Spontaneous and depolarization-induced efflux of hypotaurine from mouse cerebral cortex slices: Comparison with taurine and gaba

The spontaneous and potassium- or veratrine-stimulated efflux of [³⁵S]hypotaurine from superfused cerebral cortex slices of adult mice was compared with the release of [³H]taurine and [³H]GABA. Initially GABA was the fastest released. Hypotaurine was, however, eventually released fastest, since its spontaneous efflux did not slow down during superfusions as did taurine and GABA effluxes. More than 60 % of all preloaded labelled amino acids still remained in the slices after 80-min superfusions. The effluxes of all three amino acids were stimulated by potassium and veratrine depolarizations: GABA efflux most and hypotaurine efflux least. The veratrine-stimulated release of taurine was long-lasting, while all other responses started and ended abruptly. With respect to efflux properties hypotaurine resembled more GABA than taurine.     

Taurine and hypotaurine transport in neuroblastoma cells: effects of cations

The cation requirements of [³H]taurine and [³⁵S]hypotaurine uptake by cultured neuroblastoma C1300 cells were compared in Krebs-Ringer-Hepes-glucose medium. The uptakes were strictly sodium-dependent at both low and high taurine and hypotaurine concentrations. The omission of Ca²⁺ or Mg²⁺ ions affected uptakes only marginally. The optimal K⁺ concentration was equal to the physiological concentration, whereas abnormally high K⁺ levels inhibited similarly taurine and hypotaurine uptake. The sodium dependence curves of both uptakes were sigmoidal in character at low and high taurine and hypotaurine concentrations. Hill plots suggest that two Na⁺ ions are coupled with the transfer of one taurine or hypotaurine molecule into neuroblastoma cells. With respect to cation requirements taurine and hypotaurine transports are similar in cultured neuroblastoma cells and display features considered typical of the uptake of a neurotransmitter amino acid.     

Effects of cations on taurine,hypotaurine, and GABA uptake in mouse brain slices

The effects of cations on taurine, hypotaurine and GABA uptake were studied in mouse brain slices under identical experimental conditions. The uptakes were all strictly sodium-dependent. The omission or excess of K⁺ inhibited similarly taurine, hypotaurine and GABA uptake. The effects of omission of Ca²⁺ or Mg²⁺ were less pronounced. In both normal-sodium and low-sodium media all uptakes were saturable, consisting of both low-and high-affinity transport components. TheK _m constants for both low-and high-affinity transport components of hypotaurine and GABA increased in low-sodium medium, suggesting that sodium ions are necessary for their attachment to possible carrier sites in plasma membranes. In the case of taurine, however, the translation rate rather than the affinity of carrier sites was affected in Na⁺-free media. More than two sodium ions may be involved in the transport of one hypotaurine and one GABA molecule, whereas the coupling ratio between sodium and taurine was at least three. In its cation dependence hypotaurine uptake thus resembled more GABA uptake than taurine uptake.     

Glutamate release from cerebellar granule cells differentiating in culture: Modulation of the K+-stimulated release by inhibitory amino acids

The properties of l-[³H]glutamate release with an emphasis on the modulation by inhibitory amino acids of the potassium-induced release were studied with cerebellar granule cells from 7-day-old rats cultured for 7 or 14 days. Spontaneous glutamate release from cells grown for 7 days was fast, being slightly enchanced in Na⁺-free medium. l-Glutamate, kainate and quisqualate stimulated the release whereas N-methyl-d-aspartate and taurine were without any effect. The potassium-evoked glutamate release was Ca²⁺-dependent and potentiated by l-glutamate and quisqualate. Stimulated release was strongly depressed by glutamatediethylester. This inhibition was antagonized by GABA but not by taurine. GABA and its structural analogues taurine, hypotaurine, β-alanine and glycine were all equally effective in depressing stimulated glutamate release. The inhibition by GABA could be blocked by GABA antagonist. Both K⁺-evoked release and the kainate-induced release of glutamate were significantly greater in 14-day-old than in 7-day-old cultures, but the other properties of release were similar. The demonstration of calcium-dependent and potassium-stimulated glutamate release from cerebellar granule cells is consonant with the proposed neurotransmitter role of glutamate in these cells. The release could be modulated by both glutamatergic substances and inhibitory amino acids, the effect of GABA probably being mediated by GABAergic receptors.     

