GABA(B) receptor-mediated presynaptic inhibition of glycinergic transmission onto substantia gelatinosa neurons in the rat spinal cord

The GABA_B receptor-mediated presynaptic inhibition of glycinergic transmission was studied from young rat substantia gelatinosa (SG) neurons using a conventional whole-cell patch clamp technique. Action potential-dependent glycinergic inhibitory postsynaptic currents (IPSCs) were recorded from SG neurons in the presence of 3 mM kynurenic acid and 10 μM SR95531. In these conditions, baclofen (30 μM), a selective GABA_B receptor agonist, greatly reduced the amplitude of glycinergic IPSCs and increased the paired-pulse ratio. Such effects were completely blocked by 3 μM CGP55845, a selective GABA_B receptor antagonist, indicating that the activation of presynaptic GABA_B receptors decreases glycinergic synaptic transmission. Glycinergic IPSCs were largely dependent on Ca²⁺ influxes passing through presynaptic N- and P/Q-type Ca²⁺ channels, and these channels contributed equally to the baclofen-induced inhibition of glycinergic IPSCs. However, the baclofen-induced inhibition of glycinergic IPSCs was not affected by either 100 μM SQ22536, an adenylyl cyclase inhibitor, or 1 mM Ba²⁺, a G-protein coupled inwardly rectifying K⁺ channel blocker. During the train stimulation (10 pulses at 20 Hz), which caused a marked synaptic depression of glycinergic IPSCs, baclofen at a 30 μM concentration completely blocked glycinergic synaptic depression, but at a 3 μM concentration it largely preserved glycinergic synaptic depression. Such GABA_B receptor-mediated dynamic changes in short-term synaptic plasticity of glycinergic transmission onto SG neurons might contribute to the central processing of sensory signals.     

Dopamine Stimulation of Active Na and Cl Absorption in Rabbit Ileum: INTERACTION WITH α2-ADRENERGIC AND SPECIFIC DOPAMINE RECEPTORS

The effects of dopamine on active intestinal ion transport have been evaluated. An epithelial sheet preparation of rabbit ileum was used in vitro with the Ussing chamber-voltage clamp technique. Dopamine, in the presence of 1 mM ascorbic acid, added to the serosal bathing solution caused a dose-dependent decrease in short-circuit current, with a half-maximal effect at 1.2 μM and maximal effect of −50 μA/cm² at 50 μM; dopamine decreased the potential difference, and increased the conductance and net Na and net Cl absorption. There was no effect on the residual ion flux. Dopamine did not alter the change in short-circuit current caused by mucosal glucose (10 mM) or serosal theophylline (10 mM). Mucosal dopamine had no effect. The effect of dopamine on short-circuit current was inhibited by the dopamine antagonists haloperidol and domperidone and the α₂-adrenergic antagonist yohimbine; there was no effect of the α₁-antagonist prazosin and the β-antagonist propranolol. In addition, the α₂-adrenergic agonist clonidine, but not the α₁-agonist methoxamine caused a dose-dependent decrease in short-circuit current. The ileal effects of dopamine did not occur via conversion into norepinephrine or release of norepinephrine from the peripheral nerves since “peripheral sympathectomy” with 6-hydroxydopamine did not alter the dopamine-induced change in ileal short-circuit current. The dopamine effects were not associated with a change in basal ileal cyclic AMP content but were associated with a decrease in total ileal calcium content as measured by atomic absorption spectrometry and as estimated by ⁴⁵Ca⁺⁺ uptake. The decrease in calcium content could be attributed to a dopamine-induced decrease in ⁴⁵Ca⁺⁺ influx from the serosal surface. Because of the presence of dopamine in ileal mucosa and these effects on ileal electrolyte transport, it is possible that dopamine may be involved in the physiologic regulation of active intestinal electrolyte absorption.     

