The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

[Ca2+]i oscillations in human sperm are triggered in the flagellum by membrane potential-sensitive activity of CatSper

STUDY QUESTION How are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated? SUMMARY ANSWER P4-induced [Ca2+]i oscillations are generated in the flagellum by membrane potential (Vm)-sensitive Ca2+-influx through CatSper channels. WHAT IS KNOWN ALREADY A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Although pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required. STUDY DESIGN, SIZE, DURATION This was a laboratory study that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1000 individual cells over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp. MAIN RESULTS AND THE ROLE OF CHANCE P4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1–2 s). K+-ionophore valinomycin (1 µM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly ‘reset’ upon manipulation of [K+]o. Pre-treatment of sperm with valinomycin ([K+]o = 5.4 mM) had no effect on the P4-induced [Ca2+] transient (P = 0.95; eight experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n = 257; P = 5 × 10−55 compared to control) and significantly reduced both the amplitude and frequency of persisting oscillations (P = 0.0001). Upon valinomycin washout, oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+], the inhibitory effect of valinomycin was reduced and was dependent on EK (P = 10−25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P < 0.01). The CatSper inhibitor RU1968 (4.8 and 11 µM) caused immediate and reversible arrest of activity in 36% and 96% of oscillating cells, respectively (P < 10−10). Quinidine (300 µM) which blocks the sperm K+ current (IKsper) completely, inhibited [Ca2+]i oscillations. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This was an in-vitro study and caution must be taken when extrapolating these results to in-vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS [Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the regulation and generation (or failure) of these oscillations. STUDY FUNDING/COMPETING INTEREST(S) E.T.-N. was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). E.M-M received travel funds from the Programa de Apoyo a los Estudios de Posgrado (Maestria y Doctorado en Ciencias Bioquimicas-Universidad Autonoma de Mexico). SGB and CLRB are recipients of a Chief Scientist Office (NHS Scotland) grant TCS/17/28. The authors have no conflicts of interest.

Omega-3 fatty acids improve flow-induced vasodilation by enhancing TRPV4 in arteries from diet-induced obese mice

Aims Previous studies have shown the intake of omega-3 polyunsaturated fatty acids is associated with low rates of obesity and ischaemic pathologies. Omega-3 also have anti-inflammatory and plaque-stabilization effects and regulate vasodilation and constriction. However, there are few studies of the role of omega-3 in flow-induced vasodilation involving Ca2+-permeable ion channel TRPV4 in high-fat diet-induced obese (DIO) mouse. Here, we determined whether omega-3 protect against vascular dysfunction induced by a high-fat diet by enhancing TRPV4 activity and subsequently improving flow-mediated vasodilation. Methods and results Flow-mediated vasodilation in second-order mesenteric arteries from mice was measured using a pressure myograph. The intracellular Ca2+ concentration in response to flow and GSK1016790A (a TRPV4 agonist) was measured by Fluo-4 fluorescence. Whole-cell current was measured by patch clamp. Cell membrane tether force was measured by atomic force microscopy. Impairment of flow-mediated vasodilation in arteries and Ca2+ influx in endothelial cells from DIO mice was restored by omega-3 treatment. The improved flow-induced vasodilation was inhibited by the TRPV4 antagonist HC067047 and in TRPV4−/− mice. Omega-3 treatment enhanced endothelial TRPV4 activity and altered cell membrane mechanic property, as indicated by enhanced GSK1016790A-induced Ca2+ influx and whole-cell current and altered membrane mean tether force in endothelial cells from DIO mice. Conclusion Omega-3 improve vascular function by improving flow-induced vasodilation via enhancing TRPV4 activity in the endothelium of obese mice which may be related to improved cell membrane physical property. Activation of TRPV4 in endothelium plays an important role in the protective mechanisms of omega-3 against vascular dysfunction in obesity by improving flow-mediated vasodilation.

