Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Programmed cell death 2 functions as a tumor suppressor in osteosarcoma

Objectives: To investigate the role of programmed cell death 2 (PDCD2) in osteosarcoma (OS), along with correlations between PDCD2 and CD4⁺/CD8⁺. Methods: Sprague-Dawley (SD) rats were randomly assigned to control group and OS group. The OS group rats were subjected to induce models of OS by transplantation with UMR106 cells. Peripheral blood was collected to test the percentages of the CD4⁺ and CD8⁺ cell subsets using flow cytometry (FCM). Western blotting was performed to determine the PDCD2 protein level. The correlations between PDCD2 and CD4⁺/CD8⁺ were analyzed by Pearson correlation coefficient. Besides, specific small interfering RNAs (siRNA) against PDCD2 and nonspecific (NS)-siRNA were transfected into UMR106 cells. Cell viability and invasive ability were determined after transfection. Results: CD4⁺ cells percentages were significantly decreased in the OS group, while CD8⁺ cells were significantly increased (P < 0.05). The PDCD2 protein levels were markedly lower than that in the control group (P < 0.05). Additionally, PDCD2 was positively correlated with CD4⁺ (R² = 0.66, P < 0.05), but was negatively correlated with CD8⁺ (R² = -0.94, P < 0.05). Moreover, the cell viability and invasion ability were significantly higher than that in the control group and the NS siRNA group after transfection with PDCD2 siRNA (P < 0.05). Conclusion: These results suggest that PDCD2 is involved in the pathogenesis of OS, and PDCD2 may play an important role in tumor suppression. These mechanisms might be related to immune response induced by CD4⁺ and CD8⁺ T cells.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

The stimulus-secretion coupling of glucose-induced insulin release

The short-term influence of low concentrations of extracellular K⁺ upon insulin release was investigated in the isolated perfused rat pancreas. In the absence of glucose, the removal of extracellular K⁺ provoked a slow and transient stimulation of insulin release. This phenomenon was enhanced and prolonged at either low (2.8 mM) or high (16.7 mM) concentrations of glucose, and was abolished in the absence of extracellular Ca²⁺. Removal of extracellular K⁺ also accelerated and augmented the initial phase of glucose-induced insulin release. The late phase of glucose-induced insulin release was rapidly augmented by removal of extracellular K⁺, but inhibited in response to a partial decrease in extracellular K⁺ concentration. The release of insulin evoked by glucose in the absence of K⁺ differed from that seen in the presence of K⁺ by a progressive decline in secretory rate during prolonged stimulation, and by a lesser sensitivity towards a shortage in extracellular Ca²⁺. During constant exposure to glucose, the restoration of a normal K⁺ concentration provoked a short-lived offresponse, followed by a 8 min-period of inhibited insulin release prior to normalization of the secretory activity. These data indicate that K⁺ deprivation, apart from its long-term untoward effects upon islet function, causes a transient stimulation or facilitation of insulin release, possibly by mobilizing Ca²⁺ from intracellular stores.     

The beneficial effects of adjunctive recombinant human interleukin-2 for multidrug resistant tuberculosis

Introduction

Multidrug-resistant tuberculosis (MDR-TB) is a hard-to-treat disease with a poor outcome of chemotherapy. In the present study, the efficacy and safety of recombinant human interleukin-2 (rhIL-2) were investigated in patients with MDR-TB.

Material and methods

Fifty culture-confirmed patients with MDR-TB were included. Twenty-five patients were randomly assigned to the trial group (injection of 500 000 IU of rhIL-2 once every other day at the first, third, fifth and seventh months in addition to standard multidrug therapy) and another 25 patients to the control group with standard multidrug therapy. All patients were monitored clinically, and T-cell subsets were analyzed by flow cytometry.

Results

The rates of sputum negative conversion and X-ray resolution in the trial group were higher than those of the control, and the improvements were significant by completion of treatment. In addition, CD4⁺CD25⁺ T cells in the controls rose gradually during treatment. The levels at the end of the seventh month were significantly higher than before, which were also significantly different when compared with those from the trial group at the same time. However, there were no such changes associated with treatment in the trial group. No significant differences appeared in other T cell subsets.

