In vivo effects of high phenylalanine blood levels on Na+,K+-ATPase,Mg2+-ATPase activities and biogenic amine concentrations in phenylketonuria

OBJECTIVE: To evaluate the activities of Na+,K+-ATPase and Mg2+-ATPase in erythrocyte membranes from phenylketonuric (PKU) patients and to correlate the enzyme activities with their blood phenylalanine (Phe) levels, biogenic amines as well as with their precursors tyrosine (Tyr) and tryptophan (Try). DESIGN AND METHODS: Twenty three PKU patients were divided into group A (n = 12) on a restricted diet (Phe 1.57 +/- 0.52 mg/dL or 0.10 +/- 0.03 mM) and group B (n = 11) on a "loose" diet (Phe 24.45 +/- 1.50 mg/dL or 1.72 +/- 0.09 mM). The enzyme activities were measured spectrophotometrically, the amino acids with an automatic amino analyser and the biogenic amines with HPLC methods. RESULTS: In group B, plasma amino acids (Tyr, Try), their biogenic amines [adrenaline (A), noradrenaline (NA), dopamine (DA) and serotonin (5HT)], (Na+,K+)-ATPase and Mg2+-ATPase activities were found remarkably decreased (p < 0.001). CONCLUSIONS: High Phe and/or low NA, DA, 5HT plasma levels may indirectly inhibit the erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase in PKU patients. The observed enzyme inhibitions could be a very informative peripheral marker as regards the neurotoxic Phe brain effects.     

Erythrocyte membrane AChE,Na+, K+-ATPase and Mg2+ ATPase activities in mothers and their premature neonates in relation to the mode of delivery

Abstract

Aim. To investigate erythrocyte membrane AChE, Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in mothers and their full-term or premature newborns in relation to the mode of delivery. Methods. Blood was obtained from mothers pre- and post-delivery and the umbilical cord (CB) of their full-term newborns: Group A₁ (n = 16) born with vaginal delivery (VD), Group B₁ (n = 14) full-terms with scheduled cesarean section (CS), Group A₂ (n = 12) prematures with VD, Group B₂ (n = 14) prematures with CS. Total Antioxidant Status (TAS) and common laboratory tests were measured with routine methods, and the membrane enzyme activities spectrophotometrically. Results. TAS was reduced in mothers post VD and in the CB whereas remained unaltered in CS mothers and their newborns. AChE and Na⁺, K⁺-ATPase were increased in mothers post VD. AChE was lower in the CB of prematures than that of full-terms independently of the mode of delivery. Na⁺, K⁺-ATPase activity was increased in the groups of mothers post VD and decreased in prematures. The enzyme was higher in prematures with CS than that with VD. Mg²⁺-ATPase activity was unchanged. Conclusion. The increased maternal AChE and Na⁺, K⁺-ATPase activities may be due to the low TAS determined post VD, whereas their decreased activities in prematures to their immaturity.     

Contribution of tetrodotoxin-resistant persistent Na+ currents to the excitability of C-type dural afferent neurons in rats

BackgroundGrowing evidence supports the important role of persistent sodium currents (I_NaP) in the neuronal excitability of various central neurons. However, the role of tetrodotoxin-resistant (TTX-R) Na⁺ channel-mediated I_NaP in the neuronal excitability of nociceptive neurons remains poorly understood.MethodsWe investigated the functional role of TTX-R I_NaP in the excitability of C-type nociceptive dural afferent neurons, which was identified using a fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloride (DiI), and a whole-cell patch-clamp technique.ResultsTTX-R I_NaP were found in most DiI-positive neurons, but their density was proportional to neuronal size. Although the voltage dependence of TTX-R Na⁺ channels did not differ among DiI-positive neurons, the extent of the onset of slow inactivation, recovery from inactivation, and use-dependent inhibition of these channels was highly correlated with neuronal size and, to a great extent, the density of TTX-R I_NaP. In the presence of TTX, treatment with a specific I_NaP inhibitor, riluzole, substantially decreased the number of action potentials generated by depolarizing current injection, suggesting that TTX-R I_NaP are related to the excitability of dural afferent neurons. In animals treated chronically with inflammatory mediators, the density of TTX-R I_NaP was significantly increased, and it was difficult to inactivate TTX-R Na⁺ channels.ConclusionsTTX-R I_NaP apparently contributes to the differential properties of TTX-R Na⁺ channels and neuronal excitability. Consequently, the selective modulation of TTX-R I_NaP could be, at least in part, a new approach for the treatment of migraine headaches.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10194-022-01443-7.     