Spontaneous and evoked release of [3H]taurine from a P2 subcellular fraction of the rat retina

The effects of spontaneous and evoked [³H]taurine release from a P₂ fraction prepared from rat retinas were studied. The P₂ fraction was preloaded with [³H]taurine under conditions of high-affinity uptake and then examined for [³H]taurine efflux utilizing superfusion techniques. Exposure of the P₂ fraction to high K⁺ (56 mM) evoked a Ca²⁺-independent release of [³H]taurine. Li⁺ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [³H]taurine compared to the K⁺-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [³H]taurine. The spontaneous release of [³H]taurine was also Ca²⁺-independent. When Na⁺ was omitted from the incubation medium K⁺-evoked [³H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [³H]taurine was inhibited by 60% in the absence of Na⁺. Substitution of Br^– for Cl^– had no effect on the release of either spontaneous or K⁺-evoked [³H]taurine release. However, substitution of the Cl^– with acetate, isethionate, or gluconate decreased K⁺-evoked [³H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [³H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [³H]taurine by approximately 40%. These results suggest that the taurine release of [³H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [³H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [³H]taurine release.     

K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

Taurine and GABA release from mouse cerebral cortex slices: Effects of structural analogues and drugs

The effects of structural analogues, excitatory amino acids and certain drugs on spontaneous and potassium-stimulated exogenous taurine and GABA release were investigated in mouse cerebral cortex slices using a superfusion system. Spontaneous efflux of both amino acids was rather slow but could be enhanced by their uptake inhibitors. Taurine efflux was facilitated by exogenous taurine, hypotaurine, -alanine and GABA, whereas GABA, nipecotic acid and homotaurine effectively enhanced GABA release. The stimulatory potency of the analogues closely corresponded to their ability to inhibit taurine and GABA uptake, respectively, indicating that these efflux processes could be mediated by the carriers operating outwards. Glutamate induced GABA release, whereas taurine efflux was potentiated by aspartate, glutamate, cysteate, homocysteate and kainate. The centrally acting drugs, including GABA agonists and antagonists, as well as the proposed taurine antagonist TAG (6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide), had no marked effects on spontaneous taurine and GABA release. Potassium ions stimulated dosedependently both taurine and GABA release from the slices, the responses of taurine being strikingly slow but sustained. Exogenous GABA and nipecotic acid accelerated the potassium-stimulated GABA release, whereas picrotoxin and bicuculline were ineffective. The potassium-stimulated taurine release was unaltered or suppressed by exogenous taurine and analogues, differing in this respect from GABA release. The apparent magnitude of the depolarization-induced GABA release is thus influenced by the function of membrane transport sites, but the same conclusion cannot be drawn with regard to taurine. Haloperidol and imipramine were able to affect the evoked release of both taurine and GABA.     

Properties of [3H]taurine release from crude synaptosomal fractions of rat cerebral cortex

The release of previously accumulated [³H]taurine and [¹⁴C]GABA from crude synaptosomal (P₂) fractions isolated from rat cerebral cortex was studied using a superfusion system. The spontaneous efflux of [³H]taurine and [¹⁴C]GABA was stimulated by elevated concentrations of K⁺ (15–133 mM) in a concentration-dependent manner. This K⁺-stimulated release of [¹⁴C]GABA but not of [³H]taurine was enhanced in the presence of Ca²⁺. However, addition of 3 mM Ca²⁺ to the superfusion medium in the presence of the ionophore A 23187 resulted in a stimulation of the release of both [³H]taurine and [¹⁴C]GABA. These results are discussed in connection with the cellular localization of tourine in the central nervous system.     

