Modulation of striatal cyclic nucleotide phosphodieterase by calmodulin: regulation by opiate and dopamine receptor activation.

Incubation of striatal slices with morphine (10^?6 M) increased the cytosolic content of calmodulin. The double reciprocal plot of cAMP-phosphodiesterase (PDE) activity, vs cAMP concentrations which appears to be biphasic with an apparent low and high K_m-form in control slices was changed to a monophasic one with a low K_m-form. The changes in the apparent K_m for cAMP elicited by morphine (5 × 10^?7 M) could be blocked by haloperidol (10^?7 M) and naltrexone (10^?7 M). In slices prepared from deafferented caudate nuclei, morphine (5 × 10^?7 M) did not cause a change in the biphasic double reciprocal plot, but incubation of such slices with dopamine (2 × 10^?7 M) still caused the appearance of a low K_m-form of phosphodiesterase. Gel filtration (G-150) of soluble extracts indicated that a significantly greater proportion of the calmodulin was found in the fraction containing PDE activity in the morphine-treated slices than in control slices. Similar changes in PDE activity and calmodulin did not occur in cerebellar slices. These findings suggest that opiate receptors may be located on dopaminergic neurons, and that opiates may influence the regulation of PDE by calmodulin via post-synaptic dopamine receptors. Changes in PDE mediated by the increased availability of calmodulin can be used as an index of stimulation of dopamine receptors and may be related to the development of subsensitive receptor responses.     

Phosphorylation of membrane proteins in response to persistent stimulation of adenylate cyclsae-linked dopamine receptors in slices of striatum

Prolonged incubation of slices of striatum with agonists of D-1 dopamine receptors increased phosphorylation of at least 5 membrane protein bands. The extent of the increase in phosphate-incorporation depended on the concentration (10^?5 M?10^?4 M) of the agonist in the incubation medium and the duration of incubation (20 min or longer). Preincubation of slices with haloperidol (10^?6 M) greatly reduced, while ( ? )sulpiride (10^?6 M) failed to alter the increase of phosphorylation elicited by dopamine. Prolonged incubation of striatal slices with LY 141865 (10^?5 M) or isoproterenol (10^?5 M) increased the phosphate-incorporation only in one of the protein bands with an apparent molecular weight of 42,000. Incubation of striatal slices with cholera toxin increased the phosphorylation of protein bands in a similar way to those elicited by dopamine. The present results suggestthat the increased phosphorylation of certain protein bands elicited by prolonged exposure of striatal slices to D-1 dopamine receptor agonists may be associated with the desensitization of dopamine-sensitive adenylate cyclase.     

Effects of apomorphine in vitro on the uptake and release of catecholamines in crude synaptosomal preparations of rat striatum and hypothalamus.

It is currently believed that apomorphine exerts its pharmacological effects primarily by a direct stimulatory action on post-synaptic dopaminergic receptors. In the present study, uptake of ³H-dopamine into crude synaptosomal preparations of rat striatum and uptake of ³H-norepinephrine into crude synaptosomal preparations of rat hypothalamus were inhibited 50 per cent by concentrations of apomorphine of 3.6 ± 0.5 × 10^?5 M and 3.2 ± 0.2 × 10^?5 M respectively. Lineweaver-Burk plots of ³H-catecholamine uptake data revealed that apomorphine exhibited mixed inhibition kinetics for dopamine uptake into striatum (K_i = 1.91 ± 0.19 × 10^?5 M) and mixed inhibition kinetics for norepinephrine uptake into hypothalamus (K_i = 2.0 ± 0.17 × 10^?5 M). In crude synaptosomes of rat striatum, apomorphine elicited a dose-dependent release of ³H-dopamine during a 5-min incubation period at 37°. In contrast, the drug failed to produce a significant release of ³H-norepinephrine from crude synaptosomes obtained from rat hypothalamus. While these results suggest that apomorphine in high concentrations is capable of inhibiting the uptake of catecholamines and stimulating the release of dopamine at nerve endings, it is unlikely that these events contribute significantly to the behavioral effects elicited in animals after administration of the drug, since such effects occur at much lower doses.     

