Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2+.

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6's physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich-Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca(2+) is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium.

Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels.

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.

Calyculin A modulates the kinetic constants for the Na+-coupled taurine transport in Ehrlich ascites tumour cells.

The effect of the phosphatase inhibitor calyculin A (cal A) on the kinetic parameters of the Na+-coupled taurine uptake via the taurine transporter in the Ehrlich ascites tumour cells has been investigated. Preincubation with cal A (100 nM) reduces the initial taurine influx by about 20%, but has no effect on the diffusional component of the taurine influx or on the taurine release from cells suspended in isotonic or in hypotonic medium. Thus, cal A-sensitive phosphatases only affect taurine transport mediated by the Na+-dependent taurine transporter. Cal A increases the Michaelis-Menten constant for binding of taurine to the transporter from 31+/-6 to 45+/-4 microM and reduces the taurine transport capacity from 210+/-20 to 170+/-10 nmol x g dry wt(-1) x min(-1) [corrected]. The Michaelis-Menten constant for binding of Na+ to the taurine transporter is concomitantly increased from 96+/-11 to 129+/-8 mM and the Na+:taurine coupling ratio for activation of the transport cycle is reduced from 3.3+/-0.6 to 2.4+/-0.2. This suggests that cal A-sensitive phosphatases maintain a high affinity of the taurine transporter towards Na+ and taurine as well as a high taurine transport capacity in unpertubated Ehrlich cells.

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells.

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells.

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.

Physiological role of taurine--from organism to organelle.

Taurine is often referred to as a semi-essential amino acid as newborn mammals have a limited ability to synthesize taurine and have to rely on dietary supply. Taurine is not thought to be incorporated into proteins as no aminoacyl tRNA synthetase has yet been identified and is not oxidized in mammalian cells. However, taurine contributes significantly to the cellular pool of organic osmolytes and has accordingly been acknowledged for its role in cell volume restoration following osmotic perturbation. This review describes taurine homeostasis in cells and organelles with emphasis on taurine biophysics/membrane dynamics, regulation of transport proteins involved in active taurine uptake and passive taurine release as well as physiological processes, for example, development, lung function, mitochondrial function, antioxidative defence and apoptosis which seem to be affected by a shift in the expression of the taurine transporters and/or the cellular taurine content.

Cellular model for induction of drip loss in meat.

Drip loss from porcine muscle (M. longissimus dorsi) contained high concentrations of K(+) ( approximately 135 mM) and organic osmolytes, for example, taurine ( approximately 15 mM), as well as significant amounts of protein ( approximately 125 mg.mL(-1)). Thus, the drip reflects release of intramuscular components. To simulate events taking place at the time of slaughter and leading to release of osmolytes and subsequent formation of drip loss, C2C12 myotubes were exposed to anoxia and reduction in pH (from 7.4 to 6.0). Anoxia and acidification increased the cellular Ca(2+) concentration ([Ca(2+)](i)) at a rate of 22-32 nM.min(-)(1). The anoxia-induced increase in [Ca(2+)](i) was mainly due to influx via sarcolemmal Na(+) channels. As mammalian cells swell and release lysophospholipids during anoxia, C2C12 cells and primary porcine muscle cells were exposed to either hypotonic shock or lysophosphatidylcholine (LPC) and the release of taurine was followed. The swelling-induced taurine efflux was blocked in the presence of the anion channel blocker (DIDS), the 5-lipooxygenase inhibitors (ETH 615-139 and NDGA) but unaffected by the presence of vitamin E. In contrast, the LPC-induced taurine release was unaffected by DIDS but abolished by antioxidants (butylated hydroxytoluene and vitamin E). Thus, stress-induced taurine release from muscles may precede by two different mechanisms, one being 5-lipooxygenase dependent and the other involving generation of reactive oxygen species. A model for the cellular events, preceding formation of drip in meat, is presented.

On the similarity between the small Cl- channel and the taurine channel activated after cell swelling in Ehrlich ascites tumor cells.