Conclusions

Exogenous IL-2 in the present regimen improves immunity status. Adjunctive immunotherapy with a long period of rhIL-2 is a promising treatment modality for MDR-TB.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Intratumoral FOXP3VEGFR2 regulatory T cells are predictive markers for recurrence and survival in patients with colorectal cancer

Previously, we have shown that CD8⁺T/FOXP3⁺ cell ratio but not FOXP3⁺ cell number alone is an independent prognostic factor for colorectal cancer. In the present study, we evaluated whether the number of intratumoral FOXP3⁺VEGFR2⁺ (itFOXP3⁺VEGFR2⁺) T cells alone could be a predictive factor for survival prognosis in patients with colorectal cancer. Distribution of regulatory T cells (Tregs) at tumor sites derived from 88 patients with primary colorectal cancer was fluorescence-immunohistochemically examined. Relatively low number of itFOXP3⁺VEGFR2⁺ cells significantly correlated with poor disease-free survival (DS) and overall survival (OS); multivariate analysis indicated that number of itFOXP3⁺VEGFR2⁺ cells is an independent predictive and prognostic factor of DS and OS while neither intratumoral FOXP3⁺ cell number nor intratumoral FOXP3⁺VEGFR2⁻ cell number alone showed significant correlation with DS or OS. These results suggest that FOXP3⁺VEGFR2⁺ may be a better predictive Treg marker than FOXP3⁺ alone for recurrence and survival in patients with colorectal cancer.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Down-regulated Na+/K+-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats

This study was aimed to examine whether the Na⁺/K⁺ adenosine triphosphatase (Na⁺/K⁺-ATPase) activity in ischemic penumbra is associated with the pathogenesis of ischemia/reperfusion-induced brain injury. An experimental model of cerebral ischemia/reperfusion was made by transient middle cerebral artery occlusion (tMCAO) in rats and the changes of Na⁺/K⁺-ATPase activity in the ischemic penumbra was examined by Enzyme Assay Kit. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 6 h, 24 h, 48 h, 3 d and 7 d after tMCAO. Enzyme Assay analyses revealed the activity of Na⁺/K⁺-ATPase was decreased in the ischemic penumbra of model rats after focal cerebral ischemia/reperfusion compared with sham-operated rats, and reduced to its minimum at 48 h, while the infarct volume was enlarged gradually. In addition, accompanied by increased brain water content, apoptosis-related bcl-2 and Bax proteins, apoptotic index and neurologic deficits Longa scores, but fluctuated the ratio of bcl-2/Bax. Correlation analysis showed that the infarct volume, apoptotic index, neurologic deficits Longa scores and brain water content were negatively related with Na⁺/K⁺-ATPase activity, while the ratio of bcl-2/Bax was positively related with Na⁺/K⁺-ATPase activity. Our results suggest that down-regulated Na⁺/K⁺-ATPase activity in ischemic penumbra might be involved in the pathogenesis of cerebral ischemia/reperfusion injury presumably through the imbalance ratio of bcl-2/Bax and neuronal apoptosis, and identify novel target for neuroprotective therapeutic intervention in cerebral ischemic disease.     

Reduction of forkhead box P3 levels in CD4+CD25high T cells in patients with new-onset systemic lupus erythematosus

