Emanuel Meller

Activation of Erk and JNK MAPK pathways by acute swim stress in rat brain regions

BackgroundThe mitogen-activated protein kinases (MAPKs) have been shown to participate in a wide array of cellular functions. A role for some MAPKs (e.g., extracellular signal-regulated kinase, Erk1/2) has been documented in response to certain physiological stimuli, such as ischemia, visceral pain and electroconvulsive shock. We recently demonstrated that restraint stress activates the Erk MAPK pathway, but not c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38MAPK, in several rat brain regions. In the present study, we investigated the effects of a different stressor, acute forced swim stress, on the phosphorylation (P) state of these MAPKs in the hippocampus, neocortex, prefrontal cortex, amygdala and striatum. In addition, effects on the phosphorylation state of the upstream activators of the MAPKs, their respective MAPK kinases (MAPKKs; P-MEK1/2, P-MKK4 and P-MKK3/6), were determined. Finally, because the Erk pathway can activate c-AMP response element (CRE) binding (CREB) protein, and swim stress has recently been reported to enhance CREB phosphorylation, changes in P-CREB were also examined.ResultsA single 15 min session of forced swimming increased P-Erk2 levels 2–3-fold in the neocortex, prefrontal cortex and striatum, but not in the hippocampus or amygdala. P-JNK levels (P-JNK1 and/or P-JNK2/3) were increased in all brain regions about 2–5-fold, whereas P-p38MAPK levels remained essentially unchanged. Surprisingly, levels of the phosphorylated MAPKKs, P-MEK1/2 and P-MKK4 (activators of the Erk and JNK pathways, respectively) were increased in all five brain regions, and much more dramatically (P-MEK1/2, 4.5 to > 100-fold; P-MKK4, 12 to ~300-fold). Consistent with the lack of forced swim on phosphorylation of p38MAPK, there appeared to be no change in levels of its activator, P-MKK3/6. P-CREB was increased in all but cortical (prefrontal, neocortex) areas.ConclusionsSwim stress specifically and markedly enhanced the phosphorylation of the MAPKKs P-MEK1/2 and P-MKK4 in all brain regions tested without apparent alteration in the phosphorylation of P-MKK3/6. Curiously, phosphorylation of their cognate substrates (Erk and JNK) was increased to a much more modest extent, and in some brain regions was not altered. Similarly, there was a region-specific discrepancy between Erk and CREB phosphorylation. Possible explanations for these findings and comparison with the effects of restraint stress will be discussed.

Selective D2 dopamine receptor agonists prevent catalepsy induced by SCH 23390, a selective D1 antagonist

SCH 23390, an apparently selective antagonist of central D1 dopamine receptors, produced profound catalepsy at low doses (0.1 mg/kg, s.c.). Pretreatment with the selective D2 receptor agonists LY 141865, RU 24213 or LY 171555, the active (-) enantiomer of LY 141865, elicited a dose-dependent inhibition of the cataleptic response. Pergolide and apomorphine were also effective. This effect was not due to altered disposition or penetration of SCH 23390 into the brain since pretreatment with a dose of LY 171555 which completely blocked catalepsy had no effect on the ID50 of SCH 23390 to inhibit 3H-cis-piflutixol binding to D1 receptors measured ex vivo. Alternative mechanisms are considered to explain the results, which offer new insights into striatal dopaminergic regulation of motor activity.

Evidence that striatal synthesis-inhibiting autoreceptors are dopamine D3 receptors

The activation constants (KA; dose required to occupy 50% of receptors) for reversal of gamma-butyrolactone (GBL)-induced elevation of striatal L-3,4-dihydroxyphenylalanine (L-DOPA) levels via stimulation of presynaptic dopamine receptors were determined for apomorphine and two dopamine D3 receptor-selective agonists, quinpirole and LY163502 (quinelorane). The KA values correlated significantly with the affinities (Ki) of the agonists for the D3 (r = 0.999, P < 0.05) but not the D2 (r = -0.13) receptor, suggesting that striatal synthesis-inhibiting autoreceptors are of the D3 rather than the D2 subtype.

