Calcium-sensing receptor stimulates luminal K+-dependent H+ excretion in medullary thick ascending limbs of Henle's loop of mouse kidney

The calcium-sensing receptor (CaSR) is known well as a sensor of extracellular calcium for regulating parathyroid hormone secretion. CaSR is located along all nephron segments in the kidney. While hypercalcemia strongly enhances urinary acidification, the relationship between CaSR and acid-base metabolism in the kidney is still uncertain. In the present study, we examined whether CaSR activation caused acid secretion in the medullary thick ascending limb (mTAL), which is one of the major nephron segments involved in both mineral and acid-base regulation. The effects of a potent calcimimetic neomycin (Neo) on intracellular pH (pHi) were analyzed in the in vitro miroperfused mouse mTALs. The mTALs were incubated with 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluoresceine-acetoxymethylester (BCECF-AM) for microfluorescent pHi measurements. In HCO(3)(-)/CO(2)-buffered solution, the steady-state pHi was 7.17 +/- 0.01 (n = 19). Basolateral Neo at 0.4 mM in basolateral side significantly alkalinized the mTAL cells to 7.28 +/- 0.02 (n = 19), while Neo in the lumen had no effect on pHi. Neo in the basolateral side alkalinized the mTALs in the absence of ambient Na(+) and the presence of H(+)-ATPase inhibitor bafilomycin in the lumen, indicating that the effect of Neo is unrelated to Na(+)-dependent acid-base transporters such as Na(+)-H(+) exchangers and Na(+)-HCO(3)(-) cotransporter, or to luminal H(+)-ATPase. In contrast, the effect of Neo on pHi was inhibited by K(+) removal or treatment with specific H(+)-K(+)-ATPase (HKa) inhibitors, ouabain and Sch-28080, in the lumen. Our results suggest that hypercalcemia induces urinary acidification partly by stimulating luminal K(+)-dependent H(+)-excretion via CaSR in mouse mTALs.     

Intracellular Mg2+ and magnesium depletion in isolated renal thick ascending limb cells.

Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura-2, was used to characterize intracellular Mg2+ concentration ([Mg2+]i) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Mg2+]i was 0.52 +/- 0.02 mM, which was approximately 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2+]i, 0.48 +/- 0.02, in the normal range. However, cells cultured in nominally magnesium-free media possessed [Mg2+]i, 0.27 +/- 0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19 +/- 0.03 and low Mg, 0.35 +/- 0.01 nmol.mg-1 protein.min-1) as assessed by 28Mg uptake. Mg(2+)-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2+ refill rate was assessed by fluorescence. [Mg2+]i returned to normal basal levels, 0.53 +/- 0.03 mM, with a refill rate of 257 +/- 37 nM/s. Mg2+ entry was not changed by 5.0 mM Ca2+ or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ approximately La3+ approximately Gd3+ approximately Zn2+ approximately Be2+ at 2 mM. Intracellular Ca2+ and 45Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2+. Mg2+ uptake was inhibited by nifedipine (117 +/- 20 nM/s), verapamil (165 +/- 34 nM/s), and diltiazem (194 +/- 19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366 +/- 71 nM/s). These antagonists and agonists were reversible with removal and [Mg2+]i subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2+ and highly regulated. These entry pathways are likely involved with renal Mg2+ homeostasis.     

Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO(3)(-) absorption in renal thick ascending limb

The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.     

Electroneutral K+/HCO3- cotransport in cells of medullary thick ascending limb of rat kidney.

The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H+ secretion and basolateral HCO3- transport into the peritubular space. To characterize HCO3- transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3-/CO2-containing solutions and then abruptly diluted into HCO3-/CO2-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3-, was inhibited by 10(-4) M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl- or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl- removal from or addition of 2 mM barium into the external medium did not affect HCO3(-)-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3- transport was electroneutral. The HCO3(-)-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion. Finally, as measured by use of a K(+)-selective extracellular electrode, a component of K+ efflux out of the cells was HCO3- dependent and DIDS sensitive. The results provide evidence for an electroneutral K+/HCO3- cotransport in rat MTAL cells.     

Basolateral membrane H/OH/HCO3 transport in the rat cortical thick ascending limb. Evidence for an electrogenic Na/HCO3 cotransporter in parallel with a Na/H antiporter.