Na+/Ca2+ exchange-mediated calcium entry in human lymphocytes.

Regulation of cytosolic Ca2+ and cytosolic Na+ is critical for lymphocyte cation homeostasis and function. To examine the influence of cytosolic Na+ on Ca2+ regulation in human peripheral blood lymphocytes, Ca2+ entry and cytosolic Ca2+ (measured with fura-2) were monitored in cells in which cytosolic Na+ was increased and/or the Na+ gradient was decreased by reduction of external Na+ concentration. Ouabain-treated cells (0.1 mM for 30 min at 37 degrees C), suspended in Na(+)-free medium, showed a 30-65% increase in Ca2+ uptake compared to cells in 140 mM Na+ medium. Enhanced Ca2+ influx was entirely dependent on ouabain pretreatment and reversal of the Na+ gradient. Na pump inhibition or Na ionophore addition and subsequent exposure to Na(+)-free medium resulted in a sustained elevation of cytosolic Ca2+. As preincubation of cells in Ca(2+)-free medium further enhanced the ouabain-dependent increase in cytosolic Ca2+, the effects of the microsomal Ca(2+)-ATPase inhibitor thapsigargin on Ca2+ influx and cytosolic Ca2+ were studied. Thapsigargin stimulated Ca2+ entry following ouabain pretreatment and reversal of the Na+ gradient; the effects of thapsigargin were retained in the presence of LaCl3, a potent inhibitor of store-dependent calcium influx pathways. These results show lymphocytes demonstrate Na+/Ca2+ exchange activity and suggest the Na+/Ca2+ exchanger modulates cytosolic Ca2+ following intracellular Ca2+ store depletion.     

Antigen-dependent and -independent Ca2+ Responses Triggered in T Cells by Dendritic Cells Compared with B Cells

Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under conditions where ex vivo DCs or B cells presented known numbers of specific Ag–major histocompatibility complex (MHC) complexes to naive CD4⁺ T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag–MHC complexes presented by B cells were necessary to elicit the formation of a few T–B conjugates with small contact zones, and the resulting individual T cell Ca²⁺ responses were all-or-none. In contrast, Ag-specific T cell Ca²⁺ responses can be triggered by DCs bearing an average of 30 Ag–MHC complexes per cell. Formation of T–DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca²⁺ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T–DC adhesion precedes Ag recognition, whereas T–B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca²⁺ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC–TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.     

BKCa channels expressed in sensory neurons modulate inflammatory pain in mice

Large conductance calcium-activated potassium (BK_Ca) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BK_Ca channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BK_Ca channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BK_Ca channel activity in sensory neurons with particular pain modalities, we generated mice in which BK_Ca channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BK_Ca channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BK_Ca channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states.     

Regulation of intracellular pH by Na+/H+ exchange in human lymphocytes

Spectrofluorimetry and the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) was used to measure the intracellular pH (pHi) of suspended human lymphocytes. A linear relationship exists between pHi and the fluorescent spinal-ratio (I490nm/I435nm, emission 526 nm) between pH 6.5 and pH 7.8. At the end of each experiment the ratio was calibrated using the high [K+] nigericin technique. All solutions were HEPES buffered. The pHi in resting cells was 7.27 +/- 0.02 (n = 37) at 25 degrees C. Na+ free solution caused a pHi decrease to 6.81 +/- 0.08. The ammonium prepulse technique (25 mM NH4Cl) dropped the pHi to pH 6.80. A rapid recovery of the pHi after this acidification was observed in NaCl Ringer solution. Na+ free solution completely blocked the recovery. 1 mM amiloride led to a partial block of recovery. pHi was restored to the basal value after readdition of Na+. We conclude that in HEPES buffered solutions human lymphocytes recover pHi via a mechanism dependent on extracellular Na+ and largely accomplished by an amiloride inhibitory Na+/H+ exchanger.     

