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The endogenous taurine content, and the uptake and release of [3H]taurine were examined using the rat superior cervical ganglion. Taurine was found to be one of the most abundant amino acids in the superior cervical ganglion, and the superior cervical ganglion took up [3H]taurine from the incubation medium. Carbachol stimulated the release of [3H]taurine in a concentration-dependent manner with an EC50 of 26 microM and maximal stimulation at 100 microM. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium stimulated release with the same potency but with greater efficacy than carbachol. The nicotinic receptor antagonist hexamethonium (1 mM) inhibited carbachol-stimulated release by 74%. (+/-)-Muscarine stimulated release with an EC50 of 8 microM but with a maximal effect of only 32% of that produced by 100 microM carbachol. Oxotremorine, another muscarinic receptor agonist, was ineffective, even at 1 mM. The muscarinic receptor antagonist atropine inhibited carbachol-stimulated release by 30% at 10 microM. These results show that [3H]taurine release from rat superior cervical ganglion can be stimulated by cholinergic receptor agonists. Release is mediated predominantly by a nicotinic receptor and partially by a muscarinic receptor.

Astroglial cells are known to release taurine in response to stimulation by a variety of stimuli including beta-adrenergic receptor agonists such as isoproterenol (IPR). The effects of changing osmolarity and extracellular [K+] on IPR-stimulated taurine release were studied with LRM55 cells, a continuous astroglial cell line. IPR-stimulated taurine release decreased almost 8% for each 1% increase in osmolarity, indicating that IPR-stimulated release is highly regulated by the osmolarity of the medium. IPR-stimulated taurine release was greatly enhanced when external [K+] was increased isosmotically by substituting KCl for NaCl but was strongly suppressed when external [K+] was increased hyperosmotically by adding KCl to the medium. Both IPR-stimulated and K(+)-stimulated taurine release depended on external [Cl-]; IPR-stimulated release declined approximately in parallel to K(+)-stimulated release as [Cl-] in the medium was reduced. The high sensitivity of IPR-stimulated release to factors that change cell volume (osmolarity, external [K+], external [Cl-]) is consistent with the idea that IPR, elevated [K+], and reduced osmolarity all elicit taurine release via a single tension-controlled mechanism.

Taurine is stored in and released from astroglial cells. We have investigated whether taurine is stored in multiple subcellular compartments and whether taurine is released from a distinct pool. Taurine compartmentation was examined by determining the subcellular distribution of labelled taurine and by comparing the specific radioactivities of taurine in cells with that of the taurine released into the superfusion medium during receptor stimulation. Three observations indicate that taurine is found in a single subcellular pool. First, labelled taurine was localized exclusively within a freely exchangeable pool and not sequestered in membrane-bound compartments. Second, endogenous and newly acquired [14C]taurine was released identically by the beta-adrenergic agonist isoproterenol (IPR). Third, measurements of the specific radioactivity of taurine indicate that accumulated [14C]taurine mixes homogeneously with endogenous taurine and both are simultaneously released from the same pool. Chemical analysis revealed net uptake of taurine from the medium during loading and a net loss during IPR-stimulated release. Taurine was selectively released by IPR; five other amino acids found in the superfusion medium were unaffected. One possible mechanism capable of explaining these results is that receptor-mediated taurine release from astroglia occurs by a selective transporter moving taurine from a single cytoplasmic pool to the extracellular space.

The effects of [K+]o on taurine release from glial cells were studied with primary cultures of cerebellar astrocytes and with LRM55 cells, a continuous glial cell line. The characteristics of K(+)-stimulated taurine release were virtually identical in the 2 cell types. Both cerebellar astrocytes and LRM55 cells released taurine when stimulated with high-K+ medium prepared by isosmotically substituting KCl for NaCl, but neither cell type released taurine when stimulated with hyperosmotic high-K+ medium prepared by adding solid KCl to control medium. The membrane potential of LRM55 cells was measured by intracellular recording and was insensitive to changes in [K+]o below 20 mM. LRM55 cells released taurine when stimulated with nondepolarizing concentrations of K+ (13-22 mM) if the isosmotically prepared high-K+ medium was used, but the cells did not release taurine when treated with a depolarizing concentration of K+ (50 mM) if hyperosmotic high-K+ medium was used. The time course of K(+)-stimulated taurine release was quite slow, having a time to peak of 10-15 min. Small changes (2.5-10%) in the osmolarity of the medium strongly affected taurine release by cerebellar astrocytes and LRM55 cells. K(+)-stimulated taurine release from both cell types was inhibited when the osmolarity was increased with sucrose or NaCl and was enhanced when the osmolarity was reduced. Similarly, baseline taurine release was suppressed by small elevations in osmolarity and increased by reduced osmolarity.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.

