Chemical lesion and drug induced supersentivity and subsensitivity of caudate dopamine receptors

In order to elucidate the mechanism of denervation supersensitivity, the effects of 6-hydroxydopamine lesion, placed in the substantia nigra, were examined on rat brain caudate adenylate cyclase and ³H-haloperidol binding to membrane dopamine receptors. In addition, the effects of chronic administration of L-DOPA, bromocriptine and piribedil were also investigated on ³H-haloperidol binding and dopamine, K⁺ isoproterenol (IPNE) and 2-Cl-adenosine stimulated formation of cyclic AMP in caudate slices. 6-Hydroxydopamine lesions resulted in significantly greater stimulation of adenylate cyclase by dopamine at various concentrations tested. The haloperidol binding sites were increased by 28% on lesioned side caudate without changes in dissociation constants (K_D). Three weeks after treatment with L-DOPA, bromocriptine or piribedil, the ³H-haloperidol binding sites were decreased by 40% with no change in K_D. The stimulatory effect of dopamine on cyclic AMP formation was also abolished, although there was no change in IPNE, K⁺, or 2-Cl-adenosine stimulated cyclic AMP formation in caudate slices, suggesting a specific effect of dopamine agonists on dopamine receptors. The results of these studies suggest a close relationship between at least some populations of dopamine receptors and adenylate cyclase in the caudate nucleus.     

Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate. Effect of adenosine 3',5'-cyclic monophosphate and putative neurotransmitters.

Adenosine 3',5'-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of 32P into proteins endogenous to a homogenate of rat caudate nucleus when 4 micrometer [gamma-32P] ATP is used as substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 micrometer no significant increase in protein phosphorylation was seen. Incubation of the homogenate with 400 micron ATP and 100 micron dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.     

Hydroxyl radical attack on dopamine

Hydroxyl radicals were generated in the presence of 1 mM dopamine (3,4-dihydroxyphenylethylamine) at pH 7.2 (50 mM phosphate buffer) by the following two mechanisms: 1) a classic Fenton-type reaction between hydrogen peroxide and a ferrous chelate (ferrous diethylenetriaminepentaacetate) and 2) the cyclical redox reactions of iron-EDTA/ascorbate. Three ring-monohydroxylated products of dopamine were detected by high performance liquid chromatography with electrochemical detection: 2-hydroxydopamine, 5-hydroxydopamine, and 6-hydroxydopamine in an approximate ratio of 3:2:1. Scavengers of hydroxyl radicals (dimethyl sulfoxide, mannitol, ethanol) suppressed the yields of products in a concentration-dependent manner. The formation of nonphysiologic hydroxylated forms of dopamine can provide a probe for the formation of hydroxyl radicals in dopamine neurons.     

Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate: Effect of adenosine 3′,5′-cyclic monophosphate and putative neurotransmitters

Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of ³²P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?³²P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.     

Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.     

The effect of cyclo (Leu-Gly) on chemical denervation supersensitivity of dopamine receptors induced by intracerebroventricular injection of 6-hydroxydopamine in mice

Chemical denervation supersensitivity of postsynaptic dopamine receptors was induced in mice by intracerebroventricular injection of 6-hydroxydopamine. Fourteen days after the 6-hydroxydopamine injection, mice exhibited greater spontaneous locomotor activity and hypothermic response when challenged intraperitoneally with apomorphine. Whole brain levels of dopamine were reduced by 80%. Daily subcutaneous administration of cyclo (Leu-Gly) (50 μg/mouse/day) for 14 days inhibited the development of dopamine receptor supersensitivity induced by 6-hydroxydopamine as evidenced by the blockade of an apomorphine induced locomotor and hypothermic effect. Cyclo (Leu-Gly) did not alter the depletion of brain dopamine induced by 6-hydroxydopamine. These data suggest that cyclo (Leu-Gly) can block the development of dopamine receptor supersensitivity induced by 6-hydroxydopamine without protecting the neurons from dopamine depletion.     