Relationship between Platelet Secretion and Prothrombin Cleavage in Native Whole Blood

To determine the relationship between platelet secretion and prothrombin conversion in whole blood, the release of platelet factor 4 and the generation of a X_a-specific cleavage product of prothrombin, fragment 1 + 2, were measured during the coagulation of whole blood. There was a parallel increase in the concentration of the two proteins. Over the first 5 min of incubation, platelet factor 4 concentration increased 6 ng/ml per min, and after 6-7 min, the rate of release increased to 750 ng/ml per min. Over the initial 5-7 min of incubation, fragment 1 + 2 concentration increased 1.5 pmol/ml per min with a subsequent increase of 45 pmol/ml per min. Incubation with 10 μM prostaglandin E₁ or 15 μM prostaglandin I₂ inhibited secretion of platelet factor 4 and delayed the onset of the rapid phase of fragment 1 + 2 generation by 8 min, while stimulation of platelet secretion with 1 μg/ml collagen suspension enhanced production of fragment 1 + 2. The addition of either 10 μM epinephrine or 100 ng/ml collagen suspension to whole blood did not affect either platelet factor 4 release or fragment 1 + 2 generation, although the combination of 3 μM epinephrine and 100 ng/ml collagen suspension enhanced platelet release and prothrombin cleavage.     

Selective determination of Ag+ in the presence of Cd2+, Hg2+ and Cu2+ based on their different interactions with gold nanoclusters

In this work, a fluorescence method was developed for selective detection of Ag⁺ in the presence of Cd²⁺, Hg²⁺, and Cu²⁺ based on gold nanoclusters (AuNCs). That is, bovine serum albumin (BSA) templated AuNCs with double emission peaks were synthesized using BSA as a protective agent. AuNCs with uniform distribution and average size between 2.0 and 2.2 nm were synthesized using a green and simple method, and showed bright orange-red fluorescence under ultraviolet light. AuNCs have two emission peaks at 450 nm and 630 nm with an excitation wavelength of 365 nm. Under alkaline conditions, Cd²⁺ can combine with the surface sulfhydryl groups of BSA–AuNCs to form Cd–S bonds, which cause AuNCs to aggregate, resulting in an increase in fluorescence intensity at 630 nm. Conversely, due to the d¹⁰–d¹⁰ metal affinity interaction, the addition of Hg²⁺ can reduce the fluorescence peak at 630 nm. Ag⁺ was reduced to Ag⁰ by gold nuclei in AuNCs, forming a stable hybrid Au@ AgNCs species with blue-shifted and enhanced fluorescence. Finally, the paramagnetic behavior of Cu²⁺ combined with BSA causes the excited electrons of the gold cluster to lose their energy via ISC, eventually leading to simultaneous quenching of the two emission peaks. The results show that the limit of detection (LOD) of Ag⁺, Hg²⁺, Cd²⁺ and Cu²⁺ is 1.19 μM, 3.39 μM, 1.83 μM and 5.95 μM, respectively.

A fluorescence method was developed for selective detection of Ag⁺ in the presence of Cd²⁺, Hg²⁺, and Cu²⁺ based on gold nanoclusters. The limit of detection for Ag⁺, Hg²⁺, Cd²⁺ and Cu²⁺ is 1.19 μM, 3.39 μM, 1.83 μM and 5.95 μM, respectively.     

Rapid quantitation of multiple ions released from HeLa cells during emodin induced apoptosis by low-cost capillary electrophoresis with capacitively coupled contactless conductivity detection

Change in cation concentration, including that of potassium and sodium, is characteristic of apoptosis, therefore it is significant to detect cation concentration changes. In this work a rapid, sensitive, and practical method was developed for the determination of Na⁺ and K⁺ concentration in HeLa cells during emodin induced apoptosis by a low-cost capillary electrophoresis device with capacitively coupled contactless conductivity detection (CE-C⁴D). Under the optimized conditions, both ions were baseline separated in 4 min with 40 mM MES/40 mM His containing 1 mM 18-crown-6 as the separation buffer at pH 6.0. The limit of detections (LODs) and limit of quantifications (LOQs) were 0.47–1.15 μM and 1.58–3.86 μM, respectively. The precision for migration times and peak areas was below 0.56% and 3.74%, respectively. The data proved that the concentration of cations in cells can be accurately quantified. It was found that the K⁺ concentration decreased from 82.2 μM to 52.7 μM, and the Na⁺ concentration increased from 62.4 μM to 127.2 μM during the process of apoptosis when the cell density was 1 × 10⁵ cells per mL. The low-cost CE-C⁴D provides a convenient way to decipher the interaction of Na⁺ and K⁺ in the regulation of cell apoptosis.