Mechanisms involved in basolateral H/OH/HCO3 transport in the in vitro microperfused rat cortical thick ascending limb were examined by the microfluorometric determination of cell pH using (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein. The mean cell pH in this segment perfused with 147 mM sodium and 25 mM HCO3 at pH 7.4 was 7.13 +/- 0.02 (n = 30). Lowering bath HCO3 from 25 to 5 mM (constant PCO2 of 40 mmHg) acidified the cells by 0.31 +/- 0.02 pH units at a rate of 0.56 +/- 0.08 pH units/min. Removal of bath sodium acidified the cells by 0.28 +/- 0.03 pH units at a rate of 0.33 +/- 0.04 pH units/min. The cell acidification was stilbene inhibitable and independent of chloride. There was no effect of bath sodium removal on cell pH in the absence of CO2/HCO3. Depolarization of the basolateral membrane (step increase in bath potassium) independent of the presence of chloride. Cell acidification induced by bath sodium removal persisted when the basolateral membrane was voltage clamped by high potassium/valinomycin. Although these results are consistent with a Na/(HCO3)n greater than 1 cotransporter, a Na/H antiporter was also suggested: 1 mM bath amiloride inhibited the cell pH defense against an acid load (rapid ammonia washout), both in the presence and absence of CO2/HCO3, and inhibited the cell acidification induced by bath sodium reduction from 50 to 0 mM. In conclusion, an electrogenic Na/(HCO3)n greater than 1 cotransporter in parallel with a Na/H antiporter exist on the basolateral membrane of the rat cortical thick ascending limb.     

Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.     

Effects of MK-196 and furosemide on rat medullary thick ascending limbs of Henle in vitro

The effects of the diuretic agents, MK-196 (6,7-dichloro-2-methyl-1-oxo-2-phenyl-5-indanyloxy acetic acid) and furosemide on function of medullary thick ascending limb (tALH) isolated from the rat kidney were studied. tALH were dissected from whole kidneys of male Sprague-Dawley rats which were first perfused in vitro in a recirculating system for 10 to 15 min with a solution containing collagenase. Standard techniques for dissection and perfusion of tALH were followed. Control transepithelial voltage averaged +5.5 +/- 0.5 mV (n = 25). Drug concentration causing 50% inhibition of the voltage (EC50) was determined. EC50 for both MK-196 and furosemide in rat tALH was 8 X 10(-6) M (EC50 for MK-196 in rabbit tALH ws 7 X 10(-5) M). In other experiments, net chloride transport (JCl) in rat tALH averaged 142 +/- 19 pmol.mm-1.min-1 (n = 12), a value nearly twice that reported for the rabbit. In the rat, MK-196 (2 X 10(-4) M) reduced the voltage by 79 +/- 2% and JCl by 76 +/- 13% (n = 6). The same concentration of furosemide inhibited the voltage by 78 +/- 8% and the JCl by 104 +/- 4%(n = 5). Results indicate that: 1) rat tALHs dissected from collagenase-treated kidneys have functional characteristics similar to those reported for the rabbit tALH; and 2) the rat tALH is equally sensitive to MK-196 and furosemide, drugs which act to selectively inhibit active chloride transport.     

Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways

We have developed an immunoadsorption technique for quantitating EGTA-resistant gelsolin/actin complexes in macrophages extracted with Triton X-100. We report here that the proportion of gelsolin complexed irreversibly to actin is low in freshly harvested macrophages. The amount of the EGTA-resistant complex increases spontaneously during incubation of the cells in suspension at 37 degrees C, or after exposure to the Ca2+ ionophore ionomycin. On the other hand, exposure of suspended cells to the chemotactic oligopeptide, FMLP, or plating of the cells onto tissue culture dishes causes the EGTA-resistant complex to dissociate rapidly. Plating even prevents Ca2+ ionomycin-treated cells with elevated intracellular Ca2+ from inducing this complex. Therefore, our results suggest that macrophages possess a mechanism, not directly involving Ca2+, for dissociating actin/gelsolin EGTA-resistant complexes. This mechanism may be a Ca2+-independent signal for leukocyte activation.     

Endogenous prostaglandin E2 mediates inhibition of rat thick ascending limb Cl reabsorption in chronic hypercalcemia.

