The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl^– transport and the effects of acetylcholine (ACh) and electrochemical Cl^– potential changes across the basolateral plasma membrane on intracellular Cl^– activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl^–-selective microelectrodes. In the resting state, the basolateral membrane potential (V _m) was about –40 mV and intracellular Cl^– activity was about 35 mmol/l. Addition of ACh (10^–910^–6 mol/l) hyperpolarizedV _m and decreased the Cl^– activity in a dose-dependent manner. ACh (10^–6 mol/l) hyperpolarizedV _m by 20 mV and decreased the cytosolic Cl^– activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl^– concentration to 1/9 control depolarizedV _m and decreased cytosolic Cl^– activity at a rate of 1.9 mmol/l · min. AV _m hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl^– activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedV _m by 8 mV with little change in Cl^– activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl^– activity induced by low-Cl^– Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl^– activity. Furosemide (1 mmol/l) slightly hyperpolarizedV _m and decreased Cl^– activity at a slow rate but affected Cl^– movements induced by ACh or low-Cl^– Ringer only slightly. Cl^– uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl^– permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl^– conductance pathway and a furosemide-sensitive Cl^– uptake mechanism.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Hypoproduction of erythropoietin contributes to anemia in chronic cadmium intoxication: clinical study on Itai-itai disease in Japan

Itai-itai disease is a condition caused by longterm exposure of the inhabitants of Toyama prefecture, Japan, to cadmium intoxication. The characteristic clinical features of this disease include renal tubular dysfunction, osteomalacia, and anemia. In order to clarify the pathogenesis of the anemia, the red blood cell count, hemoglobin concentration, hematocrit, serum iron level, total ironbinding capacity, serum ferritin level, serum erythropoietin level, creatinine clearance, fractional excretion of β₂-microglobulin, and bone marrow morphology were determined in ten patients with Itai-itai disease. Low serum iron or ferritin levels were not observed, and bone marrow aspiration did not reveal any specific hematological disorders. A close relationship was observed between the decrease in the hemoglobin level and the progression of renal dysfunction. Low serum erythropoietin levels were detected despite the presence of severe anemia. These results suggest an important role of renal damage in the anemia which develops in Itai-itai disease.     

Human Platelets Effectively Kill K-562 Cells, a Chronic Myelogenic Leukemia Cell Line, in vitro

The cytotoxic effect of isolated human platelets on leukemic cells has been examined in order to investigate the role of platelets in host defense systems. K-562 cells (a chronic myelogenic leukemia cell line) showed significant change in their morphology and were killed when they were incubated with platelets in serum-free medium for several hours at 37°C, a condition where no killing of normal peripheral lymphocytes occurred. Some protease inhibitors inhibited the cytotoxicity of platelets against K-562 cells. Our results suggest that platelets may be involved in host defense against neoplasia and that certain proteases are implicated in the cytotoxic effect of platelets.     

FK506 induces biphasic Ca2+ release from microsomal vesicles of rat pancreatic acinar cells

The effect of the immunosuppressant drug FK506 on microsomal Ca2+ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 microM) was added to the microsomal vesicles at a steady state of ATP-dependent 45Ca2+ uptake, FK506 caused a dose-dependent and a biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was released at FK506 concentrations up to 10 microM (Km=0.47 microM), and 60% of total 45Ca2+ uptake was released at FK506 concentrations over 10 microM (Km=55 microM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR, 0.5 microM) increased the FK506 (< or =10 microM)-induced 45Ca2+ release (Ozawa T, Biochim Biophys Acta 1693: 159-166, 2004). Preincubation with heparin (200 microg/ml) resulted in significant inhibition of the FK506 (30 microM)-induced 45Ca2+ release. Subsequent addition of inositol 1,4,5-trisphosphate (IP3, 5 microM) after FK506 (100 microM)-induced 45Ca2+ release did not cause any release of 45Ca2+. These results indicate that two types of FK506-induced Ca2+ release mechanism operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca2+ release, which involves activation of the ryanodine receptor, and a low-affinity mechanism of Ca2+ release, which involves activation of the IP3 receptor.     

