Effect of phospholipases and proteases on the [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) binding to A1 adenosine receptors from pig cerebral cortex.

The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that 1) there is co-solubilization of receptor and phospholipids; 2) the phospholipid requirements are different for the coupled and the uncoupled receptor; 3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and 4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.     

The effect of tetrahydrobiopterin on the in situ phosphorylation of tyrosine hydroxylase in rat striatal synaptosomes

Tetrahydrobiopterin (BH₄), the obligatory cofactor of the aromatic amino acid hydroxylases, decreased the in situ³²P-phosphorylation of tyrosine hydroxylase (TH) in rat striatal synaptosomes. Incubation of pre-³²P-labeled synaptosomes with BH₄ in the presence of a permeant analogue of cAMP decreased the cAMP-stimulated level of³²P label incorporation into TH by about 50%, as determined by immunoprecipitation and autoradiography of SDS-polyacrylamide gels. The extent of inhibition mirrored changes in intrasynaptosomal BH₄ levels and varied both as a function of BH₄ concentration and length of incubation. A similar decrease in the amount of TH³²P-labeling was observed with the precursor of BH₄, sepiapterin. This effect, in turn, was reversed by the inhibitor of sepiapterin reductase, N-acetyl-serotonin. Finally, exposure of pre-³²P-labeled synaptosomes to the inhibitor of protein phosphatase 2A, okadaic acid, blocked the response to BH₄. Collectively, the data suggest that BH₄ stimulates the dephosphorylation of TH in situ and thus may play a dual role both as a cofactor for catalysis and a regulator of hydroxylase activity.Special issue dedicated to Dr. Bernard W. Agranoff.     

Activation of CFTR Cl(-) channel by tyrphostins via a protein tyrosine kinase-independent pathway in forskolin-stimulated renal epithelial A6 cells

We studied effects of tyrphostin A23 (an inhibitor of protein tyrosine kinase; PTK) and tyrphostin A63 (an inactive analog of tyrphostin A23) on forskolin-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and Cl(-) secretion in renal epithelial A6 cells. Tyrphostin A23 and A63 had no effects on the basal CFTR Cl(-) channel and Cl(-) secretion. However, under the forskolin-stimulated condition, tyrphostin A23 and A63 stimulated Cl(-) secretion by activating CFTR Cl(-) channels. These observations suggest that: 1) tyrphostin A23 and A63 stimulate the cAMP-activated CFTR Cl(-) channel via a PTK-independent, structure-dependent mechanism, and 2) tyrphostin A23 and A63 do not stimulate the basal CFTR Cl(-) channel. These lead us to an idea that: 1) cAMP might cause a conformational change of CFTR Cl(-) channel which is accessible by tyrphostins, and 2) tyrphostins would stimulate translocation of the cAMP-modified channel to the apical membrane by binding to the channel.     

The influence of glucose, other monosaccharides, and ascorbic acid on tyrosine hydroxylase activity of rat striatal synaptosomes.

We investigated the action of glucose, other monosaccharides, and ascorbic acid on the activity of tyrosine hydroxylase in rat striatal synaptosomes. We found that glucose at 0.2 mM maximally activated enzyme activity by as much as 100 percent and caused half-maximal activation at 0.036 mM. Mannose, fructose and galactose also stimulated tyrosine hydroxylase activity, half-maximal activation occurring at 0.036, 8, and 50 mM, respectively; arabinose was inactive up to 100 mM. Ascorbic acid did not stimulate enzyme activity at 0.1 and 1 mM, and at 10 mM was inhibitory.The activating effect of glucose on tyrosine hydroxylase activity was blocked by 2-deoxyglucose and by glucosamine. We interpret the action of glucose to be dependent upon its metabolism and to be indirect, probably due to the maintenance of the cofactor in the reduced form in the synaptosomes.     

Activation of nociceptin/orphanin FQ-NOP receptor system inhibits tyrosine hydroxylase phosphorylation, dopamine synthesis, and dopamine D(1) receptor signaling in rat nucleus accumbens and dorsal striatum

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA-targeted post-synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho-Ser40-TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D(1) receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D(1) receptor-stimulated cAMP formation, but failed to affect either adenosine A(2A) or DA D(2) receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D(1)-induced phosphorylation of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D(1) receptors, N/OFQ curtailed DA D(1) receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D(1) receptor signaling.     