The hypothesis that endogenous PGE2 mediates defective thick ascending limb (TAL) Cl reabsorption (percent delivered load: FRCl%) in rats with vitamin D-induced chronic hypercalcemia (HC) was tested by measuring FRCl% in loop segments microperfused in vivo in HC and control rats treated acutely with indomethacin (Indo) or its vehicle, and obtaining the corresponding outer medullary [PGE2]. Microperfusion conditions were developed in which FRCl% was exclusively furosemide sensitive. To determine the cellular mechanism, tubules were perfused acutely with forskolin (FSK), cAMP, or the protein kinase C inhibitor staurosporine (SSP). Outer medullary [PGE2] in HC rats was 9 to 10 times greater than control and could be normalized by Indo. FRCl% was 20% lower in HC rats infused with vehicle, and Indo, FSK, and cAMP returned FRCl% to normal despite sustained HC. Indo or FSK had no effect on FRCl% in control rats and Indo did not prevent inhibition of FRCl% by luminal PGE2 (1 microM). Luminal SSP (10(-7), 10(-8) M) in HC did not return FRCl% to control values. We conclude that impaired TAL FRCl% in HC occurs at a pre-cAMP site and is due to endogenous PGE2 and not to HC.     

Synthesis and structural characterization of CO2-soluble oxidizers [Bu4N]BrO3 and [Bu4N]ClO3 and their dissolution in cosolvent-modified CO2 for reservoir applications

CO₂ utilization in upsteam oil and gas applications requires CO₂-soluble additives such as polymers, surfactants, and other components. Here we report the facile synthesis of CO₂-soluble oxidizers composed of judiciously selected organic cations paired with oxidizing anions. [Bu₄N]BrO₃ and [Bu₄N]ClO₃ are prepared using a double displacement synthetic strategy, whereby the crystalline product is readily obtained in high yield and structurally characterized using single-crystal X-ray diffraction. The facility of the approach is demonstrated through the preparation of several additional alkylammonium bromate compounds. Static solubility studies using a high-pressure cell with viewing windows showed that tetrabutylammonium compounds could be solubilized using cosolvent-modified CO₂. Using 4 mol% ethanol as cosolvent, >3 mM [Bu₄N]BrO₃ could be dissolved in CO₂, while ∼0.75 mM [Bu₄N]ClO₃ could be dissolved in the same solvent system. The solubility properties of [Bu₄N]BrO₃ along with its thermal stability up to ∼200 °C suggest that it is a promising oilfield oxidizer that can be utilized in subterranean CO₂ applications.

Bromate and chlorate salts were hydrophobically modified with tetrabutylammonium to yield oxidizers that are soluble in CO₂-cosolvent mixtures.     

Effects of atrial natriuretic peptide and vasopressin on chloride transport in long- and short-looped medullary thick ascending limbs.

Recent studies have suggested a selective effect of atrial natriuretic peptide (ANP) in regulating NaCl reabsorption in juxtamedullary nephrons. We examined (a) functional differences between medullary thick ascending limbs from long and short loops of Henle (lMAL and sMAL, respectively) and (b) the interaction of ANP and arginine vasopressin (AVP) on Cl- transport (JCl) in these two segments. AVP-, glucagon-, and calcitonin-stimulated cAMP accumulation was higher in lMAL than in sMAL. 10(-10) M AVP increased JCl in lMAL but not in sMAL. ANP-stimulated cGMP production was higher in lMAL than in sMAL. 10(-10) and 10(-8) M ANP inhibited AVP-stimulated JCl in lMAL by 26-30% (from 70.3 +/- 11.4 to 51.7 +/- 13.6 pmol/mm per min and from 88.1 +/- 10.1 to 61.8 +/- 11.7 pmol/mm per min, respectively), and this effect was mimicked by 10(-5) to 10(-4) M cGMP. This effect of ANP in lMAL could account for a large part of the ANP-induced natriuresis and diuresis in vivo, in that the rate of NaCl reabsorption in MAL is the largest among distal nephron segments, providing the chemical potential energy for the renal countercurrent multiplication system.     

Effects of inhibition of RBC HCO3-/Cl- exchange on CO2 excretion and downstream pH disequilibrium in isolated rat lungs.

