Tyrosine Hydroxylase Inactivation Following cAMP-Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho-diesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co-substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos-phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.     

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [³²P]P_i and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. ³²P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of ³²P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of ³²P into tyrosine hydroxylase, with 50 μM veratridine producing a 5-fold increase in ³²P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 μM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca²⁺. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of ³²P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.     

Calcium-dependent activation of tryptophan hydroxylase by ATP and magnesium

Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg⁺⁺ in the presence of subsaturating concentrations of the cofactor 6-methyltetrahydropterin (6MPH₄). The activation of tryptophan hydroxylase under these conditions results from a reduction in the apparent K_m for 6MPH₄ from 0.21 mM to 0.09 mM. The activation requires Mg⁺⁺ and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg⁺⁺ on enzyme activity was enhanced by μM concentrations of Ca⁺⁺ and totally blocked by EGTA. These data suggest that tryptophan hydroxylase can be activated by a cyclic nucleotide independent protein kinase which requires low calcium concentrations for the expression of its activity.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

Tyrosine hydroxylase of sheep brain: some catalytic and chemical properties of the detergent-solubilized, partially purified enzyme

Abstract– Detergent-solubilized tyrosine hydroxylase from the caudate nucleus of the sheep was purified 3-fold by affinity chromatography on 3-iodotyrosine modified agarose. Supplementation of the standard assay with 1 mM Fe²⁺ resulted in only slight stimulation of the enzymic activity. The enzyme was, however, markedly inhibited by certain complexing agents specific for either Fe²⁺ or Fe³⁺. Double reciprocal plots of the kinetic data for a representative complexing agent, bathophenanthroline, showed the inhibition to be competitive with O₂ (apparent K_m 0.15 mM) and noncompetitive with either l -tyrosine or the synthetic tetrahydropterin cofactor DMPH₄ (apparent K_m's 0.12 and 0.29 mM, respectively). The combined inhibition and kinetics studies strongly suggest that brain tyrosine hydroxylase is an iron enzyme. Furthermore, the prosthetic iron very likely participates directly in catalytic function, presumably by binding molecular oxygen. The activity of ammonium sulphate-precipitated enzyme was found to be stimulated 2-fold by Fe²⁺, catalase or peroxidase, while untreated enzyme was markedly less affected by these agents. Since the only ostensible difference between the two preparations was the extensive aggregation present in the former case, the change in physical state evoked by ammonium sulphate precipitation appeared to be somehow related to this peculiar property of the enzyme.     

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus,hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of ³H₂O from L-4-³H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of ³H₂O from L-4-³H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank ³H₂O produced from L-4-³H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank ³H₂O produced from L-3,5-³H-tyrosine. The K_m values of tyrosine hydroxylase for phenylalanine determined by the production of ³H₂O from L-4-³H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of ³H₂O formed from L-4-³H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.     

Phosphorylation and Activation of Tryptophan Hydroxylase by Exogenous Protein Kinase A

Abstract: The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in V_max without alterations in the K_m for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.     

INHIBITION OF GUINEA-PIG BRAIN TYROSINE HYDROXYLASE BY CATECHOLS AND BIOPTERIN

—The inhibition by catechols and biopterin of tyrosine hydroxylase from guineapig caudate nuclei has been examined. Inhibitory constants of 10–20 μm were obtained for dopamine and noradrena-line and 150–250 μm for l -DOPA and dihydroxyphenylacetic acid. When examined under similar conditions homovanillic acid was found not to be inhibitory. Using an acetone dried powder as the source of tyrosine hydroxylase no change in K_m or V_max was observed when cyclic AMP or Ca²⁺ were added to the medium. Enzyme mechanisms and a possible explanation of the mechanisms controlling catechol synthesis are discussed.     

