Changes in cell volume and ion gradients across the plasma membrane play a pivotal role in the initiation of apoptosis. Here we explore the kinetics of apoptotic volume decrease (AVD) and ion content dynamics in wild-type (WT) and multidrug-resistant (MDR) Ehrlich ascites tumor cells (EATC). In WT EATC, induction of apoptosis with cisplatin (5 muM) leads to three distinctive AVD stages: an early AVD(1) (4-12 h), associated with a 30% cell water loss; a transition stage AVD(T) ( approximately 12 to 32 h), where cell volume is partly recovered; and a secondary AVD(2) (past 32 h), where cell volume was further reduced. AVD(1) and AVD(2) were coupled to net loss of Cl(-), K(+), Na(+), and amino acids (ninhydrin-positive substances), whereas during AVD(T), Na(+) and Cl(-) were accumulated. MDR EATC was resistant to cisplatin, showing increased viability and less caspase 3 activation. Compared with WT EATC, MDR EATC underwent a less pronounced AVD(1,) an augmented AVD(T), and a delay in induction of AVD(2). Changes in AVD were associated with inhibition of Cl(-) loss during AVD(1), augmented NaCl uptake during AVD(T), and a delay of Cl(-) loss during AVD(2). Application of the anion channel inhibitor NS3728 inhibited AVD and completely abolished the differences in AVD, ionic movements, and caspase 3 activation between WT and MDR EATC. Finally, the maximal capacity of volume-regulated anion channel was found to be strongly repressed in MDR EATC. Together, these data suggest that impairment of AVD, primarily via modulation of NaCl movements, contribute to protection against apoptosis in MDR EATC.

Background and purpose: Positive modulators of small conductance Ca 2 þ -activated K þ channels (SK1, SK2, and SK3) exert hyperpolarizing effects that influence the activity of excitable and non-excitable cells. The prototype compound 1-EBIO or the more potent compound NS309, do not distinguish between the SK subtypes and they also activate the related intermediate conductance Ca 2 þ -activated K þ channel (IK). This paper demonstrates, for the first time, subtype-selective positive modulation of SK channels. Experimental approach: Using patch clamp and fluorescence techniques we studied the effect of the compound cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) on recombinant hSK1-3 and hIK channels expressed in HEK293 cells. CyPPA was also tested on SK3 and IK channels endogenously expressed in TE671 and HeLa cells. Key results: CyPPA was found to be a positive modulator of hSK3 (EC 50 ¼ 5.6 7 1.6 mM, efficacy 90 7 1.8 %) and hSK2 (EC 50 ¼ 14 7 4 mM, efficacy 71 7 1.8 %) when measured in inside-out patch clamp experiments. CyPPA was inactive on both hSK1 and hIK channels. At hSK3 channels, CyPPA induced a concentration-dependent increase in the apparent Ca 2 þ -sensitivity of channel activation, changing the EC 50 (Ca 2 þ ) from 429 nM to 59 nM. Conclusions and implications: As a pharmacological tool, CyPPA may be used in parallel with the IK/SK openers 1-EBIO and NS309 to distinguish SK3/SK2-from SK1/IK-mediated pharmacological responses. This is important for the SK2 and SK1 subtypes, since they have overlapping expression patterns in the neocortical and hippocampal regions, and for SK3 and IK channels, since they co-express in certain peripheral tissues.

SK channels are Ca2؉ -activated K ؉ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca 2؉ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca 2؉ -activated K ؉ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.In hippocampal pyramidal neurons voltage-independent, Ca 2ϩ -activated K ϩ channels are responsible for the generation of two distinct afterhyperpolarizing currents, I AHP 5 and sI AHP (1-4). I AHP is characterized by a time constant of decay of ϳ100 ms and by its sensitivity to the bee venom toxin, apamin, and to the scorpion toxins, scyllatoxin and tamapin (5-7). sI AHP is characterized by a slower time course (in the range of seconds), by its lack of sensitivity to apamin or any other classical K ϩ channel blocker, and by its modulation by several neurotransmitters (1-3, 8). Based on their kinetic and pharmacological features and on the results obtained from genetically manipulated mice, SK channels mediate I AHP , whereas the molecular correlate for sI AHP is still unknown (2-4, 9, 10). In addition to the use of selective blockers, an important contribution to the elucidation of the physiological role of SK and IK channels has arisen from the use of a small organic compound that enhances channel activity, the benzimidazolinone 1-EBIO (11-15). 1-EBIO enhances the activity of SK channels in the presence of the physiological activator, intracellular Ca 2ϩ , by increasing the apparent sensitivity of SK channels to Ca 2ϩ (14). As a consequence, 1-EBIO increases the amplitude of SK-mediated AHP currents and their duration in a variety of neurons, leading to profound changes in neuronal activity and firing patterns (14, 16 -18). Although 1-EBIO has been a useful tool to elucidate the function of SK channels in their native context, it has some important li...

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by a polyglutamine expansion within the Ataxin-2 (Atxn2) protein. Purkinje cells (PC) of the cerebellum fire irregularly and eventually die in SCA2. We show here that the type 2 small conductance calcium-activated potassium channel (SK2) play a key role in control of normal PC activity. Using cerebellar slices from transgenic SCA2 mice we demonstrate that SK channel modulators restore regular pacemaker activity of SCA2 PCs. Furthermore, we also show that oral delivery of a novel selective positive modulator of SK2/3 channels (NS13001) alleviates behavioural and neuropathological phenotypes of aging SCA2 transgenic mice. We conclude that SK2 channels constitute a novel target for SCA2 treatment and that the developed selective SK2/3 modulator NS13001 holds promise as a potential therapeutic agent for treatment of SCA2 and possibly other cerebellar ataxias.

This study investigated the impact of women's body mass index (BMI) on the outcome after consecutive IVF/intracytoplasmic sperm injection cycles in 487 patients initiating treatment with 5-year follow-up. The total number of cycles was 1417. In total 103 (21.1%) were overweight (BMI 25-29.9 kg/m²) and 59 (12.1%) were obese (BMI ≥ 30 kg/m²). Number of initiated cycles/woman (P=0.01), number of cancelled cycles/woman (P < 0.01) and the total dose of gonadotrophin used/cycle (P < 0.01) rose with increasing BMI. A negative linear association between BMI and the number of retrieved oocytes (B=-0.243, P < 0.001) and an inverse U-shaped relationship between BMI and the number of developed embryos was seen, with less embryos available among underweight and obese women (P=0.03). The number with positive serum human chorionic gonadotrophin/cycle decreased significantly with increasing BMI (P < 0.01). The ongoing pregnancy rate/cycle among the obese women was lower (20.8% versus 28.3% in normal-weight women; P=0.04). Live-birth rate per cycle was 15.2% versus 21.5%. Multiple logistic regression analysis showed that the only independent predictors of live birth were women's age (P=0.037), women's BMI (P=0.034) and men's age (P=0.040).

SK channels are small conductance Ca 2ϩ -activated K ϩ channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism.

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by ∼20% in modestly, and ∼50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P2 analogue or a PtdIns(4,5)P2-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P2. It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization.

The DOSC measure was associated with aging-related outcomes in a midlife Danish population, and is, thus, well suited for future epidemiological research on social inequalities in health and aging.