Adenosine 3',5'-cyclic monophosphate phosphodiesterase in guinea-pig lung--properties and effect of adrenergic drugs

The hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP) by adenosine 3',5'-monophosphate phosphodiesterase (PDE) was studied in whole homogenates and cell fractions of guinea-pig lung. Approximately 60 per cent of the activity of the whole homogenate was contained in the 10,000 g supernatant. This cell fraction contained PDE with two separate affinities for cAMP: PDE I with low affinity. K_m ? 3.1 × 10^?4 M; V_max ? 90 nmoles cAMP hydrolyzed/mg protein/20 min and PDE II with high affinity K_m ? 5.7 × 10^?5 M; V_max ? 35 nmoles cAMP hydrolyzed/mg protein/20 min. Both forms required magnesium which could be replaced by manganese and cobalt but not by copper. Aminophylline and theophylline inhibited both PDE forms. Calcium only inhibited PDE I but not PDE II. Adrenergic drugs inhibited PDE I and at 0.1 mM decreased in potency in the following order: isoproterenol  epinephrine > norepinephrine > methoxamine. Butoxamine was ineffective on PDE I. Only norepinephrine inhibited PDE II but not to the same extent as its effect on PDE I. Chelation of added magnesium and endogenous calcium was not responsible for the inhibition of PDE I by the adrenergic drugs used. Propranolol (0.1 mM) and phentolamine (0.05 to 0.001 mM) alone did not effect PDE I. Propranolol (0.1 mM) failed to reverse the inhibitory effect of isoproterenol (0.1 mM) and epinephrine (0.1 mM). Thus a β-adrenergic mechanism could not explain the inhibitory effect of these agents. Phentolamine (0.05 to 0.001 mM) reversed the inhibition by epinephrine bitartrate but not by epinephrine hydrochloride. These data are inconclusive to support an α-adrenergic mechanism for the inhibition of cAMP phosphodiesterase by epinephrine.     

Evidence for the methylation of apomorphine by catechol-O-methyl-transferase in vivo and in vitro

Pretreatment with either pyrogallol, tropolone or 8-hydroxyquinoline enhanced markedly the mean stereotyped behavior scores after apomorphine treatment in the rat. Experiments in vitro, using rat liver or brain catechol-O-methyltransferase (COMT) preparations and ¹⁴C-methyl-S-adenosyl-l-methionine, demonstrated that apomorphine was methylated by this enzyme system. The apparent K_m values for dopamine and apomorphine were 2.6 × 10^?4 M and 1.4 × 10^?3 M, respectively, for liver COMT. Pyrogallol and tropolone inhibited the methylation of apomorphine in vitro competitively when the apomorphine concentration was varied. These results suggest that methylation by COMT may represent an important metabolic pathway for the deactivation of apomorphine in vivo.     

Effects of morphine on the rat striatal dopamine metabolism.

Concomitant with total suppression of the spontaneous unitary discharges of neurons in the rat corpus striatum, intracarotidly injected morphine (5 mg/kg) was also found to increase the levels of dopamine, homovanillic acid and cyclic AMP by 80, 65 and 46 per cent respectively, measured 5 min after injection. This provides further support to the hypothesis that the nigrostriatal dopaminergic pathway is stimulated by acutely administered morphine. Morphine (10^?5-10^?3 M) did not alter the activity of striatal tyrosine aminotransferase. The drug, added in vitro (10^?6-10^?4 M) or by intracarotid injection (5 mg/kg) did not affect the activity of striatal tyrosine hydroxylase. Moreover, morphine (10^?4 M) did not interfere with the inhibitory effects of dopamine (10^?6-10^?4 M) on striatal tyrosine hydroxylase. However, it significantly potentiated the stimulatory effects of cyclic AMP on this enzyme. Morphine (10^?5-10^?4) was also found to have no effect on the spontaneous or K⁺-stimulated release of dopamine from striatal homogenate and synaptosomes. However, in the presence of 5 × 10^?5 M and 10^?4 M morphine, the uptake of dopamine by striatal homogenate was inhibited by 14 and 33 per cent respectively. With synaptosomal preparations, dopamine uptake was inhibited by 17 per cent in the presence of 10^?4 M morphine—the inhibition being competitive with dopamine with an apparent K_i of 0.41 mM. The inhibition of dopamine uptake caused by 10^?4 M morphine in either preparation was not reversed by the addition of 10^?4 M naloxone. It was concluded that the increase in dopaminergic activity following acute treatment of morphine is probably due to (1) prolongation of the effect of dopamine on the post-synaptic neurons resulting in increased production of cyclic AMP which in turn potentiates dopamine synthesis and (2) decrease in presynaptic cystosol dopamine which is normally a feedback inhibitor of tyrosine hydroxylase thus leading to increased synthesis of dopamine.     