To determine the effects of the speed of the erythrocyte membrane chloride shift on pulmonary gas transfer, CO2 exchange and the kinetics of pH equilibration were measured with isolated rat lungs perfused with 20% suspensions of human erythrocytes. The lungs were ventilated with room air, and the inflowing perfusate blood gases were similar to those in mixed venous blood in vivo. All experiments were performed at 37 degrees C. Rates of CO2 excretion were determined by measuring the fraction of CO2 in mixed expired gas in a steady state. The time-course of extracellular pH equilibration in the effluent perfusate was measured in a downstream stopflow pH electrode apparatus. CO2 excretion was reduced by approximately 16% when the lungs were perfused with suspensions containing erythrocytes whose HCO-3/Cl- exchange rates was inhibited, compared with CO2 excretion when the lungs were perfused with normal erythrocyte suspensions. A fall of 0.06 in effluent perfusate extracellular pH was noted during perfusion with inhibited erythrocyte suspensions, in contrast to no observable downstream pH change during perfusion with normal erythrocyte suspensions. These results are in close agreement with the predictions of a theoretical model. Our observations suggest that CO2 transfer in capillary beds will be adversely affected in vivo when the rate of the erythrocyte HCO-3/Cl- exchange is abnormally low. Since a number of commonly used drugs are known to inhibit the chloride shift in human erythrocytes, these findings may have important clinical implications, especially in patients with impaired lung function.     

Correction: Synthesis and structural characterization of CO2-soluble oxidizers [Bu4N]BrO3 and [Bu4N]ClO3 and their dissolution in cosolvent-modified CO2 for reservoir applications

Correction for ‘Synthesis and structural characterization of CO₂-soluble oxidizers [Bu₄N]BrO₃ and [Bu₄N]ClO₃ and their dissolution in cosolvent-modified CO₂ for reservoir applications’ by Katherine L. Hull et al., RSC Adv., 2020, 10, 44973–44980, DOI: 10.1039/D0RA09563J.

The authors regret that the value for the solubility of [Bu₄N]BrO₃ in the last sentence of the Results and discussion section was given incorrectly.In the sentence beginning “Notably, the solubility of [Bu₄N]BrO₃ achieved…” on page 44978, the corrected sentence should read “Notably, the solubility of [Bu₄N]BrO₃ achieved (>0.12 wt%) with ethanol cosolvent significantly exceeds the typical concentrations utilized in the application (∼0.03 wt%)”.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced renal cell apoptosis

It has been demonstrated recently that rabbit renal proximal tubule cells (RPTC) express a novel Ca(2+)-independent phospholipase A(2) (iPLA(2)) whose activity localizes to the endoplasmic reticulum (ER-iPLA(2)) and is similar to group VIB PLA(2). In this study, the expression of group VIB PLA(2) was examined and the role of ER-iPLA(2) in cisplatin-induced apoptosis was determined. Cisplatin induced both time- and concentration-dependent RPTC apoptosis as determined by p53 nuclear localization, annexin V staining, caspase 3 activity, and chromatin condensation. Inhibition of ER-iPLA(2) with bromoenol lactone (5 microM) reduced cisplatin-induced annexin V binding 40%, chromatin condensation 55%, and caspase 3 activity 42%, but had no effect on p53 nuclear localization. Treatment of RPTC with the protein kinase C stimulator phorbol 12-myristate 13-acetate increased the activity of ER-iPLA(2) 2-fold and increased cisplatin-induced RPTC apoptosis. These studies demonstrate that group VIB PLA(2) is expressed in RPTC and suggest that RPTC ER-iPLA(2) is the rabbit homolog of group VIB PLA(2). These data also demonstrate that ER-iPLA(2) acts downstream of p53 and upstream of caspase 3 to mediate cisplatin-induced RPTC apoptosis. Finally, ER-iPLA(2) seems to be regulated by protein kinase C.     

Heterogeneous photocatalytic performances of CO2 reduction based on the [Emim]BF4 + TEOA + H2O system

The photochemical reduction of CO₂ was studied in a 1-ethyl-3-methylimidazolium tetrafluoroborate, triethanolamine and water ([Emim]BF₄ + TEOA + H₂O) system under visible light irradiation. The integration of CdS and the Co–bpy complex, which acted as a photocatalyst and cocatalyst, respectively, was employed as an efficient catalytic system for the CO₂-to-CO conversion. The utilization of [Emim]BF₄ and water took advantage of their green properties. The amount of CO production showed that the test medium containing 10 vol% H₂O was favourable for the catalytic performance of the CO₂ reduction. In order to further study the factors that influenced the current system, the physical and spectroscopy properties were characterized by altering the composition ratio of the ingredients. Relevant parameters, including the viscosity, conductivity, solubility and coordination, were adjusted using the ratio of the H₂O/[Emim]BF₄ addition, resulting in a different catalytic performance. All of these attempts led to an optimal reaction condition for the CO₂ reduction process.