Change in cation concentration, including that of potassium and sodium, is characteristic of apoptosis, therefore it is significant to detect cation concentration changes.     

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane–associated ATP-sensitive K⁺ (K_ATP) channels of pancreatic β cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of K_ATP channels on loss of mouse β cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of K_ATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1^–/– islets was less susceptible to oxidative stress induced by the oxidant H₂O₂. This was likely, at least in part, a result of the reduced ability of H₂O₂ to hyperpolarize plasma membrane potential and reduce cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) in the Sur1^–/– β cells. Remarkably, Sur1^–/– β cells were less prone to apoptosis induced by H₂O₂ or an NO donor than WT β cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1^–/– cells to H₂O₂-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca²⁺]_c. Sur1^–/– mice were less susceptible than WT mice to streptozotocin-induced β cell destruction and subsequent hyperglycemia and death, which suggests that loss of K_ATP channel activity may protect against streptozotocin-induced diabetes in vivo.     

An investigation of the metabolic activity,isozyme contribution,species differences and potential drug–drug interactions of PI-103, and the identification of efflux transporters for PI-103-O-glucuronide in HeLa1A9 cells

PI-103 is a phosphatidylinositol 3-kinase inhibitor that includes multiple receptor affinity modifications, and it is also a therapeutic drug candidate primarily for human malignant tumors. However, its metabolic fate and potential drug–drug interactions involving human cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT) enzymes remain unknown. In this study, our results demonstrated that the intrinsic clearance (CL_int) values of oxidated metabolite (M1) in human liver microsomes (HLM) and human intestine microsomes (HIM) were 3.10 and 0.08 μL min⁻¹ mg⁻¹, respectively, while PI-103 underwent efficient glucuronidation with CL_int values of 15.59 and 211.04 μL min⁻¹ mg⁻¹ for mono-glucuronide (M2) by HLM and HIM, respectively. Additionally, reaction phenotyping results indicated that CYP1A1 (51.50 μL min⁻¹ mg⁻¹), 1A2 (46.96 μL min⁻¹ mg⁻¹), and UGT1A1 (18.80 μL min⁻¹ mg⁻¹), 1A7 (8.52 μL min⁻¹ mg⁻¹), 1A8 (8.38 μL min⁻¹ mg⁻¹), 1A9 (34.62 μL min⁻¹ mg⁻¹), 1A10 (107.01 μL min⁻¹ mg⁻¹) were the most important contributors for the oxidation and glucuronidation of PI-103. Chemical inhibition assays also suggest that CYP1A2 and UGT1A1, 1A9 play a predominant role in the metabolism of PI-103 in HLM. Significant activity correlations were detected between phenacetin-N-deacetylation and M1 (r = 0.760, p = 0.004) as well as β-estradiol-3-O-glucuronide and M2 (r = 0.589, p = 0.044), and propofol-O-glucuronidation and M2 (r = 0.717, p = 0.009). Furthermore, the metabolism of PI-103 revealed marked species differences, and dogs, rats, mice and mini-pigs were not the appropriate animal models. Gene silencing of breast cancer resistance protein (BCRP) or multidrug resistance-associated protein (MRPs) transporter results indicated that M2 was mainly excreted by BCRP, MRP1 and MRP4 transporters. Moreover, PI-103 displayed broad-spectrum inhibition towards human CYPs and UGTs isozymes with IC₅₀ values ranging from 0.33 to 6.89 μM. Among them, PI-103 showed potent non-competitive inhibitory effects against CYP1A2, 2C19, 2E1 with IC₅₀ and K_i values of less than 1 μM. In addition, PI-103 exhibited moderate non-competitive inhibition against UGT1A7, 2B7, and moderate mixed-type inhibition towards CYP2B6, 2C9 and UGT1A3. Their IC₅₀ and K_i values were 1.16–6.89 and 0.56–5.64 μM, respectively. In contrast, PI-103 could activate the activity of UGT1A4 in a mechanistic two-site model with a K_i value of 13.76 μM. Taken together, PI-103 was subjected to significant hepatic and intestinal metabolism. CYP1A1, 1A2 and UGT1A1, 1A7, 1A8, 1A9, 1A10 were the main contributing isozymes, whereas BCRP, MRP1 and MRP4 contributed most to the efflux excretion of M2. Meanwhile, PI-103 had a potent and broad-spectrum inhibitory effect against human CYPs and UGTs isozymes. These findings could improve understanding of the metabolic fates and efflux transport of PI-103. The inhibited human CYP and UGT activities could trigger harmful DDIs when PI-103 is co-administered with clinical drugs primarily cleared by these CYPs or UGTs isoforms. Additional in vivo studies are required to evaluate the clinical significance of the data presented herein.