Tyrosine hydroxylase activity in the central nervous system of the crayfish,Pacifastacus leniusculus (Crustacea,Decapoda)

Summary Tyrosine hydroxylase, responsible for the formation ofl-dopa froml-tyrosine, has been identified in the central nervous system of the crayfish,Pacifastacus leniusculus (Crustacea, Decapoda). It requires pterine as cofactor and is inhibited by a number of known tyrosine hydroxylase inhibitors; iron-chelators, tyrosine analogues and also by the catecholamines, dopamine and noradrenaline. Iron enhances the activity of the enzyme. It differs from the vertebrate tyrosine hydroxylase in having a more alkaline pH optimum and a higher affinity for the pterine cofactor. Kinetic studies were performed andK _m andV _max values are presented. Dopa formed was identified and quantitatively measured by high pressure liquid chromatography (HPLC) and electrochemical detection.     

The use of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin in the analysis of nonphosphorylated and phosphorylated rat striatal tyrosine hydroxylase at pH 7.0

The kinetics of tyrosine hydroxylase from the desalted high-speed supernatants of rat striatal homogenates were examined at pH 7.0 using different concentrations of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin, ranging from 4 μM to 1.5 mM. All analyses were performed using two different buffering solutions and their appropriate reducing systems for maintaining cofactor in the reduced state. In the presence of phosphate buffer the results show that tyrosine hydroxylase exists in two kinetically different forms with apparent K_m values for the cofactor of 16 μM (low K_m) and 2.3 mM (high K_m). Similar results were obtained using MOPS buffer. A comparative analysis of the appropriate V_max values indicates that tyrosine hydroxylase as obtained by our standard preparation procedures is predominately (95%) in the high K_m form. When the striatal supernatant was exposed to phosphorylating conditions and subsequently analyzed it appeared that the enzyme now existed totally in the low K_m form with very little change in the overall V_max. A comparison of the results using the two different buffering systems, phosphate and MOPS, revealed that following phosphorylation a large percentage of enzyme was maintained in the phosphorylated state only when using phosphate buffer. In light of the present results, we can for the first time suggest a functional significance not only for the two apparently different kinetic forms of the enzyme but also for a supporting role for phosphorylation. In vivo dopamine synthesis may be accomplished to a significant extent by the phosphorylated form of the enzyme while the non-phosphorylated form may constitute a relatively inactive reservoir which can be recruited for increased dopamine synthesis by phosphorylation.     

The influence of neuropeptides on amine synthesis: Cholecystokinin-8 and neurotensin reduce striatal tyrosine hydroxylase activity

Cholecystokinin-8 and neurotensin, neuropeptides found in relatively high concentrations in some catcholaminergic neurons and/or terminal or cell body areas, reduced native and polyanion-activated rat striatal tyrosine hydroxylase activity, but not that of the enzyme activated by phosphorylating conditions. Substance P, γ₃-melanocyte-stimulating hormone, [d-ala², d-leu⁵]-enkephalin, or thyrotropin-releasing hormone had no effect on the native enzyme or on either activated form. Cholecystokinin-8 and neurotensin may interact with polyanion-activated or membrane bound-activated tyrosine hydroxylase to modulate its activity.     

PARTIAL PURIFICATION AND PROPERTIES OF CHOLINE KINASE (EC 2.7.1.32) FROM RABBIT BRAIN: MEASUREMENT OF ACETYLCHOLINE

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min^?1 mg^?l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS0₄, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm ) to phosphorylcholine [³²P] in the presence of ATP [y³²P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two K_m values for choline at pH 9.5; 32 μm and 0.31 mm . At pH 7.4, the higher K_m was not observed and enzyme activity was maximal with 0.1 mm choline. The K_m for ATP was 1.1 mm . Enzyme activity was inhibited by ATP (20 mm ), AMP, ADP, cytidine diphosphocholine (1 or 10 mm ), and activated by choline esters (1.0 mm ), NaCl or KCl(200 mm ).     

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.     