The photochemical reduction of CO₂ was studied in a 1-ethyl-3-methylimidazolium tetrafluoroborate, triethanolamine and water ([Emim]BF₄ + TEOA + H₂O) system under visible light irradiation.     

The amiodarone derivative KB130015 [2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] induces an Na+-dependent increase of [Ca2+] in ventricular myocytes

KB130015 [KB; 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] is a novel amiodarone derivative designed to retain the antiarrhythmic effects without the side effects. Unlike amiodarone, KB slows Na(+) current inactivation and could, via an increase in [Na(+)](i), potentially lead to Ca(2+) overload. Therefore, we studied the effects of KB on Na(+) and Ca(2+) handling in single pig ventricular myocytes using the whole-cell ruptured patch-clamp technique and K(5)fluo-3 as [Ca(2+)](i) indicator. KB at 10 microM did not prolong action potential duration but slightly increased the early plateau; spontaneous afterdepolarizations were not observed. The amplitude of the [Ca(2+)](i) transient was larger (434.9 +/- 37.2 versus 326.8 +/- 39.8 nM at baseline, n = 13, P < 0.05), and the time to peak [Ca(2+)](i) was prolonged. During voltage-clamp pulses, [Ca(2+)](i) transient peak was also larger (578.1 +/- 98.9 versus 346.4 +/- 52.6 nM at baseline, P < 0.05). Although L-type Ca(2+) current was reduced (by 21.9% at +10 mV, n = 9, P < 0.05), sarcoplasmic reticulum Ca(2+) content was significantly enhanced with KB. Forward Na(+)/Ca(2+) exchange was significantly decreased after KB application, but reverse mode of the Na(+)/Ca(2+) exchanger was significantly larger, suggesting an increase in [Na(+)](i) with KB. This was confirmed by a 2-fold increase of the [Na(+)]-dependent current generated by the Na/K-ATPase (from 0.17 +/- 0.02 to 0.38 +/- 0.06 pA/pF, P < 0.05). In conclusion, as predicted from the slowing of I(Na) inactivation, KB130015 leads to an increase in [Na(+)](i) and consequently in cellular Ca(2+) load. This effect is partially offset by a decrease in I(CaL) resulting in a mild inotropic effect without the signs of Ca(2+) overload and related arrhythmias usually associated with Na(+) channel openers.     

Synthesis of tetrahydrochromenes and dihydronaphthofurans via a cascade process of [3 + 3] and [3 + 2] annulation reactions: mechanistic insight for 6-endo-trig and 5-exo-trig cyclisation

Substituted tetrahydrochromenes and dihydronaphthofurans are easily accessible by the treatment of β-tetralone with trans-β-nitro styrene derived Morita–Baylis–Hillman (MBH) acetates through a formal [3 + 3]/[3 + 2] annulation. The reaction proceeds through a cascade Michael/oxa-Michael pathway with moderate to good yields. A DFT study was carried out to account for the formation of the corresponding six and five-membered heterocycles via 6-endo-trig and 5-exo-trig cyclization.

A [3 + 3] and [3 + 2] annulation strategy using nitrostyrene derived MBH primary and secondary nitro allylic acetate for the construction of tetrahydrochromenes and dihydronaphthofurans at room temperature.