Metabolic activity and disposition characteristics of PI-103.     

Evidence for the Involvement of Na/Ca Exchange in Glucose-induced Insulin Release from Rat Pancreatic Islets

Glucose-induced inhibition of Ca⁺⁺ extrusion from the β-cell may contribute to the rise in cytosol Ca⁺⁺ that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na⁺ gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of ⁴⁵Ca⁺⁺. Insulin release and ⁴⁵Ca⁺⁺ efflux were then measured during perifusion. In Ca⁺⁺-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited ⁴⁵Ca⁺⁺ efflux. Initially 1 mM ouabain inhibited ⁴⁵Ca⁺⁺ efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca⁺⁺, 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca⁺⁺ was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca⁺⁺ removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca⁺⁺ efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.     

A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels,Ca2+ Signaling,and Gene Expression in T Lymphocytes

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K⁺ channels (K_V and K_Ca, respectively), resulting in depolarization of the membrane potential. As a result, Ca²⁺ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca²⁺ signals after thapsigargin stimulation, as well as oscillatory Ca²⁺ signals, but not the Ca²⁺ transient after TCR stimulation. K⁺ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked K_V and K_Ca channels. Progesterone effectively blocked a broad spectrum of K⁺ channels, reducing both Kv1.3 and charybdotoxin–resistant components of K_V current and K_Ca current in T cells, as well as blocking several cloned K_V channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na⁺ channel, an inward rectifier K⁺ channel, or on lymphocyte Ca²⁺ and Cl⁻ channels. We propose that direct inhibition of K⁺ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.     

Metal ion size profoundly affects H3glyox chelate chemistry

The bisoxine hexadentate chelating ligand, H₃glyox was investigated for its affinity for Mn²⁺, Cu²⁺ and Lu³⁺ ions; all three metal ions are relevant with applications in nuclear medicine and medicinal inorganic chemistry. The aqueous coordination chemistry and thermodynamic stability of all three metal complexes were thoroughly investigated by detailed DFT structure calculations and stability constant determination, by employing UV in-batch spectrophotometric titrations, giving pM values (pM = −log[Mⁿ⁺]_free when [Mⁿ⁺] = 1 μM, [L] = 10 μM at pH 7.4 and 25 °C) – pCu (25.2) > pLu (18.1) > pMn (12.0). DFT calculated structures revealed different geometries and coordination preferences of the three metal ions; notable was an inner sphere water molecule in the Mn²⁺ complex. H₃glyox labels [^52gMn]Mn²⁺, [⁶⁴Cu]Cu²⁺ and [¹⁷⁷Lu]Lu³⁺ at ambient conditions with apparent molar activities of 40 MBq μmol⁻¹, 500 MBq μmol⁻¹ and 25 GBq μmol⁻¹, respectively. Collectively, these initial investigations provide insight into the effects of metal ion size and charge on the chelation with the hexadentate H₃glyox and indicate that further investigations of the Mn²⁺–H₃glyox complex in ^52g/55Mn-based bimodal imaging might be worthwhile.

The bisoxine hexadentate chelating ligand, H₃glyox was investigated for its affinity for Mn²⁺, Cu²⁺ and Lu³⁺ ions; all three metal ions are relevant with applications in nuclear medicine and medicinal inorganic chemistry.     