Region-specific effects of acute and repeated restraint stress on the phosphorylation of mitogen-activated protein kinases

The mitogen-activated protein kinases (MAPKs) are a family of signal transduction mediators that regulate a host of cellular activities, including cell growth and proliferation, and differentiation and survival, via sequential phosphorylation and activation of a cassette of three protein kinases. MAPKs are also recruited when the brain undergoes synaptic plasticity and remodeling (e.g., during induction of long-term potentiation, learning and memory consolidation). The activities of some of these kinases are altered in response to various acute stimuli such as ischemic insult, visceral pain and electroconvulsive shock. In the present study we used immunoblotting techniques to examine the effects of acute and repeated restraint stress on the phosphorylation state of three MAPKs, the extracellular signal-regulated kinase Erk1/2, c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK, in different brain regions. A single exposure to 30 min of restraint stress-elevated phospho-Erk1/2 (P-Erk1/2) levels in all three brain regions examined (hippocampus, medial prefrontal cortex and cingulate cortex), but did not alter the phosphorylation pattern of the other two MAPKs in any region. In marked contrast, exposure to restraint for 11 days (30 min/day) reduced the levels of all three MAPKs, but only in the prefrontal cortex. The results are compared to the reported effects of acute and chronic stress on other biochemical and functional measures.

Enhanced behavioral stereotypies elicited by intrastriatal injection of D1 and D2 dopamine agonists in intact rats

Five components of behavior elicited by dopamine (DA) agonists (locomotor hyperactivity, sniffing, oral activity, grooming and paw nibbling) were evaluated after bilateral infusion of the selective D1 agonist fenoldopam (SKF 82526; 2.5-10 micrograms), the selective D2 agonist quinpirole (LY 171555; 5-40 micrograms) and the muscarinic cholinergic antagonist scopolamine (5-20 micrograms) into the ventral striatum of awake, unrestrained rats. Simultaneous bilateral infusion of various dose combinations of fenoldopam (2.5-10 micrograms) and quinpirole (5-20 micrograms) elicited dramatic increases in stereotyped behaviors relative to the effects produced by corresponding doses of each drug alone. Stereotyped sniffing and paw nibbling (self-directed oral activity) were markedly enhanced, whereas conventional oral behaviors (licking, chewing and/or biting) were either slightly or not at all increased. These potentiated responses were reduced or blocked by concomitant infusion of either the selective D1 antagonist SCH 23390 (1 and 5 micrograms) or the selective D2 antagonist sulpiride (0.15 microgram). Scopolamine (10 micrograms) only slightly increased the effects of quinpirole (5 micrograms) on both sniffing and oral behaviors, whereas it dramatically potentiated the effects of fenoldopam (2.5 micrograms) on oral activity; sniffing was only slightly increased. The effects of both drug combinations were almost completely antagonized by infusion of either SCH 23390 (1 microgram) or sulpiride (0.1 microgram). The results demonstrate that the synergistic effects of co-activation of D1 and D2 receptors observed after systemic administration are mediated at least in part by an interaction at the level of the striatum. Differences and similarities between the behaviors expressed after various treatments are discussed.

Receptor reserve at striatal dopamine autoreceptors: Implications for selectivity of dopamine agonists

The dose response curve for apomorphine reversal of gamma-butyrolactone (GBL)-induced L-DOPA accumulation in rat striatum was shifted almost 6-fold to the right after partial irreversible blockade (83%) of dopamine (DA) autoreceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ); however, the maximal response was not reduced. In contrast, the major effect of a similar degree of irreversible blockade (86%) on the dose-response curve for the autoreceptor-selective agent EMD 23,448 was a reduction in maximal response (60% of control), indicating that EMD 23,448 is a partial agonist. A large receptor reserve therefore exists at the DA autoreceptor, which may explain in part why many DA agonists are more potent in models pre- than postsynaptic receptor activation.