The ability to synthesize diverse molecules utilizing nitro allylic MBH acetates in various cascade reactions has received considerable interest.¹ A few molecules synthesized using nitro allylic acetates have shown promising cytotoxic, trypanocidal and AchE inhibition² activity in pharmaceutical and medicinal chemistry. Nitro allylic MBH acetates have been used as main precursors in organocatalysis³ and heterocyclic chemistry,⁴ and as bicyclic skeletons⁵ for the construction of elegant building blocks like tetrahydro-pyranoquinolinones,⁶ sulfonyl furans,⁷ pyranonaphthoquinones,⁸ arenopyrans/arenylsulfanes,⁹ triazoles,^10a tetrasubstituted furans,^10b fused furans,^10c,d tetrasubstituted pyrroles,^11a benzofuranones,^11b and tetrahydropyrano scaffolds/pyranocoumarins.¹² The nitro allylic MBH-acetates can also undergo asymmetric benzylic^13a and allylic alkylation^13b reactions as well as kinetic resolution [KR]^13c,d under normal conditions. These acetates undergo a range of cascade [2 + 3],¹⁴ [3 + 2]¹⁵ and [3 + 3]¹⁶ ring annulation reactions using different substrates. They have been widely utilized in [3 + 2]^17a and [3 + 3]^17b annulation reactions due to their unique nature of 1,2-/1,3-biselectrophilic reactivity to form either five or six membered rings depending on the nature of nucleophiles employed in the reaction^17c,d These adducts are also stable under NHC catalytic conditions to yield cyclopentanes.¹⁸Peng-Fei Xu et al. (Scheme 1, eqn (a)) synthesised tetrahydropyranoindoles through organocatalytic asymmetric C–H functionalization of indoles via [3 + 3] annulation through 6-endo trig cyclization.¹⁹ The Namboothiri group recently developed a metal free regioselective synthesis of α-carbolines via [3 + 3] annulation involving secondary MBH acetate (Scheme 1, eqn (b)).²⁰ Previously, our group carried out a [3 + 3] cyclization reaction of β-naphthol with primary MBH acetate to study the scope of S_N2′ vs. S_N2 reaction.²¹ With our ongoing interest in using nitro styrene derived MBH adducts²² explored the reactivity of primary and secondary MBH acetate with β-tetralone 1 as our model reaction. Initially, the reaction carried out using β-tetralone 1 with primary MBH-acetate 2, predominantly gave a tetrahydrochromene 3via [3 + 3] annulation involving 6-endo trig cyclization through Michael/oxa-Michael cascade process. The possible dihydronaphthofuran product was not observed under the present conditions as primary MBH acetate 2 acts as 1,3-biselectrophile instead of 1,2-biselectrophile (Scheme 1, eqn (c)).Open in a separate windowScheme 1[3 + 3] and [3 + 2] annulation reactions using 1°- and 2°-nitro allylic MBH acetate.On the other hand, the reaction of β-tetralone 1 with secondary MBH acetate 4 gave dihydronaphthofuran instead of the possible tetrahydrochromene product due to the 1,2-biselectrophile nature of secondary MBH acetate (Scheme 1, eqn (d)). The formation of dihydronaphthofuran 5 occurs in an S_N2′ fashion via [3 + 2] annulation involving 5-exo-trig cyclization through Michael followed by intramolecular oxa-Michael reaction with the elimination of HNO₂. Subsequently, we have carried out a DFT calculation to prove the formation of tetrahydrochromene 3 using primary MBH acetate 2 and dihydronaphthofuran 5 in the case of secondary MBH acetate 4.Initially, we carried out the optimization conditions for constructing tetrahydrochromenes 3a using β-tetralone 1 with MBH nitro allylic primary acetate 2a with different bases and solvents. Reaction with organic base, i.e. DABCO using a polar aprotic solvent such as acetonitrile at room temperature gave the desired product in 27% (23 (CCDC-2149875) (

Inhibition of Ca2+-independent phospholipase A2 decreases prostate cancer cell growth by p53-dependent and independent mechanisms

The mechanisms by which Ca(2+)-independent phospholipase A(2) (iPLA(2)) mediates cell growth in p53-positive LNCaP and p53-negative PC-3 prostate cancer cell lines were studied. Exposure of cells to the iPLA(2) selective inhibitor bromoenol lactone (BEL; 0-20 microM) induced concentration- and time-dependent decreases in cell growth based on 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide staining and cell number. Decreased cell growth was not caused by cell death as BEL exposure did not alter nuclear morphology or increase annexin V (apoptotic cell marker) or propidium iodide (necrotic cell marker) staining after 48 h. Decreased growth correlated to a G(1)/G(0) arrest in LNCaP cells and aG(2)/M arrest in PC-3 cells. In LNCaP cells, G(1) arrest was preceded by time- (0-48 h) and concentration-dependent (0-10 microM) increases in the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21. Increases in p53 expression preceded increases in p21 expression by 8 h. In LNCaP cells, BEL treatment decreased the expression of the p53 antagonist Mdm2, while increasing Akt phosphorylation. BEL treatment also increased Akt phosphorylation in PC-3 cells, but Mdm2 was not detected. The ability of BEL to increase Akt phosphorylation was inhibited by the phosphoinositide 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. BEL treatment also decreased agonist-induced activation of the epidermal growth factor receptor. These data suggest that inhibition of iPLA(2) decreases prostate cancer cell growth by p53-dependent and independent mechanisms. Furthermore, alterations in Mdm2 and epidermal growth factor receptor activation following BEL exposure suggest novel roles for iPLA(2) in prostate cancer cell signaling.