The aim of this study was to quantify and evaluate the forkhead box P3 (FoxP3) expression regulatory T cells in new-onset systemic lupus erythematosus (SLE) patients before and after treatment. Forty-four newly diagnosed and untreated SLE patients, including 24 with active disease (SLEDAI > or = 10) and 20 with inactive disease (SLEDAI < 5), were enrolled in this study. Twenty-one age- and sex-matched healthy volunteers were also included as controls. Peripheral blood samples were collected and mononuclear cells isolated. The expression of CD25 and FoxP3 in CD4(+) T cells were analysed with flow cytometry. CD4(+)CD25(+) (3.95-13.04%) and CD4(+)CD25(high) (0.04-1.34%) T cells in peripheral blood in untreated patients with new-onset active lupus were significantly lower than that in the patients with inactive lupus (7.27-24.48%, P < 0.05 and 0.14-3.07% P < 0.01 respectively) and that in healthy controls (5.84-14.84%, P < 0.05). Interestingly, the decrease in CD4(+)CD25(high) T cells was restored significantly in patients with active lupus after corticosteroid treatment. There was, however, a significantly higher percentage of CD4(+)FoxP3(+) T cells in patients with active (5.30-23.00%) and inactive (7.46-17.38%) new-onset lupus patients compared with healthy control subjects (2.51-12.94%) (P < 0.01). Intriguingly, CD25 expression in CD4(+)FoxP3(+) T cells in patients with active lupus (25.24-62.47%) was significantly lower than that in those patients with inactive lupus (30.35-75.25%, P < 0.05) and healthy controls (54.83-86.38%, P < 0.01). Most strikingly, the levels of FoxP3 expression determined by mean fluorescence intensity in CD4(+)CD25(high) cells in patients with active SLE were significantly down-regulated compared with healthy subjects (130 +/- 22 versus 162 +/- 21, P = 0.012). CD4(+)CD25(high) T cells are low in new-onset patients with active SLE and restored after treatment. Despite that the percentage of CD4(+)FoxP3(+) T cells appear high, the levels of FoxP3 expression in CD4(+)CD25(high) T cells are down-regulated in untreated lupus patients. There is a disproportional expression between CD25(high) and FoxP3(+) in new-onset patients with active SLE.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.     

Role of ionic transport in regulation of hemoglobin affinity for oxygen in diabetes mellitus

The rate of Na⁺/H⁺ exchange is increased by 24%, activities of Ca-dependent K+ channels is increased by 13%, and activity of erythrocyte Ca²⁺-ATPase decreased by 17% in patients with diabetes mellitus concomitant with essential hypertension in comparison with patients with essential hypertension without disorders of carbohydrate metabolism. Changes in activity of Na⁺/H⁺ exchange, Ca-dependent K⁺ channels, and erythrocyte Ca²⁺-ATPase and increased oxygen affinity of hemoglobin are due to increased glucose concentration in the plasma and are leveled by olifen.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based perforated patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca²⁺]_in resulting from Ca²⁺ release from caffeine-ryanodine-sensitive internal stores. In addition to Ca²⁺ release, IBMX produced a biphasic membrane current response comprised of an inward current transiently interrupted by outward current. The onset of the inward current slightly preceded the onset of the [Ca²⁺]_in transient, while the interrupting outward current developed synchronously with the [Ca²⁺]_in rise. The development of IBMX-induced outward current ultimately needed the [Ca²⁺]_in elevation. After the depletion of Ca²⁺ stores by IBMX or caffeine exposure, the subsequent IBMX challenge failed to produce both the [Ca²⁺]_in transient and outward membrane current, although the inward current remained unchanged. Both components of the IBMX-induced membrane current response had a reversal potential close to the K⁺ equilibrium potential and the IBMX-induced membrane current response disappeared while dialysing the cell interior with K⁺-free, Cs⁺-containing solutions suggesting their association with K⁺ channel activity. External administration of 10 mM tetraethylammonium chloride (TEA-Cl) evoked an inward current similar to that observed in response to IBMX; in the presence of TEA-Cl, IBMX application was almost unable to induce additional inward current. IBMX (5 mM) effectively (50%) inhibited K⁺ currents evoked by step depolarizations of membrane potential. We suggest that IBMX affects membrane permeability via activation of Ca²⁺-regulated K⁺ channels and direct inhibition of TEA-sensitive K⁺ channels.     

Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca²⁺ homeostasis. As permeation of Ca²⁺ through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca²⁺ entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na⁺ permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na⁺ entry may be converted into a distinct pattern of cellular Ca²⁺ signals by interaction with Na⁺/Ca²⁺ exchanger proteins. This review discusses current concepts regarding the link between Na⁺ entry through TRPC channels and cellular Ca²⁺ signaling.     