Taurine Release Is Enhanced in Cell-Damaging Conditions in Cultured Cerebral Cortical Astrocytes

The release of preloaded [³H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K⁺ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2-disulphonate) and SITS-(4-acetamido-4-isothiocyanostilbene-2,2-disulphonate) sensitive CI^– channels, since these blockers were able to reduce both basal and K⁺ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K⁺ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.     

Potassium-Stimulated Taurine Release and Nitric Oxide Synthase Activity During Quinolinic Acid Lesion of the Rat Striatum

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K⁺-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K⁺-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K⁺-induced taurine release. We conclude that a substantial part of K⁺-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K⁺-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.     

Osmolarity-sensitive release of free amino acids from cultured kidney cells (MDCK)

Summary The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, -alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25mm. Glutamic acid, taurine and -alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using³H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of³H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na⁺, Cl^– or Ca²⁺, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na⁺/K⁺/2Cl^– carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K⁺ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl^– channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.     

In Vitro Studies on the Putative Function of N-acetylaspartate as an Osmoregulator

Efflux and tissue content of N-acetylaspartate (NAA) and amino acids were evaluated from cultured and acutely prepared hippocampal slices in response to changes in osmolarity. The osmoregulator taurine, but not NAA, was lost from both types of slices after moderate reductions in extracellular osmolarity (−60 mOsm) for 10–48 h. Hypoosmotic shock (−166 mOsm) for 5 min resulted in unselective efflux of several amino acids from acutely prepared slices. Notably, the efflux of taurine, but not NAA, was prominent also after the shock. Efflux of NAA was markedly enhanced by NMDA and high K⁺, in particular after the stimulation period. The high K⁺-mediated efflux was decreased by high extracellular osmolarity and a NMDA-receptor antagonist. The results indicate that NAA efflux can be induced by a sudden non-physiological decrease in extracellular osmolarity but not by prolonged more moderate changes in osmolarity. The mechanisms behind the efflux of NAA by high K⁺ are complex and may involve both swelling and activation of NMDA-receptors.     

Formation and accumulating of hypotaurine in rat liver regenerating after partial hepatectomy

Hypotaurine is considered to be an intermediate in the major pathway for the biosynthesis of taurine in mammals yet is rarely detected in mammalian tissue. The activity of cysteinesulfinic acid decarboxylase, the enzyme presumably responsible for the biosynthesis of hypotaurine, is frequently present in great amounts in tissue, whereas the mechanism for the conversion of hypotaurine to taurine is poorly understood, there being some doubt at present if an enzyme exists for such a purpose. This paper reports the accumulation of hypotaurine in the liver of rats regenerating after partial hepatectomy. Further, the formation and accumulation of [³⁵S]hypotaurine from [³⁵S]methionine under the same conditions was observed. No hypotaurine was detected in liver of sham-operated control animals, even after the intraperitoneal injection of authentic hypotaurine. These observations suggest that rat liver normally possesses a mechanism for the rapid conversion of hypotaurine to taurine and that this mechanism is impeded in liver regenerating after partial hepatectomy.     

Lysophosphatidylcholine Induces Taurine Release from HeLa Cells

The putative role of lysophospholipids in activation and regulation of the volume-sensitive taurine efflux was investigated in HeLa cells using tracer technique. Lysophosphatidylcholine (LPC, 10 μm) with oleic acid increased taurine efflux during hypotonic and isotonic conditions. Substituting palmitic or stearic acid for oleic acid enhanced taurine release during isotonic conditions, whereas ethanolamine, serine or inositol containing lysophospholipids were ineffective. High concentrations of LPC (25 μm) induced Ca²⁺ influx, loss of adenosine nucleotides, taurine and the Ca²⁺-sensitive probe Fura-2, and thus reflected a general breakdown of the membrane permeability barrier. Low concentrations of LPC (5–10 μm) solely induced taurine efflux. The LPC-induced taurine release was unaffected by anion channel blockers (DIDS, MK196) and the 5-lipoxygenase inhibitor ETH 615-139, which all blocked the volume sensitive taurine efflux. Furthermore, LPC-induced taurine release was reduced by antioxidants (NDGA, vitamin E) and the protein tyrosine kinase inhibitor genistein. The swelling-induced taurine efflux was in the absence of LPC unaffected by vitamin E, blocked by genistein, and increased by H₂O₂ and the protein tyrosine phosphatase inhibitor vanadate. It is suggested that low concentrations of LPC permeabilizes the plasma membrane in a Ca²⁺-independent process that involves generation of reactive oxygen species and tyrosine phosphorylation, and that LPC is not a second messenger in activation of the volume sensitive taurine efflux in HeLa cells. Received: 17 December 1999/Revised: 13 April 2000     

Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice

Summary. In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K⁺-stimulated release of [³H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA_B and GABA_C receptors. The involvement of GABA_A receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K⁺-evoked taurine release. Furthermore, several GABA_B receptor effectors were able to inhibit K⁺-stimulated taurine release in the adults, while the GABA_C receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds. Received June 28, 1999, Accepted August 31, 1999     

Sodium-dependent uptake of taurine in encapsulated Staphylococcus aureus strain M

A novel uptake system for the unusual sulfonated amino acid taurine was discovered in the prokaryote, encapsulated Staphylococcus aureus strain M. This strain has been shown previously to contain taurine in its capsular polysaccharide. Taurine uptake by whole cells incubated in buffer showed a saturable dependency upon Na⁺ and taurine uptake was itself a saturable process, stimulated by glucose, and markedly affected by temperature. No evidence was found for the inducibility of taurine uptake. In the presence of 10 mM NaCl Lineweaver-Burk plots revealed a K_m of 42 μM and V_max of 4.6 nmol/min per mg dry weight for taurine uptake at 37°C. Increasing concentrations of Na⁺ decreased the K_m of the system and appeared to increase the V_max. Of various other cations tested only Li⁺ supported marked taurine uptake. Excess unlabelled taurine did not cause efflux of radioactivity taken up. Taurine was taken up into cold trichloroacetic acid-soluble material and did not chromatograph as taurine, indicating rapid metabolism during or closely following uptake. Taurine uptake appeared to occur via a highly specific system because amino acids representing the major known groups of amino acid transport systems in S. aureus did not inhibit taurine uptake, and uptake was only slightly diminished by the structurally closely related compounds hypotaurine and 3-amino-1-propane sulfonic acid. Sulfhydryl group reagents, electron transport inhibitors, an uncoupler and inhibitors of Na⁺-linked transport processes inhibited taurine uptake. A variety of other metabolic inhibitors had little effect on taurine uptake.     

Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]i and calmodulin

Cell swelling results in regulatory activation of multipleconductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement ofextracellular and intracellularCa²⁺ in volume-dependent[³H]taurine effluxfrom primary cultured astrocytes and compare theCa²⁺ sensitivity of this efflux inslow (high K⁺ medium induced) andfast (hyposmotic medium induced) cell swelling. NeitherCa²⁺-free medium norCa²⁺-channel blockers prevented thevolume-dependent[³H]taurine release.In contrast, loading cells with the membrane-permeable Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed[³H]taurine efflux by65-70% and 25-30% underhigh-K⁺ and hyposmotic conditions,respectively. Fura 2 measurements confirmed that BAPTA-AM, but notCa²⁺-free media, significantlyreduced resting intracellular Ca²⁺concentration([Ca²⁺]_i).The calmodulin antagonists trifluoperazine and fluphenazine reversiblyand irreversibly, respectively, inhibited thehigh-K⁺-induced[³H]taurine release,consistent with their known actions on calmodulin. In hyposmoticconditions, the effects were less pronounced. These data suggest thatvolume-dependent taurine release requires minimal basal[Ca²⁺]_iand involves calmodulin-dependent step(s). Quantitative differences inCa²⁺/calmodulin sensitivity ofhigh-K⁺-induced and hyposmoticmedium-induced taurine efflux are due to both the effects of theinhibitors on high-K⁺-induced cellswelling and their effects on transport systems and/or signalingmechanisms determining taurine efflux.

