ALBERT

Notes: Abstract. The putative role for Ca2+ entry and Ca2+ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P3 content, as measured in extracts from [2-3H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca2+-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca2+-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca2+ (1% only at 0.1-mm Ca2+ and in Ca2+-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca2+ upon the volume-induced K+ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca2+ chelator BAPTA when the BAPTA-loading is performed in K+ equilibrium medium; (vi) an inhibition of the swelling-induced K+ loss (about 50%) at 1-mm Ba2+, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 μm), reported inhibitors of the K+ loss induced by Ca2+-mobilizing agonists. Thus, Ca2+signaling by Ca2+ release or Ca2+ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells.

Notes: Abstract. Amiloride-sensitive, Na+-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na+ influx is also observed. This indicates that hypertonic challenge activates a Na+/H+ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO− 3, but not in nominally HCO− 3-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage.

Notes: Abstract. Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+] i . The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+] i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD4 (100 nm), the highest measured increase in [Ca2+] i in Ehrlich cells suspended in Ca2+-containing medium is 260 ± 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+] i . Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+] i in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+] i in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+] i in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4,5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+] i , indicating that a U73122-sensitive phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores.

Notes: Abstract. Stimulation with leukotriene D4 (LTD4) (3–100 nm) induces a transient increase in the free intracellular Ca2+ concentration ([Ca2+] i ) in Ehrlich ascites tumor cells. The LTD4-induced increase in [Ca2+] i is, however, significantly reduced in Ca2+-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD4 concentration (3 nm) does not result in any detectable increase in [Ca2+] i . Addition of LTD4 (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD4-induced (100 nm) acceleration of the RVD response is also seen in Ca2+-free medium and also at 3 nm LTD4, indicating that LTD4 can open K+- and Cl−-channels without any detectable increase in [Ca2+] i . Buffering cellular Ca2+ with BAPTA almost completely blocks the LTD4-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca2+] i level after BAPTA-loading or buffering of [Ca2+] i seems to inhibit the LTD4-induced stimulation of the RVD response even though the LTD4-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca2+] i . The LTD4 receptor antagonist L649,923 (1 μm) completely blocks the LTD4-induced increase in [Ca2+] i and inhibits the RVD response as well as the LTD4-induced acceleration of the RVD response. When the LTD4 receptor is desensitized by preincubation with 100 nm LTD4, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD4 plays a role in the activation of the RVD response. LTD4 seems to activate K+ and Cl− channels via stimulation of a LTD4 receptor with no need for a detectable increase in [Ca2+] i .