Dopamine D2 Receptors Attenuate Phosphatidylisitol 4,5-Bisphosphate in Synaptosomal Membranes from Rat Neostriatum

The effects of dopamine on [32P]ATP-labelled phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid were analyzed by TLC in synaptosomal membranes of the rat neostriatum. The incorporation of 32P into these compounds was found to be stable within 1 min and was maintained during the 30 min of incubation. Dopamine (0.1-10 microM) was found to attenuate the levels of phosphatidylinositol 4,5-bisphosphate without affecting the levels of phosphatidylinositol 4-phosphate or phosphatidic acid. The maximal decrease (-35 +/- 4%) was reached at 10 microM of dopamine after 30 min of incubation. The dopamine (0.1 microM)-induced decrease was blocked by the D2 selective antagonist raclopride (1 microM), but not by the D1 selective antagonist SCH 23390 (1 microM). These findings indicate the existence of an intramembrane coupling of dopamine D2 receptors to phosphoinositide turnover and may underlie some of the physiological effects of D2 receptor stimulation.     

Reversible decrease in dopaminergic 3H-agonist binding after 6-hydroxydopamine and irreversible decrease after kainic acid

Six days after the unilateral intrastriatal injection of 30 ug 6-hydroxydopamine (6-OHDA) the number of stereospecific 3H-dopamine and 3H-apomorphine binding sites (Bmax) was reduced by 50-60% in the caudate nucleus ipsilateral to the lesion. The dopamine content of the lesioned caudate nucleus was also reduced to 2% of the contralateral side or of sham-operated controls. The preincubation of depleted homogenates with added dopamine reversed the effects of 6-OHDA on the Bmax of 3H-agonists. A similar pattern of depletion, decrease in binding and in vitro reversal by dopamine was observed after a single injection of reserpine (4.0 mg/kg, im.). The intrastriatal injection of kainic acid also lowered the Bmax of 3H-agonists by 65% without altering dopamine content. Preincubation of homogenates of kainic acid-lesioned caudate nuclei with 355 nM (endogenous) dopamine did not reverse the decrease in binding. We conclude that treatments which deplete endogenous dopamine, including the lesion of nigrostriatal terminals, induce a reversible change in the parameters of 3H-agonist binding whereas the destruction of intrinsic caudate neurons with kainic acid results in an irreversible loss of receptors.     

Serotonin stimulates protein phosphorylation in the cestode Hymenolepis diminuta.

1. Serotonin stimulated the incorporation of 32P from [gamma-32P] ATP into crude membrane preparations (P2) of Hymenolepis diminuta in a dose-dependent manner (EC50 of approximately 0.79 microM). 2. This response was seen with several serotonin agonists, and was inhibited by several serotonin antagonists, which were identical to the previously described activation and inhibition of serotonin-sensitive adenylate cyclase. 3. Cyclic AMP produced a dose-dependent stimulation of 32P incorporation into the P2 fraction, with an EC50 of approximately 2.51 microM. 4. The targets for the serotonin stimulated incorporation of 32P were found to be in trypsin-labile proteins with Mr's of 134,000, 110,000, 82,000, 80,000 and 31,000.     

Hydroxylated serotonin and dopamine as substrates and inhibitors for human cytosolic SULT1A3

Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is known to play an important role in the regulation and homeostasis of monoamine neurotransmitters. The current study was designed to examine the occurrence of the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine by human cytosolic SULTs and to investigate the inhibitory effects of these hydroxylated derivatives on the sulfation of their unhydroxylated counterparts, serotonin and dopamine. A systematic study using 11 known human cytosolic SULTs revealed SULT1A3 as the responsible enzyme for the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine. The pH-dependence and kinetic constants of SULT1A3 with 7-hydroxyserotonin or 6-hydroxydopamine as substrate were determined. The inhibitory effects of 7-hydroxyserotonin and 6-hydroxydopamine on the sulfation of serotonin and dopamine were evaluated. Kinetic analyses indicated that the mechanism underlying the inhibition by these hydroxylated monoamine derivatives is of a competitive-type. Metabolic labeling experiments showed the generation and release of [³⁵S]sulfated 7-hydroxyserotonin and [³⁵S]sulfated 6-hydroxydopamine when SK-N-MC human neuroblastoma cells were labeled with [³⁵S]sulfate in the presence of 7-hydroxyserotonin or 6-hydroxydopamine. Upon transfection of the cells with siRNAs targeted at SULT1A3, diminishment of the SULT1A3 protein and concomitantly the sulfating activity toward these hydroxylated monoamines was observed. Taken together, these results indicated clearly the involvement of sulfation in the metabolism of 7-hydroxyserotonin and 6-hydroxydopamine. By serving as substrates for SULT1A3, these hydroxylated monoamines may interfere with the homeostasis of endogenous serotonin and dopamine.     

Neostriatal dopamine receptor loss and behavioral deficits in the senescent rat

This study was carried out to evaluate the behavioral implications of previously reported declines in striatal dopamine receptors sensitive to [³H]-neuroleptic specific binding. Rotational behavior was examined following right intrastriatal dopamine (DA) injections in nialamide pretreated rats that had been previously unilaterally lesioned in the left substantia nigra with 6-hydroxydopamine. Results showed that following DA injections old rats exhibited significant deficits in rotational behavioral response strength when compared to young rats. Results are discussed in terms of relating behavioral alterations in stereotypic behavior that occur with senescence to changes in striatal D₂ receptors.     

The dopamine receptor adenylate cyclase complex: Evidence for post recognition site involvement for the development of supersensitivity

The dopamine receptor adenylate cyclase complex of a rat striatal membrane preparation became more responsive to dopamine following the injection of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle or following the subcutaneous implantation of morphine pellets. Moreover, the membrane cyclase system was more responsive to activation by GTP, guanyl-5-yl-imidodiphosphate and Mn-ATP. These observations suggest that both 6-OHDA and morphine induce similar biochemical changes in striatum and that the increased responsiveness arises, in part, from modification of the nucleotide regulatory and/or catalytic components of adenylate cyclase.     

Studies on axonal transport in dopaminergic neurons: effect of neuropharmacologic lesions

Axonal transport of [³H]protein to the nucleus accumbens, olfactory tubercle, septal region, caudate nucleus, and hypothalamic region was investigated in rats after unilateral injection of [³H]lysine into the substantia nigra. Co-injection of 2 μg of colchicine with the [³H]lysine depressed the recovery of [³H]protein from forebrain structures by over 70 percent without altering incorporation into midbrain protein, whereas 1 or 2 μg of cycloheximide decreased the incorporation of labelled lysine into both midbrain and forebrain protein by 69 to 76 percent. Partial 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra decreased striatal dopamine levels by 78 percent and reduced axonal protein transport by 47 to 82 percent. Injecting the [³H]lysine 2 mm dorsal to the substantia nigra decreased transport by 95 percent. Unilateral kainic acid-induced lesions of the caudate, which decreased striatal glutamic acid decarboxylase activity by 44 percent and spared striatal dopamine content, did not alter the transport of [³H]protein. Thus, axonal transport of protein in dopamine-containing systems is dependent upon the site of injection of labelled precursor and upon the integrity of a 6-OHDA sensitive pathway. Further, transport is sensitive to inhibitors of both microtubule assembly and protein synthesis, and insensitive to intrastriatal kainic acid lesions.     