Cytosolic and membrane-bound cyclic nucleotide phosphodiesterases from guinea pig cardiac ventricles

Cyclic nucleotide phosphodiesterase (PDE) activities were characterized in the cytosolic and post-nuclear membrane preparations of guinea pig cardiac ventricles. The cytosolic PDE activities were stimulated 5-fold by calmodulin (CaM) on both substrates (1 μM) and 1.2-fold by cGMP (5 μM) on cAMP hydrolysis. Conversely, in the membrane preparation, CaM only stimulated PDE activities 1.2- to 1.4-fold, but cGMP induced a 3-fold increase of the hydrolysis of cAMP. In both the cytosolic and the membrane preparations, the hydrolysis of cAMP was inhibited by 100 μM of either the PDE III inhibitor SK & F 94120 (27% and 31% respectively) or the PDE IV inhibitor rolipram (14% and 23% respectively). Four peaks were resolved from the cytosolic preparation by chromatography. Peak A and peak B hydrolyzed both cAMP and cGMP and were stimulated respectively by CaM and cGMP. Peak C and peak D selectively hydrolyzed cAMP. Peak C had an apparent K_m value for cAMP of 3.3 μM and was inhibited by PDE IV inhibitors. Peak D showed an apparent K_m value for cAMP of 0.43 μM and was inhibited by cGMP and by cardiotonic inhibitors of PDE III. Similar potencies of these inhibitors were observed in the membrane preparation. These results suggest that in guinea pig cardiac ventricles: (1) PDE I (CaM-activated) is almost exclusively cytosolic; (2) PDE II (cGMP-stimulated), PDE III (cGMP-inhibited and cardiotonic-sensitive) and PDE IV (rolipram-sensitive) are present in cytosolic and membrane preparations; (3) PDE III and PDE IV differ in their apparent K_m values for cAMP. The latter observation could explain the differential effects of PDE III and PDE IV inhibitors in the regulation of cardiac contraction.     

On the mechanism of presynaptic autoreceptor-mediated inhibition of transmitter synthesis in dopaminergic nerve terminals