反向模式钠钙离子交换体抑制剂降低造影剂诱导的肾小管上皮细胞凋亡

目的 探讨反向模式钠钙离子交换体抑制剂KB-R7943是否对造影剂肾损伤具有保护作用.方法 培养大鼠肾小管上皮细胞分别与不同浓度KB-R7943(10-5,10-6 mol/L)作用12 h后,加入造影剂作用1 h.采用LDH检测细胞损伤,倒置显微镜观察细胞形态变化,流式细胞仪检测细胞凋亡,共聚焦显微镜测定细胞内钙和反应氧产物水平,RT-PCR检测钠钙离子交换体mRNA表达.相同渗透压甘露醇作对照.数据以均数±标准差(x±s)表示,统计采用方差分析和q检验,简单直线相关分析两者相关性,以P<0.05为差异具有统计学意义.结果 造影剂作用1 h诱导了明显细胞损伤和细胞凋亡,细胞内钙和反应氧产物增加,KB-R7943降低了细胞内钙和反应氧产物水平,同时降低了细胞损伤和细胞凋亡并呈剂量效应;钠钙离子交换体mRNA表达无变化.结论 KB-R794对造影剂诱导的肾小管上皮细胞损伤具有保护作用.     

Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.

BACKGROUND: Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. METHODS: FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. RESULTS: In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. CONCLUSIONS: The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.     

Calcium channel antagonist verapamil modulates human spermatozoal functions

The effect of calcium channel blocker, verapamil (0.5–50 μM), has been studied in vitro in relation to certain spermatozoal functions in human ejaculates. Disruptive changes in the head and tail region of the spermatozoa, separation of heads from tails and coiling of the tail were observed. Motility was considerably reduced, while the pattern of motility also changed from rapid, linear progression to slow or sluggish linear or non-linear movement and finally to non-progressive motility, or even immotility. Verapamil significantly inhibited the influx of extracellular Ca²⁺. The study of kinetic effects further revealed a reduction in the maximum uptake velocity, but no change in the apparent substrate affinity constant. A highly significant decrease in Ca²⁺-dependent ATPase activity was also noted. In order to see whether this drug had any cytotoxic effect, presumably through lipid peroxides, thiobarbituric acid-reactive substances were measured. Verapamil produced an increase in the formation and release of malonyldialdehyde. The level of membrane cholesterol and phospholipid in the spermatozoa was also lowered considerably. The potential of such a calcium channel blocking agent in the designing of a male contraceptive programme is discussed.     

Overexpression of the Na+/Ca2+ exchanger influences ouabain‐mediated spontaneous Ca2+ activity but not positive inotropy

Administration of digitalis in heart failure (HF) increases quality of life but does not carry a prognostic benefit. Digitalis is an indirect inhibitor of the Na⁺/Ca²⁺ exchanger (NCX), which is overexpressed in HF. We therefore used the cardiac glycoside ouabain in Ca²⁺ imaging experiments and patch‐clamp experiments in isolated ventricular myocytes from nonfailing transgenic NCX overexpressor mice (OE). In field‐stimulated myocytes, ouabain (1–100 μm ) increased the amplitude of the Ca²⁺ transient in OE and wild‐type (WT) similarly. Ouabain‐mediated spontaneous Ca²⁺‐activity was significantly more pronounced in OE compared to WT myocytes at higher concentrations (100 μm). Also, at very high concentrations (1000 μm ) of ouabain, the number of cells with hypercontraction leading to cell death was higher in OE. Ouabain (10 μm ) shortened the action potential duration in both genotypes. Our findings suggest that the proarrhythmic but not the inotropic effects of cardiac glycosides are enhanced by increased NCX expression. This may offer an explanation for the observed lack of prognostic benefit but increased quality of life in HF, which is accompanied by NCX upregulation.     

Na+/H+ exchange in human lymphocytes and platelets in chronic and subacute metabolic acidosis.