Modulation of inhibitory and excitatory fast neurotransmission in the rat CNS by heavy water (D2O)

The effects of heavy water (deuterium oxide, D2O) on GABAergic and glutamatergic spontaneous and evoked synaptic transmission were investigated in acute brain slice and isolated “synaptic bouton” preparations of rat hippocampal CA3 neurons. The substitution of D2O for H2O reduced the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in a concentration-dependent manner but had no effect on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, for evoked synaptic responses in isolated neurons, the amplitude of both inhibitory and excitatory postsynaptic currents (eIPSCs and eEPSCs) was decreased in a concentration-dependent manner. This was associated with increases of synaptic failure rate ( Rf) and paired-pulse ratio (PPR). The effect was larger for eIPSCs compared with eEPSCs. These results clearly indicate that D2O acts differently on inhibitory and excitatory neurotransmitter release machinery. Furthermore, D2O significantly suppressed GABAA receptor-mediated whole cell current ( IGABA) but did not affect glutamate receptor-mediated whole cell current ( IGlu). The combined effects of D2O at both the pre- and postsynaptic sites may explain the greater inhibition of eIPSCs compared with eEPSCs. Finally, D2O did not enhance or otherwise affect the actions of the general anesthetics nitrous oxide and propofol on spontaneous or evoked GABAergic and glutamatergic neurotransmissions, or on IGABA and IGlu. Our results suggest that previously reported effects of D2O to mimic and/or modulate anesthesia potency result from mechanisms other than modulation of GABAergic and glutamatergic neurotransmission.

Impact of Na+/Ca2+ Exchangers on Therapy Resistance of Ovary Carcinoma Cells

Background/Aims: According to previous observations, enhanced store-operated Ca2+-entry (SOCE) accomplished by the pore forming ion channel unit Orai1 and its regulator STIM1 contribute to therapy resistance of ovary carcinoma cells. Ca2+ signaling is further shaped by Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether therapy resistance is further paralleled by altered expression and/or function of Na+/Ca2+-exchangers. Methods: In therapy resistant (A2780cis) and therapy sensitive (A2780sens) ovary carcinoma cells transcript levels were estimated from RT-PCR, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free extracellular perfusate by Na+ free and Ca2+ containing (2 mM) extracellular perfusate, as well as cell death from PI -staining in flow cytometry. Results: The transcript levels of NCX3, NCKX4, NCKX5, and NCKX6, slope and peak of Δ[Ca2+]i as well as Ica were significantly higher in therapy resistant than in therapy sensitive ovary carcinoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i and significantly augmented the cisplatin-induced cell death of therapy resistant ovary carcinoma cells without significantly modifying cisplatin-induced cell death of therapy sensitive ovary carcinoma cells. Conclusion: Enhanced Na+/Ca2+-exchanger activity may contribute to the therapy sensitivity of ovary carcinoma cells.

Voltage dependence of the Ca2+-activated K+channel KCa3.1 in human erythroleukemia cells

We have isolated a K+-selective, Ca2+-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K+gradients of ∼9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above −40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability ( Po) of KCa3.1, with no increase in channel number. The voltage modulation of Powas an increasing monotonic function of voltage. In the absence of elevated Ca2+, voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.

A mutation in the β-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the β-subunit of ENaC (βV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αβγENaC or mutant αβV348MγENaC in Xenopus laevis oocytes. The βV348M mutation stimulated amiloride-sensitive whole-cell current (Δ Iami) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability ( Po). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline Po of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the βV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different βV348 mutants in oocytes suggests that the βV348M mutation increases channel Po by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the βV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

Effects of insulin on Na and K transporters in the rat CCD

We tested the effects of insulin (2 nM, 30–60 min) on principal cells of isolated split-open rat cortical collecting ducts (CCD) using whole-cell current measurements. Insulin addition to the superfusate of the tubules enhanced Na pump (ouabain-sensitive) current from 18 ± 3 to 31 ± 3 pA/cell in control and from 74 ± 9 to 126 ± 11 pA/cell in high K-fed animals. It also more than doubled ROMK (tertiapin-Q-sensitive) K+ currents in control CCD from 320 ± 40 to 700 ± 80 pA/cell, although it did not affect this current in tubules from K-loaded rats. Insulin did not induce the appearance of amiloride-sensitive Na+ current in control animals, while in high K-fed animals the currents were similar in the presence (140 ± 30) and the absence (180 ± 70 pA/cell) of insulin. Intraperitoneal injection of insulin plus hypertonic dextrose decreased Na excretion, as previously reported. However, injection of dextrose alone, or the nonmetabolized sugar mannose, had similar effects, suggesting that they were largely the result of vascular volume depletion rather than specific actions of the hormone. In summary, we find no evidence for acute upregulation of the epithelial Na channel (ENaC) by physiological concentrations of insulin in the mammalian CCD. However, the hormone does activate both the Na/K pump and apical K+ channels and could, under some conditions, enhance renal K+ secretion.