The effect of apomorphine (APO) upon dopamine (DA) synthesis and release from rat striatal slices was studied. The synthesis of DA was measured by incubating the slices in Krebs-Ringer phosphate (KRP) medium of variable ionic composition containing l-tyrosine[¹⁴C-U] as DA precursor. A superfusion system was used to study both spontaneous and K⁺-induced release of labeled DA from striatal slices. The addition of APO directly to the normal KRP medium markedly blocked the formation of [¹⁴C]DA from [¹⁴C]tyrosine with an ic₅₀ of 1.8 × 10^?7 M. Haloperidol (4 × 10^?7 M), a known DA antagonist, produced a shift to the right of the concentration-response curve for APO inhibition on DA synthesis, whereas the DA antagonist (+)butaclamol (4 × 10^?7 M) completely reversed the inhibition caused by APO (2 × 10^?7 M). DA uptake blockers, such as benztropine (2 × ^?6 M) or cocaine (1 × 10^?5 M), did not affect the ability of APO to inhibit DA synthesis. Furthermore, the α₂-adrenergic agonist clonidine produced only a mild inhibition and the β-adrenergic agonist isoproterenol produced no inhibition of [¹⁴C]DA formation. APO was able to inhibit DA formation both in the absence of calcium in the incubation medium or in the presence of high external calcium concentrations (4, 8 and 24 mM) which depress the rate of DA synthesis. Incubation conditions that cause an increase of free intraneuronal calcium concentrations, such as Na⁺-free medium, the presence of ouabain (1 × 10^?4 M), or K⁺ depolarization, dramatically abolished or impaired the ability of APO to inhibit DA synthesis in striatal slices. It was not possible to demonstrate any change in spontaneous and K⁺ (27 mM)-induced release of DA in the presence of APO concentrations that produced a marked inhibition of DA synthesis. The results reported in this work indicate that tissue slices can be used as a valuable experimental tool to study the inhibitory effect of APO on DA synthesis, and that this effect occurs through an interaction of APO with presynaptic DA autoreceptors located in striatal dopaminergic nerve terminals. The results obtained are not in keeping with the hypothesis that this autoreceptormediated inhibition of DA synthesis occurs through regulation of calcium influx into the DA nerve terminals. However, the possibility is raised that a sensitivity to high intraneuronal calcium concentration exists during the events that mediate APO-DA autoreceptor interaction and DA synthesis inhibition. It is further suggested that DA-synthesis-modulating autoreceptors do not participate in the modulation of DA release.     

Analysis of the effects of apomorphine and bulbocapnine in relation to the proposed dopamine receptor

On rat isolated vas deferens, apomorphine was found to be l/5th as active as dopamine. Reserpine or cocaine pretreatment failed to reduce the response to apomorphine. Low concentrations of apomorphine which do not cause contraction of the tissue, antagonize the effects of dopamine. The effects of dopamine were antagonized equally by phentolamine or apomorphine. On rabbit aortic strips 4 × 10^?4M apomorphine repeated at 45 min intervals induces pronounced tachyphylaxis. During the tachyphylactic period dopamine was not inhibited to a significantly greater extent than was phenylephrine. Bulbocapnine tested against dopamine and phenylephrine yielded identical pA₂ values of 6. Only at 10^?5M was bulbocapnine observed to produce a preferentially greater blockade of dopamine. Dopamine or isoprenaline-induced relaxations of aortic strips were not blocked by bulbocapnine. Dopamine and 3 times 10^?6M bulbocapnine increased the chronotropic effects in atria. Apomorphine and higher doses of bulbocapnine produced rate decelerating effects. On atria low doses of apomorphine were equally effective in reducing the effects of dopamine or histamine. These results are discussed in light of the proposal that the effects of apomorphine and bulbocapnine involve dopamine receptor interactions. In all three tissues there was no clear cut evidence of specific dopamine receptors.     

Inhibition of guinea pig aortic sarcolemmal Ca2+-Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone

Milrinone is a positive inotrope/vasodilator that inhibits cardiovascular low K_m cAMP phosphodiesterase (PDE) and not Na⁺?K⁺ ATPase activity. To explore other possible mechanisms of action, we quantitated the effects of milrinone on Ca²⁺-stimulated Mg²⁺ ATPase activity in guinea pig aortic smooth muscle plasma membranes. Milrinone inhibited Ca²⁺- stimulated activity, but not basal activity, in aortic microsomes. Maximum inhibition (70%) occurred at 1 μM, which coincided with the inflection of a parabolic dose-response curve. In a sarcolemmal-enriched (F1) aortic preparation, 1 μM cAMP, 1 μM Cl-930 (another low K_m cAMP PDE inhibitor), and 100 μM W-7 (a calmodulin antagonist) all inhibited Ca² -stimulated Mg²⁺ ATPase activity. This F1 preparation contained cAMP PDE activity which was inhibited by 1 μM milrinone (26%) and 1 μM Cl-930 (40%) but not by 100 μM W-7. The inhibition of F1 Ca²⁺ -Mg²⁺ ATPase activity by 1 μM milrinone could be diminished by increasing the concentration of CaCl₂ in reaction mixtures. In sum, these studies show that milrinone can inhibit vascular sarcolemmal Ca²⁺ -stimulated Mg²⁺ ATPase activity. However, inhibition may be direct or direct or may be secondary to cAMP PDE inhibition in vascular sarcolemma, since inhibition also occurs with cAMP and another low-K_m cAMP PDE inhibitor, Cl-930.     