Stimulation of Monovalent Cation Active Transport by Low Concentrations of Cardiac Glycosides: ROLE OF CATECHOLAMINES

The stimulatory effect of low concentrations of ouabain on the Na-K pump in isolated guinea pig left atria was studied in vitro by assessing active transport of the K⁺ analog Rb⁺. Active transport of Rb⁺ was stimulated 20±8% (SEM, P < 0.05) above control values by 3 nM ouabain, but was inhibited by concentrations >10 nM. Preincubation with the β-adrenergic antagonist propranolol (1 μM) completely blocked stimulation of active transport of Rb⁺ by 3 nM ouabain. Norepinephrine, 10 nM, increased Rb⁺ active transport 29±10% (P < 0.02) above control values. The β-adrenergic agonist l-isoproterenol, 10 nM, increased active transport of Rb⁺ by 33±10% (P < 0.01) above control levels. This stimulatory effect was abolished if tissues were first exposed to propranolol. Tyramine (0.1 μM), a stimulator of endogenous catecholamine release, increased active transport of Rb⁺ 26±12% (P < 0.05) above control values. Rb⁺ active transport was not significantly changed when left atrial tissues were incubated with α-adrenergic agonists or antagonists. Ouabain stimulation of Rb⁺ active transport was prevented by in vivo depletion of myocardial endogenous catecholamines by either reserpine or 6-hydroxydopamine. These findings indicated that in myocardial tissue, Na-K pump stimulation by low concentrations of ouabain is mediated at least in part through β-adrenergic effects of endogenous catecholamines.     

Zinc(ii)-based coordination polymer encapsulated Tb3+ as a multi-responsive luminescent sensor for Ru3+, Fe3+, CrO42−, Cr2O72− and MnO4−

A zinc(ii)-based coordination polymer (CP), namely [Zn(modbc)₂]_n (Zn-CP) (modbc = 2-methyl-6-oxygen-1,6-dihydro-3,4′-bipyridine-5-carbonitrile), has been synthesized and characterized. Single-crystal structural determination reveals that Zn-CP is a two-dimensional framework structure with tetranuclear homometallic Zn₄(modbc)₄ units cross-linked by modbc. The excellent luminescence as well as good stability of Zn-CP do not enable it to have selective sensing capability for different ions. After encapsulation of Tb³⁺ in Zn-CP, the as-obtained fluorescent functionalized Tb³⁺@Zn-CP maintained excellent luminescence as well as stability, which made it a highly selective and sensitive multiresponsive luminescent sensor for Ru³⁺, Fe³⁺, CrO₄²⁻, Cr₂O₇²⁻, and MnO₄⁻ with high sensitivity, good anti-interference performance, and quick response time (∼10 s). The detection limits are 0.27 μM, 0.57 μM, 0.10 μM, 0.43 μM and 0.15 μM, respectively. A possible sensing mechanism was discussed in detail.

A composite, Tb³⁺@Zn-CP, for sensing Ru³⁺, Fe³⁺, CrO₄²⁻, Cr₂O₇²⁻ and MnO₄⁻ with fast response times was reported.     

Effect of inhibition of the electrogenic Na+/K+ pump on the mechanical activity in the rat uterus