Role of macrophages in a mouse model of postoperative MRSA enteritis

BACKGROUND: We have established a mouse model for fatal postoperative enteritis due to Staphylococcus aureus to analyze mechanisms of bacterial translocation and determine reasons for the lethality of this infection. In the present study the role of macrophages was ascertained in protection against S. aureus induced enteritis. MATERIALS AND METHODS: Mice were pretreated with cyclophosphamide (CY), an immunosuppressant, and then infected directly into the jejunum with methicillin-resistant S. aureus (MRSA) isolated from a patient. In other groups of mice liposome-encapsulated dichloromethylene diphosphate (Cl2MDP) was administered to deplete macrophages in vivo. Other experimental groups received lipoteichoic acid (LTA), which was used to inhibit the ability of macrophages to bind MRSA, and additionally, to analyze dependence of bacterial clearance on the macrophage scavenger receptor. The ability of macrophages to bind MRSA was compared with survival rates in this mouse model of fatal postoperative enteritis. RESULTS: Injection of liposome-encapsulated Cl2MDP decreased survival rate of mice infected intraintestinally with MRSA in a dose-dependent manner. Cyclophosphamide also decreased survival rate of MRSA-infected mice and was found to correlate with its ability to decrease the number of macrophages in the spleen. Intravenous LTA administration did not affect total splenocyte numbers or the number of splenic macrophages but decreased the ability of macrophages to bind MRSA and adversely affected survival of mice infected with MRSA. CONCLUSIONS: Macrophages play a critical role in protection against MRSA administered directly into the jejunum. LTA recognition sites (probably type A scavenger receptors) on macrophages are required for binding and phagocytosing MRSA.     

Nonspecific augmentation of lymph node T cells and I-E-independent selective deletion of VβbTl4+ T cells by Mtv-2 in the DDD mouse

DDD/1 (DDD) mice were characterized by marked paucity of T cells in lymph nodes (LN). In DDD-Mtv-2/Mtv-2 (DDD-Mtv-2) congenics, T cells were 4- to 18-fold increased depending on ages but B cells doubled at the most. Thymus weight also increased. In DDDfDDD-Mtv-2, DDD neonatally infected with Mtv-2-derived exogenous MMTV (MMTV-2), neither LN cells nor thymus weight increased. The V_β 5⁺ and V_β 8⁺ Tcell contents in LN were practically the same among three strains. The Mtv-2-induced expansion of LN Tcells was polyclonal and appeared indigenous to DDD mice. Both Mtv-2 and MMTV-2 induced progressive age-dependent deletion of V_β 14⁺CD4⁺ LN cells. Mtv-2 but not MMTV-2 caused deletion of V_β 14⁺CD8+ LN cells and mature V_β 14⁺CD4⁺ thymocytes. Thus, Mtv-2- and MMTV-2-induced V_β 14⁺ T cell deletion may reflect intrathymic and peripheral elimination, respectively. The absence of I-E gene expression in DDD indicates that V_β 14⁺ T cell deletion advances independently of I-E molecules in this experimental system.     

Mechanism of uphill chloride transport of the mouse lacrimal acinar cells: Studies with Cl−-sensitive microelectrode

The mechanism of uphill Cl^– accumulation by mouse lacrimal acinar cells was studied using double-barrelled Cl^–-selective microelectrodes. When measured in standard tris-buffered saline solution, the membrane potential (V _m) was –39.2±0.4 mV and intracellular Cl^– activity (A _Cl ⁱ ) was 34.6±0.7 mmol/l which was 1.4 times higher than the equilibrium level. In Na⁺-free solution,A _Cl ⁱ decreased from 34 mmol/l to 19 mmol/l in 100 min, a level that was close to the equilibrium activity. Return to the standard solution restored the normal level ofA _Cl ⁱ in 5 min. In the presence of furosemide (1 mmol/l), Cl^– uptake induced by Na⁺-readmission was inhibited by 44%. Superfusion with a K⁺-free solution gradually decreasedA _Cl ⁱ until it was close to the equilibrium level after 75 min; superfusion with a high-K⁺ (29.5 mmol/l) solution increasedA _Cl ⁱ significantly. In the presence of ouabain (1 mmol/l), switching the superfusing solutions from K⁺-free to high-K⁺ and from high-K⁺ to K⁺-free at timed intervals of 15 min caused, respectively, an increase (+9 mmol/l) and a decrease (–7 mmol/l) inA _Cl ⁱ . These changes inA _Cl ⁱ were inhibited by furosemide respectively by 61% and 24%. In the presence of furosemide, DIDS (1 mmol/l) or furosemide plus DIDS, the initial rate of Cl^– uptake after cessation of acetylcholine (ACh 1 mol/l) stimulation was inhibited by 47%, 37% or 74%, respectively. Present results show that the characteristics of the uphill chloride uptake by the mouse lacrimal acinar cells are consistent with those of Na⁺–K⁺–Cl^– cotransport. The additional inhibitory effect of DIDS to furosemide inhibition suggests an involvement of anion exchange transport, in parallel with the cotransport, in uphill Cl^– uptake into the cells.