     

The effect of taurine on the efflux of calcium from locust mitochondria

The effect of taurine has been studied on ⁴⁵Ca²⁺ efflux from mitochondria obtained from the flight muscle and thoracic ganglia of the desert locust. Mitochondria from both tissues readily accumulated ⁴⁵Ca²⁺ and this uptake was stimulated by the presence of phosphate ⁴⁵Ca²⁺ accumulation was abolished by ruthenium red (5 μ M). Only in the presence of 10 mM Na⁺ and either ruthenium red (5 μ M) or EGTA (500 μ M), was an efflux of ⁴⁵Ca²⁺ observed. Taurine (20 mM) abolished the Na⁺-dependent ⁴⁵Ca²⁺ efflux but had no effect in the absence of Na⁺. These results suggest that taurine may contribute to the control of the concentration of intracellular free calcium.     

Characteristics of taurine release in slices from adult and developing mouse brain stem

Summary. Taurine has been thought to function as a regulator of neuronal activity, neuromodulator and osmoregulator. Moreover, it is essential for the development and survival of neural cells and protects them under cell-damaging conditions. Taurine is also involved in many vital functions regulated by the brain stem, including cardiovascular control and arterial blood pressure. The release of taurine has been studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release have not been systematically characterized in the brain stem. The properties of release of preloaded [³H]taurine were now characterized in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice, using a superfusion system. In general, taurine release was found to be similar to that in other brain areas, consisting of both Ca²⁺-dependent and Ca²⁺-independent components. Moreover, the release was mediated by Na⁺-, Cl⁻-dependent transporters operating outwards, as both Na⁺-free and Cl⁻ -free conditions greatly enhanced it. Cl⁻ channel antagonists and a Cl⁻ transport inhibitor reduced the release at both ages, indicating that a part of the release occurs through ion channels. Protein kinases appeared not to be involved in taurine release in the brain stem, since substances affecting the activity of protein kinase C or tyrosine kinase had no significant effects. The release was modulated by cAMP second messenger systems and phospholipases at both ages. Furthermore, the metabotropic glutamate receptor agonists likewise suppressed the K⁺-stimulated release at both ages. In the immature brain stem, the ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) potentiated taurine release in a receptor-mediated manner. This could constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.     

Calcium Modulates Osmosensitive Taurine Efflux in HeLa Cells

The role of Ca²⁺ in the signaling transduction pathway involved in osmosensitive taurine efflux in HeLa cells was studied using radiotracer efflux techniques. Taurine efflux induced by extracellular hypotonicity was decreased by 85% by removal of extracellular Ca²⁺ and simultaneous depletion of intracellular Ca²⁺ stores with thapsigargin. Extracellular Ca²⁺ removal, thapsigargin treatment, or addition of Gd³⁺ all decreased taurine efflux by ～50%. To explore the putative signal transduction pathways involved in swelling-induced taurine efflux, HeLa cells were exposed to PP1, an inhibitor of the Src family of tyrosine kinases, the phospholipase C inhibitor U73122, the IP₃ receptor antagonist 2-APB, and the generic protein kinase C inhibitor chelerythrine. All of these treatments caused ～50% inhibition of taurine release in Ca²⁺-rich extracellular medium and ～85%–90% in Ca²⁺-free conditions. The inhibitors of the conventional protein kinase C isoforms BIM-1 and Gö6976 reduced taurine efflux to a lesser extent. Acute (10-min) exposure to the phorbol ester tetradecanoyl phorbol acetate (TPA) increased taurine efflux in 25%, whilst overnight exposure had an inhibitory effect decreasing efflux by 22%. A working model for activation of osmosensitive taurine efflux in HeLa cells involving different Ca²⁺ signaling pathways is presented.