The swelling-activated Cl- efflux, mediated by the mini Cl- channel (3-7 pS), and the swelling-activated taurine efflux in Ehrlich cells are both inhibited by the anion channel blocker MK196 (indacrinone). The swelling-activated taurine efflux is also inhibited by DIDS and following depolarization of the cell membrane, but it is significantly stimulated by arachidonic acid in the micromolar range. The swelling-activated Cl- efflux is, in contrast, only slightly affected by DIDS, unaffected by the membrane potential, and completely blocked by arachidonic acid in the micromolar range. It is suggested that the swelling-activated mini Cl- channel and the taurine channel in the Ehrlich cells represent two distinct types of channels.

Cell volume regulation: physiology and pathophysiology.

Cell volume perturbation initiates a wide array of intracellular signalling cascades, leading to protective and adaptive events and, in most cases, activation of volume-regulatory osmolyte transport, water loss, and hence restoration of cell volume and cellular function. Cell volume is challenged not only under physiological conditions, e.g. following accumulation of nutrients, during epithelial absorption/secretion processes, following hormonal/autocrine stimulation, and during induction of apoptosis, but also under pathophysiological conditions, e.g. hypoxia, ischaemia and hyponatremia/hypernatremia. On the other hand, it has recently become clear that an increase or reduction in cell volume can also serve as a specific signal in the regulation of physiological processes such as transepithelial transport, cell migration, proliferation and death. Although the mechanisms by which cell volume perturbations are sensed are still far from clear, significant progress has been made with respect to the nature of the sensors, transducers and effectors that convert a change in cell volume into a physiological response. In the present review, we summarize recent major developments in the field, and emphasize the relationship between cell volume regulation and organism physiology/pathophysiology.

Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants.

The involvement of group VI Ca(2+)-independent PLA(2)s (iPLA(2)-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0-6 h) increase in bromoenol lactone (BEL)-sensitive iPLA(2) activity, which was suppressed by specific short interfering (si)RNA knockdown of iPLA(2)-VIA. OGD was associated with an increase in iPLA(2)-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA(2)-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA(2)-VIA homolog to catalytically inactive 50-kDa iPLA(2)-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this approximately 50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA(2)-VIA seemed to predominantly reside at the endoplasmatic reticulum, where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA(2)-VIA knockdown or pharmacological inhibition (10 microM BEL), whereas iPLA(2)-VIA overexpression had no effect on cell viability. Taken together, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA(2)-VIA activity that decreases cell viability. iPLA(2)-VIA activation may be modulated by changes in the levels of active and inactive iPLA(2)-VIA isoforms.

Activation of PLA2 isoforms by cell swelling and ischaemia/hypoxia.

Phospholipase A2 (PLA2) activity is increased in mammalian cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid and membrane-bound lysophospholipids. Even though arachidonic acid acts as a second messenger in its own most mammalian cells seem to rely on oxidation of the fatty acid into highly potent second messengers via, e.g. cytochrome P450, the cyclo-oxygenase, or the lipoxygenase systems for downstream signalling. Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the cell type and the physiological stress condition.

Effect of arachidonic acid, fatty acids, prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells.

Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE2 inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE2-induced Na uptake. PGE2 has no effect on the volume-activated K and Cl permeabilities. LTB4, LTC4 and LTE4 inhibit RVD insignificantly in hypotonically swollen cells. LTD4, moreover, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD4 even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.

Reactive oxygen species regulate swelling-induced taurine efflux in NIH3T3 mouse fibroblasts.

NIH3T3 mouse fibroblasts generate reactive oxygen species (ROS) and release taurine following exposure to hypotonic medium and to isotonic medium containing the lipase activator melittin. The swelling-induced taurine release is potentiated by H2O2, the calmodulin antagonist W7, and ATP, but inhibited by the antioxidant butulated hydroxytoluene (BHT), the NAD(P)H oxidase inhibitor diphenylene iodonium (DI), and the iPLA2 inhibitor bromoenol lactone (BEL). The swelling-induced ROS production is also inhibited by BHT and BEL. H2O2 does not affect the volume set point for activation of the volume-sensitive taurine efflux. The 5-lipoxygenase (5-LO) inhibitor ETH 615-139 impairs the swelling-induced taurine efflux in the absence as well as in the presence of H2O2. The melittin-induced taurine release is, in analogy with the swelling-induced taurine release, potentiated by H2O2 and inhibited by BHT, DI, BEL, ETH 615-139 and anion channel blockers. Thus, swelling- and melittin-induced cell signalling and taurine release involve joint elements. The swelling-induced taurine efflux is potentiated by the protein tyrosine phosphatase inhibitor vanadate, and the potentiating effect of H2O2 and vanadate is impaired in the presence of protein tyrosine kinase inhibitor genistein. It is suggested that (i) iPLA2 and 5-LO activity is required for the swelling-induced activation of taurine efflux from NIH3T3 cells, (ii) ROS are produced subsequent to the PLA2 activation by the NAD(P)H oxidase complex, and (iii) ROS inhibit a protein tyrosine phosphatase (PTP1B) causing a potentiation of the swelling-induced taurine release.