Measurement of tyrosine hydroxylase apoenzyme protein by enzyme-linked immunosorbent assay (ELISA): effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity and content.

A enzyme-linked immunosorbent assay has been developed for tyrosine hydroxylase (TH). The method uses a polyclonal antibody to trap TH, a monoclonal antibody to bind the immobilized TH, a biotinylated, anti-mouse immunoglobulin to bind the monoclonal antibody, and streptavidin covalently coupled to horseradish peroxidase (SA-HRP). The antigen-antibody complex is detected colorometrically following incubation with an HRP substrate. The method detects less than 1 ng (16 fmol) of TH and can be performed in 3 h. The high specificity of the assay is attributed to the use of both polyclonal and monoclonal antibodies, each of which are specific for TH. Data acquisition and reduction is rapid and linked directly to a common desktop computer. Levels of TH protein average 1 ng/microgram protein in striatum and, following treatment with the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), are decreased to a similar extent as is catalytic activity. In contrast, MPTP did not alter TH homospecific activity. The monoamine oxidase B inhibitor deprenyl blocked both the decrease in activity and the decrease in immunoreactive protein caused by MPTP.     

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.     

Activation of striatal tyrosine hydroxylase by in vivo electrical stimulation: Comparison with cyclic AMP-mediated activation

These studies were carried out to characterize the activation of rat striatal tyroxine hydroxylase produced by depolarization of the medial forebrain bundle and to evaluate the possible role of cyclic AMP as a mediator of this activation. The enzymatic properties of tyrosine hydroxylase following in vivo depolarization were compared to those produced by treatment of striatal synaptosomes with dibutyryl cyclic AMP (dbcAMP). Similar effects were observed with regard to enzyme distribution, altered sensitivity to dopamine-induced inhibition, and activity as a function of tyrosine concentration. However, differences between the two treatments were also apparent. First, treatment with dbcAMP shifted the pH optimum from 6.2 to 7.0. In contrast, electrical stimulation decreased the rate of decline in activity as the pH was increased above the optimum, but did not shift the pH optimum. Second, plots of tyrosine hydroxylase activity versus cofactor concentration revealed two enzyme forms for both control and electrically stimulated preparations. However, dbcAMP treatment converted the enzyme to a single high affinity form. These results can be explained by one of the following: (1) cyclic AMP is the sole mediator of enzyme activation, but does not produce a maximally activated enzyme following in vivo depolarization (2) cyclic AMP is only one of several mediators involved or (3) cyclic AMP is not involved in depolarization-induced activation, with activation occurring via the mediation of other intracellular messengers, such as calcium.     

N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand

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Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.     

Loss of myocardial protection from ischemic preconditioning following chronic exposure to R(-)-N6-(2-phenylisopropyl)adenosine is related to defect at the adenosine A1 receptor

Exogenously administered adenosine agonist will protect myocardium against infarction during ischemia. However, long-term exposure to adenosine agonists is associated with loss of this protection. To determine why this protection is lost, isolated, perfused rabbit hearts were studied after administration of R(-)-N⁶-(2-phenylisopropyl)adenosine (PIA), 0.25 mg/h IP, for 3-4 days to intact animals. All hearts experienced 30 min of regional ischemia and 120 min of reperfusion. Control groups 1 and 2 were untreated. In group 1 this ischemia/reperfusion was the only intervention, whereas group 2 hearts were preconditioned with a cycle of 5 min global ischemia/10 min reperfusion preceding the 30 min regional ischemia. Groups 3-5 had been chronically exposed to PIA. Group 3 hearts had 1 preconditioning ischemia/reperfusion cycle before the prolonged ischemia. Group 4 received a 5 min infusion of 0.1 mol/L phenylephrine in lieu of global ischemia, whereas group 5 was instead treated with 1 mol/L carbachol. Infarct size averaged 32% of the risk zone in group 1, whereas ischemic preconditioning limited infarction to 8.2 in group 2. Prolonged exposure of group 3 hearts to PIA resulted in the inability of preconditioning with 5 min global ischemia to protect (28.7 ± 4.4% infarction). However, protection was restored by either phenylephrine, an agonist of ₁-adrenergic receptors which couple to G_q and stimulate PKC, or carbachol, an agonist of M₂-muscarinic receptors which couple instead to G_i as do adenosine A₁ receptors (5.2 ± 1.7% and 9.2 ± 2.1% infarction, resp.). Therefore, cross tolerance to ischemic preconditioning develops after chronic PIA infusion. Since both the G_i and the PKC components of the preconditioning pathway were shown to be intact, tolerance must have been related to downregulation or desensitization of the A₁ adenosine receptor.     