Correlation between drug-induced supersensitivity of dopamine dependent striatal mechanisms and the increase in striatal content of the Ca2+ regulated protein activator of cAMP phosphodiesterase

Summary Rats were injected daily with 1.3 mol/kg of haloperidol s.c. for 10 days. From the second to the ninth day after haloperidol withdrawal the rats developed supersensitivity to the behavioral affects of apomorphine. Concomitantly, the K _a of dopamine for the activation of striatal adenylate cyclase was lowered and the striatal content of the Ca²⁺ dependent protein that activates cAMP phosphodiesterase was increased. This activator protein is stored in striatal membranes and can be released by membrane phosphorylation in cytosol. This protein increases the activity of the high K _m phosphodiesterase (Uzunov et al., 1976) but when it is bound to striatal membranes it facilitates the activation of striatal adenylate cyclase by dopamine (Gnegy et al., 1976 b). The increase in protein activator content of striatal membranes caused by haloperidol could be a primary factor in causing supersensitivity to the biochemical and behavioral effects of dopamine receptor agonists.     

Mouse neuroblastoma clone N1E-115: A suitable model for studying the action of dopamine agonists on tyrosine hydroxylase activity

The DOPA-content in neuroblastoma clone N1E-115 is higher than the dopamine or noradrenaline content. Blockade of tyrosine hydroxylase by ifα-methyl-p-tyrosine (1 × 10 su?3 M) resulted in a decrease of cellular DOPA-content to 24.9% after 4 hr. The accumulation of DOPA in these cells which is probably due to limited activity of l-aromatic amino acid decarboxylase led us to use DOPA-content as a measure of tyrosine hydroxylase (TH) activity. Dopamine and especially apomorphine were effective at low concentrations (dopamine ic₅₀1 × 10^?5 M, apomorphine 2 × 10^?7 M); lisuride had no effect on TH-activity. The low effective dose of apomorphine and the failure of lisuride to influence TH-activity are comparable to the observations in striatal synaptosomal preparations and make the N1E-115 clone a suitable model for studying the mechanism of TH-regulation. However, since haloperidol (1 × 10^?5 M) did not reverse the apomorphine-induced blockade of TH, a receptor-mediated blockade of TH seems to be improbable.     

The in vitro uptake of biogenic amines by snail (Heliz pomatia) nervous tissue.

The uptake of [³H]5-HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the suboesophageal ganglia of the snail, Helix pomatia, was studied. When tissues were incubated at 25° in mediums containing radioactive amines, tissue: medium ratios of about 30:1, 18: l, 4:1 and 5:1 for 5-HT, dopamine, noradrenaline and octopamine respectively were obtained after 20–30 min incubation. Tissues incubated at 25° in mediums containing radioactive amines for 20–30 min showed that 90% of the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]noradrenaline or [³H]octopamine. The high tissue : medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, demonstrated saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver-Burk plots, two uptake mechanisms for 5-HT at 25° were resolved, the high affinity uptake process having a K_m1valueof 8.48 × 10^?8 M and V_max1 value of 0.077 nmole/g per min, while the lower affinity process had a K_m2 value of 1.8 × 10^?6 M and a V_max2 value of 0.66 mole/g per min. At 0° a single uptake mechanism for 5-HT occurred which gave a K_m value of 0.152 × 10^?8 M and a V_m value of 0.0203 nmole/g per min. In the case of dopamine, the Lineweaver-Burk plot of 25° showed a single uptake process with values for K_m and V_max of 1.02 × 10^?7 M and 0.0673 nmole/g per min respectively. This process did not function at 0°. The effects of various agents and ions upon the accumulation processes for all amines were also studied, and the findings indicate that the same neurons may well accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the snail ganglia is a mechanism for inactivating these substances at 25° and that an uptake mechanism for 5-HT also functions at 0°. The present results are discussed from the point of view of the monoamines having transmitter functions in the snail CNS.     