The effect of acid-base disturbances on sodium/proton (Na+/H+) exchange has been examined in animal models; however, few data are available from human studies. To test the effect of metabolic acidosis on Na+/H+ exchange in man, as well as to examine the relationship between Na+/H+ exchange and cytosolic calcium ([Ca2+]i), we measured both variables in patients with decreased renal function with mild metabolic acidosis (pH 7.34 +/- 0.06), in normal control subjects (pH 7.41 +/- 0.02), and in subjects before (pH 7.40 +/- 0.01), and after (pH 7.26 +/- 0.04) ammonium chloride (NH4Cl) 15 g for 5 d. Lymphocytes and platelets were loaded with the cytosolic pH (pHi) indicator 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein and acidified to pH approximately 6.6 with propionic acid. To quantitate Na+/H+ exchange, dpHi/dt was determined at 1 min. [Ca2+]i was measured with fura-2. Na+/H+ exchange was significantly increased only in lymphocytes of patients with renal insufficiency. Neither intracellular pH (pHi) nor [Ca2+]i was different from controls. NH4Cl resulted in a significant increase in Na+/H+ exchange in lymphocytes, but not in platelets of normal subjects. Values of pHi and [Ca2+]i in either cell type remained unaffected. Since metabolic acidosis influenced Na+/H+ only in lymphocytes, but not in platelets, it is possible that protein synthesis may be involved in increasing Na+/H+ exchange.     

Afatinib triggers a Ni2+-resistant Ca2+ influx pathway in A549 non-small cell lung cancer cells

Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1–30 μM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca²⁺ influx without causing Ca²⁺ release, and the Ca²⁺ influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca²⁺ response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn²⁺ influx. Ca²⁺ influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni²⁺ (a non-specific Ca²⁺ channel blocker). Afatinib caused an increase in mitochondrial Ca²⁺ level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24–48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca²⁺ condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca²⁺ influx pathway, mitochondrial Ca²⁺ overload, and potential collapse in part contribute to afatinib-induced cell death.     

Functional correlation of ATP1A2 mutations with phenotypic spectrum: from pure hemiplegic migraine to its variant forms

BackgroundMutations in ATP1A2, the gene encoding the α2 subunit of Na⁺/K⁺-ATPase, are the main cause of familial hemiplegic migraine type 2 (FHM2). The clinical presentation of FHM2 with mutations in the same gene varies from pure FHM to severe forms with epilepsy and intellectual disability, but the correlation of these symptoms with different ATP1A2 mutations is still unclear.MethodsTen ATP1A2 missense mutations were selected according to different phenotypes of FHM patients. They caused pure FHM (FHM: R65W, R202Q, R593W, G762S), FHM with epilepsy (FHME: R548C, E825K, R938P), or FHM with epilepsy and intellectual disability (FHMEI: T378N, G615R, D718N). After ouabain resistance and fluorescence modification, plasmids carrying those mutations were transiently transfected into HEK293T and HeLa cells. The biochemical functions were studied including cell survival assays, membrane protein extraction, western blotting, and Na⁺/K⁺-ATPase activity tests. The electrophysiological functions of G762S, R938P, and G615R mutations were investigated in HEK293T cells using whole-cell patch-clamp. Homology modeling was performed to determine the locational distribution of ATP1A2 mutations.ResultsCompared with wild-type pumps, all mutations showed a similar level of protein expression and decreased cell viability in the presence of 1 µM ouabain, and there was no significant difference among the mutant groups. The changes in Na⁺/K⁺-ATPase activity were correlated with the severity of FHM phenotypes. In the presence of 100 µM ouabain, the Na⁺/K⁺-ATPase activity was FHM > FHME > FHMEI. The ouabain-sensitive Na⁺/K⁺-ATPase activity of each mutant was significantly lower than that of the wild-type protein, and there was no significant difference among all mutant groups. Whole-cell voltage-clamp recordings in HEK293T cells showed that the ouabain-sensitive pump currents of G615R were significantly reduced, while those of G762S and R938P were comparable to those of the wild-type strain.ConclusionsATP1A2 mutations cause phenotypes ranging from pure FHM to FHM with epilepsy and intellectual disability due to varying degrees of deficits in biochemical and electrophysiological properties of Na⁺/K⁺-ATPase. Mutations associated with intellectual disability presented with severe impairment of Na⁺/K⁺-ATPase. Whether epilepsy is accompanied, or the type of epilepsy did not seem to affect the degree of impairment of pump function.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10194-021-01309-4.     