Long-term modulation of tyrosine hydroxylase activity and expression by endothelin-1 and -3 in the rat anterior and posterior hypothalamus

Brain catecholamines are involved in the regulation of biological functions, including cardiovascular activity. The hypothalamus presents areas with high density of catecholaminergic neurons and the endothelin system. Two hypothalamic regions intimately related with the cardiovascular control are distinguished: the anterior (AHR) and posterior (PHR) hypothalamus, considered to be sympathoinhibitory and sympathoexcitatory regions, respectively. We previously reported that endothelins (ETs) are involved in the short-term tyrosine hydroxylase (TH) regulation in both the AHR and PHR. TH is crucial for catecholaminergic transmission and is tightly regulated by well-characterized mechanisms. In the present study, we sought to establish the effects and underlying mechanisms of ET-1 and ET-3 on TH long-term modulation. Results showed that in the AHR, ETs decreased TH activity through ET(B) receptor activation coupled to the nitric oxide, phosphoinositide, and CaMK-II pathways. They also reduced total TH level and TH phosphorylated forms (Ser 19 and 40). Conversely, in the PHR, ETs increased TH activity through a G protein-coupled receptor, likely an atypical ET receptor or the ET(C) receptor, which stimulated the phosphoinositide and adenylyl cyclase pathways, as well as CaMK-II. ETs also increased total TH level and the Ser 19, 31, and 40 phosphorylated sites of the enzyme. These findings support that ETs are involved in the long-term regulation of TH activity, leading to reduced sympathoinhibition in the AHR and increased sympathoexcitation in the PHR. Present and previous studies may partially explain the cardiovascular effects produced by ETs when applied to the brain.     

Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors.

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A(2A) receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A(2A) receptor-deficient mice (A(2A)-R(-/-)). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A(2A) receptor by striatopallidal neurones. To clarify the functions of A(2A) receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60-80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three-fold increase in extracellular glutamate concentration. This was associated with an up-regulation of D(1) and D(2) dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A(1) receptors expression were indistinguishable between A(2A)-R(-/-) and wild-type mice. Altogether these results pointed out that the lack of A(2A) receptors leads to a functional hypodopaminergic state and demonstrated that A(2A) receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.     

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.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.     

Anti-malarial activity of N(6)-substituted adenosine derivatives. Part I

The synthesis and biological evaluation of novel N(6)-substituted adenosine derivatives is reported. The first series of compounds was obtained using an established procedure for the nucleophilic substitution of a 1-(6-chloro-purin-9-yl)-beta-D-1-deoxy-ribofuranose with various amines. In addition, attachment of two different amino-functionalised spacer arms at the N(6)-position of adenosine enabled derivatisation by an innovative polymer-assisted protocol. Thus, we were able to prepare three series of substituted derivatives that displayed activity versus the multiresistant Plasmodium falciparum strain Dd2 in cell culture experiments.     

Antimalarial activity of N(6)-substituted adenosine derivatives. Part 3

A series of novel 3'-amido-3'-deoxy-N(6)-(1-naphthylmethyl)adenosines was synthesized applying a polymer-assisted solution phase (PASP) protocol and was tested for anti-malarial activity versus the Dd2 strain of Plasmodium falciparum. Further, this series and 62 adenosine derivatives were analyzed regarding 1-deoxy-d-xylulose 5-phosphate (DOXP) reductoisomerase inhibition. Biological evaluations revealed that the investigated 3',N(6)-disubstituted adenosine derivatives displayed moderate but significant activity against the P. falciparum parasite in the low-micromolar range. On the molecular level, DOXP reductoisomerase utilizing an adenosyl-containing substrate was identified as a promising metabolic target for ligands of adenosine binding motifs.