Activation of tyrosine hydroxylase in rat striatal slices by K+-depolarization--effect of ethanol.

Slices from rat corpus striatuum were incubated for 10 min at 37° in freshlyy oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K⁺ (55mM) media both in the presence and absence of ethanol (0.2 to 0.8%, w/v). Thereafter, the slices were homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 105,000 g supernatant fraction. The presence of K⁺ (55 mM) in the incubation media increased about 3-fold the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, when compared to that found in striatal slices incubated in normal KRP media. Incubation of striatal slices in a KRP-high K⁺ media also produced changes in the kinetic properties of tyrosine hydroxylase. The K_^m of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH₄) was decreased from 0.82 to 0.09 mM and the K_i of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2 to 0.8%, w/v) added directly to the KRP-high K⁺ media markedly blocked the K⁺-induced activation of tyrosine hydroxylase as well as the kinetic alterations in the enzyme observed after K⁺-depolarization of the striatal slices. In contrast, the presence of ethanol did not modify the activity and kinetic characteristics of tyrosine hydroxylase isolated from slices incubated in normal KRP media. The results reported in this work suggest that the increase in DA synthesis observed in striatal slices after K⁺-depolarization might be mediated in part via an allosteric activation of tyrosine hydroxylase. This activation appears to be mediated by an increase in the affinity of the enzyme for the pterin cofactor and a decreased affinity for the end-product inhibitor DA. Also, the blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K⁺-depolarization seems to offer a likely explanation for the inhibitory effect of ethanol on K⁺-induced increase in DA synthesis reported recently by Gysling et al. (Biochem. Pharmac.25, 157 (1976)).     

Effects of naloxone on d-amphetamine- and apomorphine-induced behavior

The effects of acute naloxone administration on d-amphetamine- and apomorphine-induced behavior were studied. Naloxone, in doses of 0.3–10 mg/kg (s.c.), antagonized the increase in ambulation and rearing induced by 1 mg/kg of d-amphetamine. When the dose of d-amphetamine was increased to 3 mg/kg, naloxone (3 mg/kg) antagonized only the increase in rearing activity. No dose (0.3–10 mg/kg, s.c.) of naloxone significantly affected d-amphetamine- or apomorphine-induced stereotyped activity. Naloxone (3 mg/kg) significantly augmented the apomorphine (1 mg/kg. s.c.)-induced increase in ambulation but attenuated the apomorphine (0.3 mg/kg)-induced increase in rearing activity. Naloxone (3 mg/kg) or apomorphine (0.03 mg/kg) significantly decreased the ambulation and rearing induced by a novel environment. In combination and in these doses, naloxone and apomorphine produced an additive effect on these behaviors. The neurochemical mechanisms by which naloxone affects d-amphetamine- and apomorphine-induced behavior were investigated. Naloxone (10^?6M) had no significant effect on [³H]spiroperidol binding in either the caudate nucleus or nucleus accumbens except for a modest inhibition (24%) of both the K_m and B_max in the accumbens microsomal fraction. Similarly, naloxone (10^?6 M) had no significant effect on [³H]dopamine(DA) uptake into either brain region nor did naloxone alter the d-amphetamine-inhibition of uptake. Using perfused tissue slices, naloxone (10^?6?10^?5 M) significantly attenuated the increase in [³H]DA release induced by d-amphetamine (10^?5 M) in both brain regions. Naloxone (1 mg/kg) had no significant effect on DA or dihydroxyphenylacetic acid (DOPAC) levels or on the DA/DOPAC ratio in the caudate nucleus or nucleus accumbens. However, naloxone did reverse the marked increases in the DA-DOPAC ratio induced by d-amphet-amine (1 mg/kg) in both brain regions.     