Halothane increases cytosolic Ca2+ and inhibits Na+/H+ exchange in L6 muscle cells

The effects of the general anesthetic halothane on the concentration of cytosolic free calcium ([Ca2+]i) and cytosolic pH (pHi), were investigated in L6 rat skeletal muscle cells. Basal [Ca2+]i was 169 +/- 8 nM, measured with the fluorescent Ca2(+)-indicator 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5- methylphenoxy)ethane-N,N,N',N'-tetra-acetate. Halothane (5.7 mM) increased [Ca2+]i to 225 +/- 15 nM in the presence of extracellular Ca2+, and from 137 +/- 6 nM to 179 +/- 9 nM in Ca2+ absence. This increase was dose-dependent. The anesthetic released about 50% of the releasable Ca2+ from intracellular stores. The resting pHi of L6 cells was 7.24 +/- 0.04, measured with the fluorescent pH indicator bis-carboxyethylcarboxyfluorescein. Halothane did not affect resting pHi, but inhibited cytoplasmic alkalinization by hypertonicity or cytoplasmic acidification: (1) The hypertonicity-induced alkalinization via activation of Na+/H+ exchange (to 7.50 +/- 0.08, initial rate 0.10 +/- 0.02 pH U/min) was inhibited with 5.7 mM halothane by 67%. (2) Acid-loaded cells (pHi 6.43 +/- 0.01 in cells) recovered towards neutrality via activation of Na+/H+ exchange (rate 0.47 pH U/min), and halothane inhibited the rate of pHi recovery by 50%. The halothane-mediated inhibition of alkalinizations after hypertonic exposure or acid-loading was also observed in bis-(o-amino-phenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells in Ca2(+)-free medium. Therefore, halothane increases [Ca2+]i and in parallel inhibits Na+/H+ exchange, compromising the ability of muscle cells to recover from imposed acidification.     

Both CD8+ and CD16+ human T cell clones can provide B cell help for immunoglobulin production

Summary The functional properties of two CD4⁺CD8⁻CD16⁻, five CD4⁻CD8⁺CD16⁻ and three CD4⁻CD8⁻CD16⁺ human T cell clones were compared. All CD4⁻ T cell clones displayed strong cytolytic activity in the lectin-dependent lytic assay against the P815 murine mastocytoma cell line, but only the CD4⁻CD8⁻CD16⁺ T cell clones exhibited lytic activity against the natural killer-sensitive K562 cell line. Upon activation with anti-CD3 monoclonal antibody, all T cell clones were able to support IgM and IgA synthesis in autologous B cells. Both CD4⁺ and CD4⁻ T cell clones required cell-to-cell interaction with the B cells in order to exert their helper activity for immunoglobulin production. However, unlike CD4⁺, CD4⁻CD8⁺CD16⁻ and CD4⁻CD8⁻CD16⁺ T cell clones provided helper function for immunoglobulin synthesis only when low T/B cell ratios were used in culture. At higher T/B cell ratios, there was a decline in the B cell helper activity of CD4⁻ T cell clones that was probably related to the expression of cytolytic capacity against the antigen-presenting B cell. These data support the notion that under certain experimental conditions even cytotoxic T lymphocytes and natural killer cells may provide B cell helper function.     

A novel and efficient method to induce allospecific CD8+ memory T lymphocytes

The aim of the current study was to establish a simple method for effectively inducing memory T lymphocytes by the intraperitoneal injection of spleen lymphocytes into mice. In total, 75 mice were divided into the following groups: an injection group administered three doses of spleen lymphocytes (1 × 10⁶, 5 × 10⁶, and 1 × 10⁷ cells), a transplantation group in which a 0.25‐cm² skin section from C57BL/6 mice was transplanted onto the back of the recipient, and a control group in which an equal volume of phosphate‐buffered saline was injected. At 1, 2, or 3 months following transplantation, the following parameters were evaluated: quantity of T lymphocytes, percentage of cluster of differentiation 8⁺ (CD8⁺) memory T cells, and proliferation index of purified CD8⁺ memory T cells. No significant differences among groups were detected at 1 month (p > .05). However, the injection group administered 1 × 10⁶ cells exhibited the highest proportion of CD8⁺ memory T cells among all groups at 2 months, and the proportions of CD8⁺ T cells were higher in the three injection groups than in the skin transplantation and control groups at 3 months. The proportions of memory T cells were higher in the injection groups administered 5 × 10⁶ or 1 × 10⁷ cells than in the skin transplantation and control groups at 3 months. The newly established method effectively induces memory T lymphocytes via the intraperitoneal injection of spleen lymphocytes in vivo and has potential applications in the field of immunotherapy.     