Summary— The effects of ouabain and K⁺-free solution were studied in estrogen-primed rat uterine strips under resting tone or repeatedly stimulated with KCl, acetylcholine or oxytocin applied for 20 minutes at 60 minute intervals. These effects were compared with those of the K⁺ channel opener cromakalim. In preparations under resting tone, ouabain (0.1 mM and 0.3 mM) induced rhythmic contractions which disappeared after 20–30 minutes whereas at a higher concentration (1 mM) it evoked a rapid, phasic response followed by a small tonic contraction. Exposure of the strip to a K⁺-free solution induced either rhythmic waves, which ceased after 8–10 minutes, or a single phasic contraction which was followed by a small and slow increase in the resting tone (54 ± 10 mg after 180 min exposure). Nifedipine (0.3 μM) abolished the rhythmic or phasic component of these responses but failed to modify the late small tonic contraction induced by ouabain 1 mM or by K⁺-free solution. Ouabain (0.1–1 mM) or K⁺-free-evoked responses disappeared after short (4 min) or prolonged (60 min) exposure to a Ca²⁺-free, 3 mM EGTA-containing solution. Cromakalim (10 nM ?0.1 mM) did not induce any variation in the resting tone either in the presence or in the absence of Ca²⁺ in the medium. In strips repeatedly stimulated with acetylcholine (0.1 mM) or oxytocin (1 μM), ouabain (0.3 mM), K⁺-free-solution and cromakalim (10 μM) reduced the amplitude of the initial, phasic response and progressively decreased the oscillatory component of the response to these agonists. Conversely, the successive responses evoked by KCl 60 mM in similar experimental conditions were not affected by ouabain or cromakalim. Ouabain (0.3 mM), K⁺-free solution and cromakalim (10 μM) decreased the Ca²⁺-independent, maintained contractions induced by acetylcholine or oxytocin after prolonged exposure to a Ca²⁺-free, EGTA-containing medium. These inhibitory effects were partially or completely reversed in the presence of the non-selective potassium channel blocker tetraethylammonium (10 mM) or in a Ca²⁺-free solution containing 60 mM K⁺. In conclusion, these results suggest that the response induced by ouabain or K⁺-free solution in estrogen-primed rat myometrium involves Ca²⁺ influx through potential-operated calcium channels but not Ca²⁺ release from intracellular stores. In addition, our results show that prolonged exposure to ouabain or K⁺-free medium decreases membrane receptor-mediated responses in rat uterus. This inhibitory effect seems to be the result, at least in part, of a decrease in the cytosolic level of K⁺, due to the inhibition of the electrogenic Na⁺ pump.     

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic β cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K⁺ (K_ATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic β cells. These β-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from β-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K_ATP channels in pancreatic β cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K_ATP channels, closed K_ATP channels, elevated intracellular calcium levels, and stimulated insulin release in β-V59M β cells, indicating that events downstream of K_ATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic β cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. β-V59M islets also displayed a reduced percentage of β cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of β-V59M mice. Their cause requires further investigation.     

Comprehensive In Vitro Analysis of Voriconazole Inhibition of Eight Cytochrome P450 (CYP) Enzymes: Major Effect on CYPs 2B6, 2C9, 2C19, and 3A

Voriconazole is an effective antifungal drug, but adverse drug-drug interactions associated with its use are of major clinical concern. To identify the mechanisms of these interactions, we tested the inhibitory potency of voriconazole with eight human cytochrome P450 (CYP) enzymes. Isoform-specific probes were incubated with human liver microsomes (HLMs) (or expressed CYPs) and cofactors in the absence and the presence of voriconazole. Preincubation experiments were performed to test mechanism-based inactivation. In pilot experiments, voriconazole showed inhibition of CYP2B6, CYP2C9, CYP2C19, and CYP3A (half-maximal [50%] inhibitory concentrations, <6 μM); its effect on CYP1A2, CYP2A6, CYP2C8, and CYP2D6 was marginal (<25% inhibition at 100 μM voriconazole). Further detailed experiments with HLMs showed that voriconazole is a potent competitive inhibitor of CYP2B6 (K_i < 0.5), CYP2C9 (K_i = 2.79 μM), and CYP2C19 (K_i = 5.1 μM). The inhibition of CYP3A by voriconazole was explained by noncompetitive (K_i = 2.97 μM) and competitive (K_i = 0.66 μM) modes of inhibition. Prediction of the in vivo interaction of voriconazole from these in vitro data suggests that voriconazole would substantially increase the exposure of drugs metabolized by CYP2B6, CYP2C9, CYP2C19, and CYP3A. Clinicians should be aware of these interactions and monitor patients for adverse effects or failure of therapy.     