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells.

The nature of the leukotriene-D4 (LTD4) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD4 treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD4 also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO3. On the other hand, LTD4 fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD4 in Ehrlich cells induces Cl-independent K loss through the Ca2+-dependent K channels. However, the effect of physiological doses of LTD4 on cell volume seems not to be as potent in Cl-free, NO3 cells when compared to Cl-containing cells, indicating that LTD4 in Ehrlich cells also provokes Cl-dependent K loss. LTD4 seems not to produce K loss through an electroneutral K+/H+ exchange system. LTD4 still produces Cl-independent K loss and cell shrinkage in the presence of the anti-calmodulin drug pimozide but not in the presence of the LTD4 receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD4-induced shrinkage. It is suggested that the LTD4-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD4 might exert its effect through another lipoxygenase product. The Ca2+-calmodulin complex is not involved in the LTD4-induced activation of K and Cl transporting systems.

Ca2+-mediated potentiation of the swelling-induced taurine efflux from HeLa cells: on the role of calmodulin and novel protein kinase C isoforms.

The present work sets out to investigate how Ca(2+) regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca(2+)-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca(2+)-free or high-K(+) media. Addition of Ca(2+)-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca(2+)-activated K(+) channels. The taurine release from control cells and cells exposed to Ca(2+) agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca(2+)-augmented taurine release. Exposure to Ca(2+)-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca(2+)-concentration has returned to the unstimulated level. The Ca(2+)-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca(2+)-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca(2+) regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).

Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells.

The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl- but inhibited by addition of MK196 (anion channel blocker) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl-, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a "Mini Cl- channel." The Cl- efflux via this Cl- channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS and inhibited by arachidonic acid and oleic acid. It is suggested that the swelling-activated "Mini Cl- channel" and the swelling-activated taurine channel in the Ehrlich cell represent two distinct types of channels.

Lysophosphatidylcholine-induced taurine release in HeLa cells involves protein kinase activity.

It has recently been demonstrated that exogenous addition of low concentrations (< 15 microM) of lysophosphatidyl choline (LPC, palmitic acid in the sn-1 position) induces a transient increase in taurine efflux from HeLa cells in a process that seems to involve generation of reactive oxygen species (ROS) and tyrosine phosphorylation (J. Membrane Biol. 176 (2000) 175-185). We now demonstrate that LPC also induces release of taurine under isotonic conditions in mouse fibroblast (NIH/3T3) and Ehrlich ascites tumor cells. Furthermore, we show that in the case of HeLa cells addition of the calmodulin antagonist W-7 (50 microM) or the calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 (10 microM) reduces the LPC-induced taurine release under isotonic conditions. Conversely, addition of a standard protein kinase C (PKC) inhibitor chelerythrine (10 microM) leads to a potentiation of the LPC-induced taurine efflux, whereas direct activation of PKC by the phorbol ester PMA has no effect. It is suggested that the putative generation of ROS following addition of LPC is modulated by calmodulin/CaMKII, and that the effect of chelerythrine is more likely related to the ROS production than to PKC inhibition.

Phosphorylation is involved in the regulation of the taurine influx via the beta-system in Ehrlich ascites tumor cells.

The role of 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the beta-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the beta-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the beta-system is stimulated by an increased level of cAMP or following addition of N6,2'-O-dibutyryl-3',5'-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the beta-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the beta-system is in State II.