Na+/Ca2+‐exchanger‐mediated Mn2+‐enhanced 1H2O MRI in hypoxic,perfused rat myocardium

Paramagnetic Mn²⁺ has emerged in the search for non‐invasive magnetic resonance imaging (MRI) techniques to monitor Ca²⁺ in diagnostic and prognostic cardiovascular disease tests because it both alters MRI contrast and behaves as a Ca²⁺ ‘surrogate’ in vivo. However, the reliance on macroscopically averaged measurements to infer microscopic processes constitutes a major limitation of MRI. This investigation circumvents this limitation and contributes an MRI‐based myocardial Ca²⁺‐transporter assay, which probes the Na⁺/Ca²⁺‐exchanger involvement in Mn²⁺ (and presumably Ca²⁺) transport by virtue of its response to pharmacological inhibition. In the model employed herein, ex vivo arrested rat hearts underwent normoxia and then hypoxia while a constant (hyperkalemic) perfusion minimized flow (and uncontrolled Ca²⁺‐channel) contributions to Mn²⁺‐enhanced MRI measurements. The results (i) demonstrate that Mn²⁺ (and presumably Ca²⁺) accumulates via Na⁺/Ca²⁺‐exchanger‐mediated transport during hyperkalemic hypoxia and further, (ii) implicate hypo‐perfusion (rather than the diminished participation of an isolated sarcolemmal Ca²⁺‐transporter) as the mechanism that underlies the reported reductions of Mn²⁺ accumulation (relative to healthy myocardium) subsequent to myocardial insults in MRI studies. Although myriad studies have employed Mn²⁺‐enhanced MRI in myocardial investigations, this appears to be the first attempt to assay the Na⁺/Ca²⁺‐exchanger with MRI under highly circumscribed conditions. MRI‐based Ca²⁺‐transporter assays, such as the Na⁺/Ca²⁺‐exchanger assay utilized here, will inevitably impact disciplines in the medical sciences and beyond. Copyright © 2007 John Wiley & Sons, Ltd.     

原因不明反复自然流产患者外周血TLR2、TLR4表达及CD4+ CD25+ Treg细胞的变化

目的探讨原因不明反复自然流产(URSA)患者外周血TLR2、TLR4的表达及CD4+CD25+调节性T(Treg)细胞变化的关系。方法用实时荧光定量PCR及流式细胞术检测35例URSA患者、24例妊娠孕妇外周血单个核细胞(PBMC)中TLR2、TLR4mRNA及其蛋白质的表达;流式细胞术检测CD4+CD25+Treg细胞的比例,并分析表达TLR2、TLR4蛋白与CD4+CD25+Treg细胞的相关性。结果 URSA组PBMC中TLR2、TLR4 mRNA分别为0.52±0.04、3.26±1.37;表达TLR2、TLR4蛋白的PBMC比例分别为(32.36±4.18)%、(41.29±3.67)%;与妊娠对照组相比,URSA组外周血TLR4 mRNA及表达TLR4蛋白的PBMC比例差异显著(t分别为19.84和21.85,P均<0.05);URSA组CD4+CD25+Treg细胞的比例为(2.75±0.62)%,显著低于妊娠对照组的(5.17±0.65)%,差异有统计学意义(t=48.40,P<0.01);URSA患者表达TLR4蛋白的PBMC与CD4+CD25+Treg细胞的比例降低呈显著相关(r=0.724,P<0.05)。结论 URSA患者外周血中低表达的CD4+CD25+Treg细胞、高表达的TLR4可能参与了URSA的发生。     

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.