Tyrosine hydroxylase regulation in rat striatal and olfactory tubercle slices--I. Activation induced by exposure to a calcium-free and high-magnesium medium.

Slices from rat corpus striatum and olfactory tubercle were incubated for 15 min at 37° in Krebs-Ringer phosphate (KRP) medium or Ca²⁺-free KRP medium both in the presence and absence of high Mg²⁺ (12 mM). Tyrosine hydroxylase activity and kinetic parameters were determined in the 20,000 g supernatant fraction prepared from slices. The omission of Ca²⁺ from the incubation medium resulted in a moderate increase in the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, and a significant increase in the activity of tyrosine hydroxylase isolated from olfactory tubercle slices. The presence of Mg²⁺ (12 mM) in the Ca²⁺-free KRP medium produced a further increase in the activity of the enzyme isolated both from striatal and olfactory tubercle slices. The per cent of stimulation of enzyme activity induced by incubating the slices in a Ca²⁺-free, high-Mg²⁺ KRP medium was maximal when assayed at pH 7.0. The Mg²⁺-induced activation of tyrosine hydroxylase was antagonized by increasing the Ca²⁺ concentration (3.75 to 15.0 mM) in the medium in which the slices were incubated. The direct addition of Mg²⁺ (5–20 mM) to striatal and olfactory tubercle homogenates also resulted in an increase in the activity of tyrosine hydroxylase. The addition of high concentrations of Ca²⁺ (10 mM) to the homogenates also resulted in an increase in enzyme activity but this effect was additive to that produced by Mg²⁺ (10 mM). Incubation of striatal slices in a Ca²⁺-free, high-Mg²⁺ KRP medium produced changes in the kinetic properties of tyrosine hydroxylase. The apparent _m of the enzyme for 6-methyl-5,6,7,8-tetrahydropterine HCl (6-MPH₄) was decreased significantly from 0.85 to 0.40 mM, with no significant change in the V_max. However, the K_i of the enzyme for dopamine (DA) was unchanged. Magnesium ions (12 mM) added to KRP-high K⁺ (55 mM) medium blocked the activation of tyrosine hydroxylase induced by K⁺-depolarization of striatal slices. However, Mg²⁺ (12 mM) addition to the incubation medium did not block but actually further increased the tyrosine hydroxylase activation that results after incubating striatal slices in a KRP medium enriched with dibutyryl cAMP (1 mM). Moreover, the stimulating effect on tyrosine hydroxylase observed when assays were conducted in the presence of Mg²⁺, ATP and cAMP remained unchanged in homogenates prepared from slices previously incubated in a Ca²⁺-free, high-Mg²⁺ KRP medium. The results reported in this work clearly demonstrate that a kinetic activation of tyrosine hydroxylase in dopaminergic nerve terminals can occur under conditions of diminished extracellular Ca²⁺ and blockade of transmitter release. Cyclic AMP does not seem to play a role in the tyrosine hydroxylase activation induced by incubation of slices in Ca²⁺-free and high-Mg²⁺ medium. The results support the hypothesis that the increase in DA biosynthesis observed in dopaminergic neurons after inhibition of impulse flow may result primarily as a consequence of a diminished entrance of Ca²⁺ into the nerve terminal.     

Choline and diethylcholine transport into a cholinergic clone of neuroblastoma cells.

The transport of choline and diethylcholine has been investigated in a cholinergic clone (S20F₃) of mouse neuroblastoma cells. Choline transport was linear for the first 20 min of incubation and was temperature dependent at low concentrations (< 1 × 10^?5 M). Diethylcholine transport was linear for the first 10 min of incubation and was also temperature dependent at low concentrations. High affinity (K_m < 1 × 10^?6 M) and low affinity (K_m > 1 × 10^?5 M) components of transport were found for both compounds. The transport system had a greater apparent affinity (lower K_m) for choline than for diethylcholine (3-fold), but maximal transport velocities were about equal. Each compound competitively inhibited the other's high affinity transport. Hemicholinium (1 × 10^?5 M) slightly inhibited high affinity choline transport but triethylcholine (1 × 10^?6?1 × 10^?4 M) did not. Choline transport was also found to be dependent on the pH and pCO₂ of the medium.     