THE ROLE OF INTRANEURONAL 5-HT AND OF TRYPTOPHAN HYDROXYLASE ACTIVATION IN THE CONTROL OF 5-HT SYNTHESIS IN RAT BRAIN SLICES INCUBATED IN K+-ENRICHED MEDIUM

Abstract— The incubation of brain stem slices from adult rats in a K⁺-enriched medium containing a 5-HT uptake inhibitor (fluoxetine) significantly increased their capacity to synthesize 5-HT from tryptophan. The K+-induced stimulation of 5-HT synthesis was at least partly dependent on the depletion of the indoleamine in tissues since: (1) a good correlation was found between the respective changes in 5-HT release and synthesis evoked by high K⁺ concentrations in the presence of various 5-HT uptake inhibitors; (2) the modifications in endogenous 5-HT levels produced by in vim treatments with drugs (reserpine, pargyline) or by incubating slices with 5-HT altered the stimulating effect of high K⁺ concentrations and fluoxetine on 5-HT synthesis; (3) the replacement of Ca²⁺ by Co²⁺ (4 mM) or EGTA (0.1 mM) in the incubating medium completely prevented the increased 5-HT release and synthesis evoked by high K⁺ concentrations and fluoxetine. The extraction of tryptophan hydroxylase from incubated tissues revealed that the increased 5-HT synthesis occurring in K⁺-enriched medium was associated with an activation of this enzyme. Kinetic analyses indicated that this activation resulted from an increase in the V_max of tryptophan hydroxylase, its apparent affinities for both tryptophan and 6-MPH₄ being not significantly affected. In contrast to the tryptophan hydroxylase from tissues incubated in normal physiological medium, the activated enzyme from tissues depolarized by K⁺ was hardly stimulated by Ca²⁺-mediated phosphorylating conditions. This led to the proposition of a hypothetical model by which the Ca²⁺ influx produced by the neuronal depolarization would trigger the activity of a Ca²⁺-dependent protein kinase capable of activating tryptophan hydroxylase. Although this sequence is still largely speculative it must be emphasized that, as expected from such a model, the regional differences in the K⁺-evoked activation of tryptophan hydroxylase in slices (cerebral cortex > brain stem > spinal cord) were parallel to those of the Ca²⁺-dependent protein phosphorylation (r= 0.92) and those of the activating effect of phosphorylating conditions on soluble tryptophan hydroxylase (r= 0.96).     

Sodium-dependent Potassium Channels in Leech P Neurons

In leech P neurons the inhibition of the Na⁺-K⁺ pump by ouabain or omission of bath K⁺ leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K⁺ and Na⁺ are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K⁺ conductance of the plasma membrane, which compensates for the reduction of the K⁺ gradient. The data presented here strongly suggest that the increased K⁺ conductance is due to Na⁺-activated K⁺ (K_Na) channels. Specifically, an increase in the cytosolic Na⁺ concentration ([Na⁺]_i) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R_in) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R_in and [Na⁺]_i followed a simple model in which the R_in decrease was attributed to K⁺ channels that are activated by the binding of three Na⁺ ions, with half-maximal activation at [Na⁺]_i between 45 and 70 mM. At maximum channel activation, R_in was reduced by more than 90%, suggesting a significant contribution of the K_Na channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K_Na channels in leech P neurons are also activated by Li⁺.     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

Mild chronic stress leads to desensitisation of presynaptic autoreceptors and a long-lasting increase in noradrenaline synthesis in rat cortical synaptosomes

An i.p. injection of normal saline combined with 1 min handling when repeated 14 times results in an increase in noradrenaline synthesis in synaptosomes prepared from the cortex of stressed rats; at 24 h synthesis acceleration is greater than at 48 h after the last stress.The activity of tyrosine hydroxylase solubilised from the hippocampus is the same in the control and the stressed group, when assayed at the optimal pH of 5.8 and with saturating concentration (2 mM) of the cofactor 6 MPH₄. However enzyme from stressed rats shows a relative increase in the activity at pH 7.4 assayed in the presence of 0.2 mM 6 MPH₄. This indicates activation, not induction, of the enzyme. 8-Br-cAMP produced the same increase in noradrenaline synthesis in cortical synaptosomes from control and stressed rats; however 50 mM K⁺ did not increase synthesis rate in stressed rats. Furthermore in synaptosomes from stressed rats neither isoprenaline (which increases noradrenaline synthesis) nor clonidine with 50 mM K⁺ (which leads to a depression of the K⁺-accelerated synthesis) had any effect on synthesis rate. The results suggest that the increased noradrenaline synthesis rate in cortical synaptosomes from stressed rats represents a Ca²⁺-dependent activation of tyrosine hydroxylase resulting from the desensitisation of alpha₂-autoreceptors.     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995