An ‘‘off–on–off’’ sensor for sequential detection of Cu2+ and hydrogen sulfide based on a naphthalimide–rhodamine B derivative and its application in dual-channel cell imaging

A novel colorimetric and fluorometric sensor with unique dual-channel emission to sequentially detect Cu²⁺ and hydrogen sulfide (H₂S) was synthesized from naphthalimide–rhodamine B through the PET and FRET mechanism. The sensor showed a selective “off–on” fluorescence response with a 120-fold increase toward Cu²⁺, and its limits of detection were 0.26 μM and 0.17 μM for UV-vis and fluorescence measurements, respectively. In addition, 1–Cu²⁺ was an efficient “on–off” sensor to detect H₂S with detection limits of 0.40 μM (UV-vis measurement) and 0.23 μM (fluorescence measurement), respectively. Furthermore, the sensor can also be used for biological imaging of intracellular staining in living cells. Therefore, the sensor should be highly promising for the detection of low level Cu²⁺ and H₂S with great potential in many practical applications.

A novel colorimetric and fluorometric sensor with unique dual-channel emission to sequentially detect Cu²⁺ and hydrogen sulfide (H₂S) was synthesized from naphthalimide–rhodamine B through the PET and FRET mechanism.     

2B4, the Natural Killer and T Cell Immunoglobulin Superfamily Surface Protein,Is a Ligand for CD48

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (K _d ≈ 16 μM at 37°C) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (K _d ≈ 8 μM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.     

Inhibition of Aminoglycoside 6′-N-Acetyltransferase Type Ib by Zinc: Reversal of Amikacin Resistance in Acinetobacter baumannii and Escherichia coli by a Zinc Ionophore

In vitro activity of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] was inhibited by ZnCl₂ with a 50% inhibitory concentration (IC₅₀) of 15 μM. Growth of Acinetobacter baumannii or Escherichia coli harboring aac(6′)-Ib in cultures containing 8 μg/ml amikacin was significantly inhibited by the addition of 2 μM Zn²⁺ in complex with the ionophore pyrithione (ZnPT).     

review article: Phospholemman: A Novel Cardiac Stress Protein

Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co‐localizes and co‐immunoprecipitates with Na⁺‐K⁺‐ATPase, Na⁺/Ca²⁺ exchanger, and L‐type Ca²⁺ channel. Functionally, when phosphorylated at serine⁶⁸, PLM stimulates Na⁺‐K⁺‐ATPase but inhibits Na⁺/Ca²⁺ exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L‐type Ca²⁺ channel. Therefore, PLM occupies a key modulatory role in intracellular Na⁺ and Ca²⁺ homeostasis and is intimately involved in regulation of excitation–contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na⁺ overload and simultaneously preserves inotropy by inhibiting Na⁺/Ca²⁺ exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na⁺‐K⁺‐ATPase, Na⁺/Ca²⁺ exchanger, and potentially L‐type Ca²⁺ channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189–196     

Utilization of a plasticized PVC optical sensor for the selective and efficient detection of cobalt(ii) in environmental samples

A novel sensitive, selective, and reversible cobalt(ii) ion optical sensor was prepared by the incorporation of 5-[o-carboxyphenylazo]2,4-dihydroxybenzoic acid [CPDB] and sodium tetraphenylborate (NaTPB) in a plasticized polyvinyl chloride (PVC) membrane containing dioctyl adipate (DOA) as a plasticizer. The influence of several parameters such as pH, base matrix, solvent mediator and reagent concentration was optimized. A comparison of the obtained results with those of previously reported sensors revealed that the proposed method, in addition to being fast and simple, provided a good linear range (0.05–45.20 μM) and low detection limit (0.015 μM). Low detection and quantification limits and excellent selectivity in the presence of interfering ions such as Fe³⁺, Cu²⁺, Ni²⁺, Ag⁺, Au³⁺, Cr³⁺, Cd²⁺, Zn²⁺, Hg²⁺, and SO₄²⁻ make it feasible to monitor Co²⁺ ion content accurately and repeatedly in environmental samples with complicated matrices. The optode was regenerated successfully using 0.3 M nitric acid (HNO₃) solution while its response was reversible with a relative standard deviation (RSD) lower than 1.9% for seven replicate determinations of 20 μM Co²⁺ in various membranes. The optode was stable and was stored for at least 15 days without observing any change in its sensitivity.

A novel sensitive, selective, and reversible cobalt(ii) ion optical sensor was prepared by the incorporation of [CPDB] and (NaTPB) in a (PVC) membrane containing (DOA) as a plasticizer.