Down-regulation of TET2 in CD3+ and CD34+ cells of myelodysplastic syndromes and enhances CD34+ cells proliferation

Aims and background: To investigate the expressions of TET2 mRNA in bone marrow CD3⁺ and CD34⁺ cells of the patients with myelodysplastic syndromes (MDS) and to study the effect of silencing TET2 by small interfering RNA (siRNA) on the biological characteristics of CD34⁺ cells. Methods: CD3⁺ and CD34⁺ cells were sorted by magnetic activated cell-sorting system from bone marrow of MDS patients and controls. The mRNA expressions of TET2 in bone marrow CD3⁺ and CD34⁺ cells of 28 MDS patients and 20 controls were detected by qPCR. The silencing effect of RNA interference (RNAi) on TET2 expression in CD34⁺ bone marrow cells of normal control was identified by qPCR and Western blot analysis. The cell cycle kinetics and cell apoptosis were then detected by flow cytometry. Results: The expression of TET2 mRNA in CD3⁺ and CD34⁺ cells was down-regulated in MDS compared with that in controls [(0.16±0.11) vs. (1.05±0.32) (P<0.001); (0.58±0.26) vs. (1.25±0.94) (P<0.005)]. The siRNA targeting TET2 suppressed the expression of TET2 in normal CD34⁺ cells. Meanwhile, the proliferation activity was significantly enhanced [G0/G1: (87.82±8.25)% vs. (92.65±7.06)% and (93.60±5.54)%, P<0.05; S: (11.50±8.31)% vs. (6.92±7.04)% and (5.95±5.53)%, P<0.05] and the apoptosis rate was declined [(21.28±9.73)% vs. (26.17±9.88)% and (26.20±9.78)%] in the cells which transfected with TET2 siRNA as compared to those in the cells transfected with scrambled siRNA and control cells. Conclusions: The TET2 expression of in CD3⁺ and CD34⁺ cells of MDS patients was decreased. Suppression of TET2 expression renders the CD34⁺ cells harboring more aggressive phenotype. This preliminary finding suggests that CD34⁺ cells lowering expression of TET2 may play an oncogenic role on myeloid tumor and CD3⁺ T cells of MDS patients may be derived from the malignant clone.     

Bumetanide protects focal cerebral ischemia-reperfusion injury in rat

Objective: Bumetanide has been reported to attenuate ischemia-evoked cerebral edema. However, whether bumetanide can protect cerebral ischemia-reperfusion injury (IRI) in vivo is unclear. In the present study, we aim to determine whether intravenously injection bumetanide can attenuate cerebral IRI and if its protection effect might be related to the modification of cerebral NKCC1 and KCC2 protein expression. Methods: Focal cerebral ischemia was induced by occluding the right middle cerebral artery (MCAO) for 2-h, followed by 3-h, 24-h or 48-h of reperfusion respectively. Brain edema, neurological deficits, and infarction volume were determined by (wet weights - dry weights)/dry weights ×100, 5-point neurological function score evaluation system, and TTC staining, respectively. The expression levels of NKCC1 and KCC2 were determined by immunohistochemical staining. Results: Reperfusion increased brain edema, neurological deficits, and infarction volume. Bumetanide decreased brain edema, attenuated the neurological defects and reduced post-ischemic cerebral infarction. Cerebral ischemia-reperfusion injury increased NKCC1 expression level and decreased KCC2 expression level. Interestingly, bumetanide down-regulated the NKCC1 protein expression level without changing the KCC2 protein expression level in rat brain cortex. Conclusion: These results suggest that bumetanide protects focal cerebral ischemia-reperfusion injury in rat, which might through the inhibition of NKCC1.     

Analysis of Ejaculates from Patients with Cystic Fibrosis

Ejaculates from two men with cystic fibrosis (CF) were examined. Both had azoospermia. A considerable decrease in volume and fructose content was noted. The absolute amounts of calcium, magnesium, and zinc per ejaculate showed normal values in one of the patients but were two to three times increased in the other compared to mean values of a control group. Thus the concentrations of these cations were increased at least fivefold in both patients.

The amount of Mg²⁺- and Ca²⁺dependent ATPase was comparable to that of controls, but values were higher than in men with oligospermia. Both the divalent cations and the Mg²⁺- and Ca²⁺ -dependent ATPase curve profiles of split ejaculate fractions were atypical. Secretory granules and vesicles were plentiful in the seminal plasma of both patients while amorphous substance was practically absent.

The present findings agree with a less affected function of the prostate gland and a dysfunction of the seminal vesicles in these patients.     

L-alanine evokes opening of single Ca2+-activated K+ channels in rat liver cells

Cellular uptake of neutral amino acids via Na⁺ cotransporters is known to be associated with an increased membrane K⁺ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K⁺ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K⁺-selective channels are opened by Ca²⁺ acting from the inside of the membrane at a concentration as low as 0.1 M.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.