of the QRS complex, indicating slowing of transmural conduction. In myocytes, exposure to acidic conditions resulted in a 17.3 ± 0.9% reduction in upstroke velocity. Analysis of fast I(Na) showed that current density was similar in epicardial and endocardial cells at normal pH (68.1 ± 7.0 vs. 63.2 ± 7.1 pA/pF, respectively). Extracellular acidosis reduced the fast I(Na) magnitude by 22.7% in epicardial cells and 23.1% in endocardial cells. In addition, a significant slowing of the decay (time constant) of fast I(Na) was observed at pH 6.6. Acidosis did not affect steady-state inactivation of I(Na) or recovery from inactivation. Analysis of late I(Na) during a 500-ms pulse showed that the acidosis significantly reduced late I(Na) at 250 and 500 ms into the pulse. Using action potential clamp techniques, application of an epicardial waveform resulted in a larger late I(Na) compared with when an endocardial waveform was applied to the same cell. Acidosis caused a greater decrease in late I(Na) when an epicardial waveform was applied. These results suggest acidosis reduces both peak and late I(Na) in both cell types and contributes to the depression in cardiac excitability observed under ischemic conditions.     

The interaction between megalin and ClC-5 is scaffolded by the Na⁺-H⁺ exchanger regulatory factor 2 (NHERF2) in proximal tubule cells

Albumin endocytosis in the proximal tubule is mediated by a number of proteins, including the scavenger receptor megalin/cubilin and the PSD-95/Dlg/ZO-1 (PDZ) scaffolds NHERF1 and NHERF2. In addition, in a number of in vitro and in vivo models, the loss of ClC-5 results in a decreased cell surface expression and whole cell level of megalin, suggesting an interaction between these two proteins in vivo. We investigated if ClC-5 and megalin interact directly, and as ClC-5 binds to NHERF2, we investigated if this PDZ scaffold was required for a megalin/ClC-5 complex. GST-pulldown and immunoprecipitation experiments using rat kidney lysate demonstrated an interaction between ClC-5 and megalin, which was mediated by their C-termini. As this interaction may be controlled by a scaffold protein, we characterised any interaction between megalin and NHERF2. Immunoprecipitation experiments indicated that megalin interacts with NHERF2 in vivo, and that this interaction was via an internal NHERF binding domain in the C-terminus of megalin and PDZ2 and the C-terminus of NHERF2. Silencing NHERF2 had no effect on megalin protein levels in the whole cell or plasma membrane. Using siRNA against NHERF2, we demonstrated that NHERF2 was required to facilitate the interaction between megalin and ClC-5. Using fusion proteins, we characterised a protein complex containing ClC-5 and megalin, which is scaffolded by NHERF2, in the absence of any other proteins. Importantly, these observations are the first to describe an interaction between megalin and ClC-5, which is scaffolded by NHERF2 in proximal tubule cells.     

Remodeling of atrial ATP-sensitive K⁺ channels in a model of salt-induced elevated blood pressure

Hypertension is associated with the development of atrial fibrillation; however, the electrophysiological consequences of this condition remain poorly understood. ATP-sensitive K(+) (K(ATP)) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure (BP) leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Elevated BP was induced in mice with a high-salt diet (HS) for 4 wk. High-resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP), and action potential duration at 90% repolarization (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS hearts compared with control hearts. ERP and APD(90) were significantly shorter in the right atrial appendage and left atrial appendage of HS hearts compared with control hearts. Perfusion with 1 μM glibenclamide or 300 μM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared with untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS animals compared with control animals. Sulfonylurea receptor 1 protein expression was increased in the left atrial appendage and right atrial appendage of HS animals (415% and 372% of NS animals, respectively). In conclusion, K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches.     

Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F⁺ cells

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO(3)(-)) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3'-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3'-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK(1)-F(+) cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.     

NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na⁺/K⁺homeostasis in Arabidopsis under salt stress

Maintaining cellular Na(+)/K(+) homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na(+)/K(+) homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na(+) contents, lower K(+) contents, and resultant greater Na(+)/K(+) ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H(2)O(2) can partially reverse the increased effects of NaCl on Na(+)/K(+) ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na(+)/K(+) ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K(+) currents were arrested, and NaCl-promoted increases in cytosolic Ca(2+) and plasma membrane Ca(2+) influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na(+)/K(+) homeostasis, thus improving the salt tolerance of Arabidopsis.     

Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series

Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.     

Regulation of apical H⁺-ATPase activity and intestinal HCO₃⁻ secretion in marine fish osmoregulation

The absorption of Cl(-) and water from ingested seawater in the marine fish intestine is accomplished partly through Cl(-)/HCO(3)(-) exchange. Recently, a H(+) pump (vacuolar-type H(+)-ATPase) was found to secrete acid into the intestinal lumen, and it may serve to titrate luminal HCO(3)(-) and facilitate further Cl(-)/HCO(3)(-) exchange, especially in the posterior intestine, where adverse concentration gradients could limit Cl(-)/HCO(3)(-) exchange. The H(+) pump is expressed in all intestinal segments and in gill tissue of gulf toadfish (Opsanus beta) maintained in natural seawater. After acute transfer of toadfish to 60 ppt salinity, H(+) pump expression increased 20-fold in the posterior intestine. In agreement with these observations was a fourfold-increased H(+)-ATPase activity in the posterior intestine of animals acclimated to 60 ppt salinity. Interestingly, Na(+)-K(+)-ATPase activity was elevated in the anterior intestine and gill, but not in the posterior intestine. Apical acid secretion by isolated intestinal tissue mounted in Ussing chambers fitted with pH-stat titration systems increased after acclimation to hypersalinity in the anterior and posterior intestine, titrating >20% of secreted bicarbonate. In addition, net base secretion increased in hypersalinity-acclimated fish and was ～70% dependent on serosal HCO(3)(-). Protein localization by immunohistochemistry confirmed the presence of the vacuolar-type H(+)-ATPase in the apical region of intestinal enterocytes. These results show that the H(+) pump, especially in the posterior intestine, plays an important role in hypersaline osmoregulation and that it likely has significant effects on HCO(3)(-) accumulation in the intestinal lumen and, therefore, the continued absorption of Cl(-) and water.