Tyrosine hydroxylase in snail (Helix pomatia) nervous tissue.

Snail nervous tissue synthesizes [¹⁴]dopamine and [¹⁴]dihydroxyphenylalanine (DOPA) from [^14C]tyrosine. The K_m value for the overall conversion of [¹⁴C]dopamine was 6 × 10^?4M. The enzyme converting [¹⁴C]tyrosine to [¹⁴C]DOPA. tyrosine hydroxylase, has the following characteristics. Approximately 85–90 per cent of the enzyme is soluble, and the enzyme of the nervous tissue, isolated by ammonium sulfate fractionation, had the highest activity in the 25–40 per cent fraction. The enzyme has a pH optimum of 6.5 in Tris-HCl, sodium acetate and potassium phosphate buffers. The enzyme requires a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine (DMPH₄), 2-amino-4-hydroxy-6-methyltetrahydropteridine (6MPH₄), and 2-amino-4-hydroxy-6-(L-erythro-1, 2-dihydroxypropyl)tetrahydropteridine (l-erythro-tetrahydrobiopterin) (BH₄) are 5 × 10^?4 M. 3.2 × 10^?4 M and 1.1 × 10^?4 M respectively. The K_m values for tyrosine at 10^?3 M BH₄ or 6MPH₄ are 1.4 × 10^?4 M and 4.2 × 10^?5 respectively. The enzyme is markedly stimulated by Fe²⁺ and catalase. The activity is drastically inhibited by dopamine, 6-hydroxydopamine, 5-hydroxytryptophan (5-HTP), noradrenaline and sodium dodecyl sulphate. Analogues of tyrosine also slightly inhibit the activity while Triton X-100, homovanillic acid, dihydroxyphenylacetic acid (DOPAC), reserpine, tyramine, pargyline and sucrose have little effect. The properties of the snail tyrosine hydroxylase are compared with those of the vertebrates.     

Dopaminergic effects of buspirone,a novel anxiolytic agent

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methyxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [³H]spiroperidol from striatal binding sites, with an ic₅₀ (1.8 × 10^?7 M), comparable to that of clozapine ($\text{ic}{}_{50}\text{= 1.4 × 10}{}^{\mathrm{?7}}\text{M}$) but considerably lower than that of haloperidol (4.7 × 10^?9 M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.     

Activity of two new potent dopaminergic agonists at the striatal and anterior pituitary levels

Two N-diphenethylamine derivatives, RU24213 and RU24926, displaced [³H]-dihydroergocryptine binding to bovine anterior pituitary membranes at K_D values of 150 and 100 nM, respectively, and were potent inhibitors of prolactin release in anterior pituitary cells in primary culture at respective ED₅₀ values of 5 and 3 nM. After administration by the oral route in the rat, both compounds (5 mg/kg), exerted potent and long-lasting inhibitory effects on plasma prolactin levels which were still reduced to 45% of control 6 hr after the administration of the compound with longer-lasting activity, RU24926. Furthermore, these two drugs were more potent than apomorphine in decreasing striatal dopamine turnover and increasing striatal acetylcholine levels in either intact rats or animals having a unilateral 6-hydroxy dopamine-induced lesion of the nigrostriatal dopaminergic pathway. The stimulatory effect of both RU24213 and RU24926 on striatal acetylcholine levels in intact rats was much longer-lasting than that of apomorphine, a significant effect of RU24926 being still observed 4 hr after intraperitoneal administration of the compound (5 mg/kg). At concentrations up to 10^?4 M, RU24213 and RU24926 were inactive on either basal or dopamine-stimulated striatal adenylyl cyclase activity. The present data indicate that these two new drugs have potent dopaminergic activity at the striatal and anterior pituitary levels and therefore have a potential therapeutic activity for the treatment of Parkinson's disease and prolactin-secreting adenomas.