Adrenergic stimulation of LRM55 glial cells results in the release of the neuroactive amino acid taurine. The present study characterizes the receptors involved in taurine release and shows that taurine release is mediated by cyclic adenosine 3',5'-monophosphate (cAMP). beta-Receptors in LRM55 cells were first characterized by [125I]iodohydroxybenzylpindolol binding. Binding was stereospecific and saturable with time and ligand concentration. Kinetic analysis of equilibrium binding at 37 degrees C revealed a single component of high affinity (Km = 113 pm; Bmax = 52.1 +/- 5.0 fmol/mg of protein). The pharmacologies of the stimulation of cAMP accumulation and taurine release were similar. The agonists isoproterenol (IPR), epinephrine (E) and norepinephrine (NE) showed a rank order of potency characteristic of a beta-adrenergic system (IPR greater than E greater than or equal to NE). The beta-antagonists alprenolol and propranolol inhibited the IPR stimulation of both processes; the alpha-antagonist phentolamine did not. The dependence of taurine release on cAMP was further suggested by the similarity of the two time courses and was demonstrated by the stimulation of taurine release by the cAMP analogue dibutyryl cAMP. Thus, one physiological response of glial cells to beta-adrenergic stimulation is the release of taurine. Receptor-activated release of taurine from glia represents a previously undescribed neuronal-glial interaction by which glia may actively regulate neuronal excitability.

Astroglial cells release taurine when stimulated by beta-adrenergic agonists and other neuroactive agents. The Ca2+-dependency of taurine release by an LRM55 astroglial cell line was investigated by removing Ca2+ from the perfusion medium and by using three inorganic and three organic Ca2+-channel blockers (Mn2+, Co2+, Cd2+, verapamil, nifedipine, and diltiazem). Spontaneous release and release stimulated by the beta-adrenergic agonist isoproterenol were not inhibited when cells were perfused with medium containing no added Ca2+ and 10 microM EGTA. Isoproterenol-stimulated taurine release was not blocked when extracellular Ca2+ was completely replaced by Mn2+, Co2+, or Cd2+, nor was it blocked by verapamil, nifedipine, or diltiazem. In fact isoproterenol-stimulated taurine release was increased by 50 microM diltiazem and when Ca2+ was replaced by Co2+. The rate of spontaneous release increased slowly and continually when Co2+ was substituted for Ca2+ but was almost unaffected by substitution of Mn2+ or Cd2+. Application of diltiazem increased spontaneous release significantly, while verapamil and nifedipine appeared to cause small increases. These results indicate that entry of Ca2+ from the extracellular medium is not required for either receptor-mediated or spontaneous taurine release from astroglial cells. Some other changes in the medium did strongly affect release. Both spontaneous and isoproterenol-stimulated release were inhibited by elevated osmotic pressure, and spontaneous release was greatly increased when Ca2+ was completely removed without substituting another divalent cation. Spontaneous release increased when antagonistic metal ions were replaced with Ca2+ and when organic channel blockers were removed.