Electrophysiological evidence for involvement of cyclic adenosine monophosphate in dopamine responses of caudate neurons

Microiontophoresis of dopamine, apomorphine, cyclic adenosine monophosphate (cyclic AMP) and monobutyryl cyclic AMP depresses the spontaneous or drug-evoked discharge of the majority of neurons in the rat caudate nucleus. Responses to dopamine are enhanced only slightly by the phosphodiesterase (PDE) inhibitors aminophylline or papaverine, both of which also directly slow caudate neurons. However, iontophoresis of the PDE inhibitor methyl isobutyl xanthine (IBMX), or combination of IBMX and papaverine produced marked potentiations of dopamine-induced inhibitions. IBMX alone has little or no direct actions. Chlorpromazine, but not the beta adrenergic blocker MJ-1999, antagonized dopamine, but not cyclic AMP responses. After destruction of the nigral striatal dopamine bundle by focal injection of 6-hydroxy-dopamine, caudate neurons show supersensitivity to dopamine and apomorphine. These results provide electrophysiological support for the hypothesis that the dopamine receptor in caudate may be functionally related to the adenyl cyclase system, as suggested by recent biochemical findings.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca2+ and calmodulin

The effects of Ca2+ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [32P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca2+. Maximal incorporation of [32P] into the protein was achieved with free Ca2+ concentrations of 90 microM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 microM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [32P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca2+ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca2+ concentrations between 10(-6) - 10(-5) M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca2+ and calmodulin.     

The decrease of NAD(P)H has a prominent role in dopamine toxicity

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Receptor-mediated phosphorylation of spermatozoan proteins

These studies are the first to report egg peptide-mediated stimulation of protein phosphorylation in spermatozoa. Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) or resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2) stimulated the incorporation of 32P into various proteins of isolated spermatozoan membranes in the presence, but not absence, of GTP. The Mr of three of the phosphorylated proteins were 52,000, 75,000, and 100,000. GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] but not GDP beta S (guanosine 5'-O-(2-thiodiphosphate] or GMP-PNP (guanylyl imidodiphosphate) also supported the peptide-mediated stimulation of protein phosphorylation. The peptides markedly stimulated guanylate cyclase activity, and GTP gamma S or GTP but not GMP-PNP served as effective substrates for the enzyme. The accumulation of cyclic AMP was not stimulated by the peptides. Subsequently, it was shown that added cyclic GMP or cyclic AMP increased 32P incorporation into the same membrane proteins as those observed in the presence of peptide and GTP. The amount of cyclic GMP (up to 3 microM) formed by membranes in the presence of peptide and 100 microM GTP equated with the amount of added cyclic GMP required to increase the 32P content of a Mr 75,000 protein selected for further study. 32P-Peptide maps of the Mr 75,000 protein indicated that the same domains were phosphorylated in response to cyclic nucleotides or to egg peptide and GTP. Intact cells were subsequently incubated with 32P to determine if the radiolabeled proteins observed in isolated membranes also would be obtained in intact cells. The 32P contents of proteins of Mr 52,000, 75,000, and 100,000 were significantly increased by the addition of resact. Peptide maps confirmed that the increased 32P incorporation obtained in a Mr 75,000 protein of isolated membranes occurred on the same protein domains as the 32P found on the Mr 75,000 protein of intact cells. These results suggest that a GTP or GTP gamma S requirement for peptide-mediated protein phosphorylation in spermatozoan membranes is mainly due to the enhanced formation of cyclic GMP, and it therefore is likely that peptide-induced elevations of cyclic nucleotide concentrations in spermatozoa are responsible for the specific increases in 32P associated with at least three sperm proteins, all apparently localized on the plasma membrane.     

Fluorescent histochemical detection of injected dopamine in lateral hypothalamus after degeneration of catecholaminergic fibres

The fluorescent histochemical technique was used to examine the presence of dopamine 1 h after injection into the lateral hypothalamus which had been depleted of catecholamines by repeated injections of 6-hydroxydopamine. There was considerably more fluorescence at the dopamine injection site which had been previously treated with 6-hydroxydopamine than at the contralateral site treated with saline. These results suggest that the destruction of catecholamine containing nerve fibres from the hypothalamus may impair the ability of brain tissue to remove amines which are produced endogenously during degeneration. This could explain why amine accumulation produced during degeneration of catecholamine-containing neurons can remain for several weeks after the parent cells, hypothesized to have produced the accumulating amine, have died.     

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.