Pharmacological and biochemical properties of isomeric yohimbine alkaloids.

The stereochemical and pharmacological properties of yohimbine and some of its isomers are briefly reviewed. Several pharmacological and physical properties of a selection of the isomers have been determined with a view to elucidating which might be important in the elaboration of the known behavioral effects produced by them. Activity is not dependent upon lipid solubility or on the ease of access to the central nervous system. The isomers are weak inhibitors of rat-brain acetylcholinesterase and weak antagonists at muscarinic cholinergic receptors. In the rat brain in vitro they do not possess significant monoamine oxidase-inhibiting properties nor do they inhibit the uptake of serotonin. They are relatively potent antagonists of 5HT on the rat isolated fundus preparation and their potency in this preparation may be related to their ability to produce behavioral and cardiovascular effects in man and dogs.

Dopamine agonist induced self-multilative biting behavior in monkeys with unilateral ventromedial tegmental lesions of the brainstem: Possible pharmacological model for Lesch-Nyhan syndrome

We have investigated the effects of various dopamine (DA) agonists on induction of abnormal involuntary movements (AIM) in a group of monkeys which had denervated nigro-striatal DA neurons for 10-14 years rendered by a unilateral surgical ventromedial tegmental (VMT) lesion of the brainstem. The surgical lesions were placed when the monkeys were 2-4 years old. The administration of mixed DA agonists, such as L-DOPA, apomorphine (Apo) and abeorphine 201-678, elicit a self-mutilative biting behavior (SMB) of the forelimb digits contralateral to the lesion, and spasticity of the contralateral hindlimb. These dysfunctions resemble, in some aspects, the neurological disturbances associated with Lesch-Nyhan syndrome. The SMB behavior was elicited by mixed DA agonists which predominantly stimulate D1, but not D2 DA receptors, and was prevented or abolished by the D1 DA antagonist SCH 23390 or by the D1 and D2 DA antagonist fluphenazine (Flu), but not by the D2 antagonist (+/-)sulpiride. These results suggest that DA agonist-induced SMB behavior is mediated by D1 and/or by both D1 and D2 DA receptor pathways. To study the relationships between HPRT, the defective enzyme in Lesch-Nyhan syndrome, and the DA neuronal systems, we have measured the effects of nigro-striatal DA degeneration and intrastriatal neuronal degeneration on HPRT activity. The unilateral 6-OHDA-induced nigro-striatal DA degeneration does not significantly alter the HPRT activity on the lesioned side of the striatum, while the quinolinic acid-induced intrastriatal neuronal degeneration significantly reduces the enzyme activity. These results suggest that HPRT is localized on intrastriatal neurons which are also known to contain DA receptors. It is postulated that HPRT deficiency in Lesch-Nyhan syndrome results in abnormal guanine nucleotide metabolism which may affect the regulation of DA receptors.

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site-and phosphospecific antibodies and site-directed mutagenesis

Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT‐20 cells expressing wild‐type or [Leu19]‐recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l‐DOPA biosynthesis rates in intact AtT‐20 cells. Treatment of either PC12 cells or AtT‐20 cells expressing wild‐type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT‐20 cells expressing wild‐type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT‐20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization‐induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.

TETRAHYDRO-β-CARBOLINES: SPECIFIC INHIBITORS OF TYPE A MONOAMINE OXIDASE IN RAT BRAIN1

Abstract— A series of serotonin and phenethylamine analogs were tested for their ability to differentially inhibit type A or B monoamine oxidase (EC 1.4.3.4) in rat hypothalamus. The most specific and potent effects were shown by several tetrahydro‐β‐carbolines which competitively inhibited the oxidative deamination of serotonin at micromolar concentrations. In contrast, effective inhibition of phenethylamine deamination was observed only at or near millimolar concentrations of these drugs. Significant reductions of type A but not type B MA0 activity were also observed after administration of 25–100 mg/kg, i.p., of tetrahydro‐β‐carboline and its 6‐methoxy derivative.