When astroglial cells are exposed to beta-adrenergic agonists for long periods of time (greater than 20 min), transient increases in taurine release and intracellular cyclic AMP (cAMP) are observed. Three phases of taurine release can be distinguished: activation, inactivation, and an elevated steady state. In this article, we present data describing the relationship between intracellular cAMP levels and inactivation of taurine release. To do this, we compared the apparent first-order rate constants for the inactivation of taurine release (ktau) with the apparent first-order rate constant for the decline of intracellular cAMP (kcAMP). We also measured ktau under experimental conditions that were chosen to provide a wide range of intracellular cAMP concentrations or to stimulate release without the involvement of the beta-adrenergic receptor and adenylate cyclase. When taurine release was stimulated with a saturating concentration of isoproterenol, the inactivation of release was significantly faster than the decline of intracellular cAMP. Furthermore, there were no significant differences in ktau measured under any of the experimental conditions used. Thus, inactivation of taurine release does not involve changes in the activity of the beta-adrenergic receptor and adenylate cyclase, i.e., desensitization, and appears to be independent of the intracellular concentration of cAMP. These results indicate that cAMP-mediated events can be regulated by mechanism(s) in addition to those that control receptor-adenylate cyclase interactions and the synthesis of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of triethyltin (TET) on the transport of taurine, glutamate, lysine, Na+, K+ (using 86Rb+ as tracer), and Cl- by LRM55 glioma cells were examined. Taurine transport was inhibited by TET at much lower concentrations (IC50 = 2.5 microM) than either glutamate or lysine transport (135 and 110 microM, respectively). TET had no significant effect on Na+, Cl-, or 86Rb+ influx at the low concentrations greater than 100 microM. The failure of low concentrations (less than or equal to 10 microM) of TET to affect ion transport indicated that inhibition of taurine transport was not secondary to effects of TET on ion movements or gradients. This conclusion was supported by the observation that neither ouabain nor furosemide, which do affect ion movement and gradients, strongly inhibited taurine transport. Uncouplers and inhibitors of oxidative phosphorylation (cyanide, 2,4-dinitrophenol, and carbonyl cyanide-m-chlorophenyl hydrazone) also had only small effects on taurine transport, suggesting that inhibition by TET was not secondary to possible effects on oxidative phosphorylation. TET had no effect on the efflux of taurine from LRM55 cells at the low concentrations that inhibit uptake, but it induced a nonspecific increase in membrane permeability at much higher concentrations (greater than 100 microM). Tri-n-propyltin and tri-n-butyltin were also potent inhibitors of taurine transport (IC50 = 2.3 and 11 microM, respectively), but trimethyltin was much less potent (144 microM).

Stimulation of beta-adrenergic receptors on LRM55 astroglial cells results in cAMP-dependent release of taurine. We have previously demonstrated that extracellular Ca2+ is not required for either spontaneous or receptor-mediated taurine release (Martin et al., 1988b). In the present series of experiments we investigated the relationship between changes in intracellular free Ca2+ ([Ca2+]i) and taurine release. [Ca2+]i was measured using the fluorescent probe fura-2 and was manipulated by changing the concentration of Ca2+ in the incubation medium and by using the Ca2+ ionophore ionomycin. [Ca2+]i was reduced from 150 +/- 95 nM (n = 46) in control medium (containing 1.1 mM CaCl2) to 46 +/- 10 nM (n = 43) in saline containing no CaCl2 and 10 microM EGTA. [Ca2+]i was rapidly elevated to greater than or equal to 1 microM in medium containing 100 microM CaCl2 and 10 microM ionomycin. Taurine release, either spontaneous or stimulated by isoproterenol, was not significantly affected by these manipulations of [Ca2+]i. [Ca2+]i did not change when cells were stimulated with 100 nM isoproterenol in either control saline containing 1.1 mM CaCl2 or in CaCl2-free saline containing 10 microM EGTA. Other secretogogs (serotonin and ethanol) did not cause changes in [Ca2+]i. These data indicate that neither spontaneous or receptor-mediated taurine release from astroglial cells is Ca2+ dependent. However, when cells were preloaded with Ca2+, allowed to recover briefly, and then stimulated with isoproterenol, it was possible to demonstrate transient increases in Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of adrenergic receptors in primary cultures of astrocytes and LRM55 glial cells stimulates taurine release. Release is inhibited by the beta-adrenergic antagonists alprenolol and propanolol but not by the alpha-adrenergic antagonist phentolamine. Receptor-mediated taurine release occurs via a specific mechanism, since efflux of other amino acids and metabolites is not affected by the beta-adrenergic agonist isoproterenol.