Dopamine Release via Protein Kinase C Activation in the Fish Retina

Calcium-dependent phospholipid-sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activated PKC in the nanomolar range. A major 38-kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (greater than 15 min). Rod-bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (alpha/beta-subtype). The PKC antibody recognized a 78-kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium- and dose-dependent [3H]DA release during a short period (2 min), with the minimal effective dose being approximately 1 nM. Other phorbols having no tumor-promoting activity, such as 4 beta-phorbol and 4 alpha-phorbol 12,13-didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1-oleoyl-2-acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG- or TPA-induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Methiothepin and Lysergic Acid Diethylamide on Serotonin Release In Vitro and Serotonin Synthesis In Vivo: Possible Relation to Serotonin Autoreceptor Function

Abstract: An in vitro system characterizing the presyn- aptic serotonin (5-HT) autoreceptor which controls the release of 5-HT from rat brain slices is described. Using this system, methiothepin (1–10 μ M) demonstrated 5-HT autoreceptor antagonist activity -by enhancing 5-HT release, while several recognized postsynaptic 5-HT receptor antagonists were inactive: mianserin, cinanserin, cyproheptadine, methysergide. The activity of methiothepin was highest in hypothalamic slices and lowest in striatal slices and was inhibited by the autoreceptor agonists lysergic acid diethylamide (LSD) and 5-methoxy- tryptamine (5-MT). The reversal of the methiothepin-enhanced 5-HT release from hypothalamic slices by LSD was not influenced by 0.3 μ M tetrodotoxin. The peripheral administration of LSD to rats has been shown to reduce 5-HT synthesis and release by a mechanism thought to involve, in part, an autoreceptor-mediated reduction in impulse flow of 5-HT neurons. In the present experiments, intraperitoneal injection of methiothepin antagonized the LSD-induced reduction in hypothalamic 5-HT synthesis (5-hydroxytryptophan accumulation) while exerting no influence by itself. Conversely, compounds which were not active as 5-HT autoreceptor antagonists in vitro (i.e., cyproheptadine, methysergide, cinanserin) did not influence the effect of LSD on 5-HT synthesis. Further, the reduction in 5-hydroxytryptophan (5-HTP) accumulation by LSD showed regional differences in inhibition by methiothepin (hypothalamus > cortex > striatum) which paralleled the autoreceptor antagonist activity of methiothepin in vitro. These data suggest that similar autoreceptor mechanisms control 5-HT release and synthesis in terminal 5-HT projection areas and that the reduction in 5-HT accumulation by LSD and the antagonism by methiothepin may represent a useful biochemical measure of 5-HT autoreceptor activity in vivo.     

Phases in potassium transport and their regulation under near-equilibrium conditions in wheat seedlings

Potassium uptake and release in roots and translocation to the shoots were studied in 14-day-old winter wheat ( Tritictum aestivum L. cv. Martonvásári 8) of different K status. Transport processes were measured in the growth solutions for 5 h ensuring near-equilibrium conditions. The uptake showed three phases: (1) at low external K⁺ concentrations it increased with increasing concentrations and culminated at 0.1 m M : (2) between 0.1 and 1 m M it decreased, and (3) it increased again above 1 m M : The release of K⁺ showed a constant low level below 1 m M while paralleling the uptake above that. The uncoupler 2,4-dinitrophenol inhibited uptake phases (1) and (2), whereas it did not affect either phase (3) or K⁺ release. Translocation showed similar patterns. It is concluded that phases (1) and (2) depend on metabolic energy while phase (3) is mostly passive. It is emphasized that different types of regulation seem to operate in the transport mechanism: i.e. limitation by transport sites, control by negative feedback and by K⁺/K⁺ exchange, respectively.     

Antide B,an antagonist of LHRH with cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine in position 5

Summary Several LHRH antagonists with trans-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine (trans-PzACAla) in the position 5 were synthesized and their antiovulatory activity was compared with the activity of the analogs containing cis-PzACAla in this position. In all cases cis-isomer produced more potent analogs. Introduction of cis-PzACAla in the position 5 of Antide gave Antide B which completely inhibits ovulation at a dose of 0.5µg/rat. Antide B releases negligible histamine (ED₅₀ = 104µg/mL), and has excellent solubility in water. Also, an improved synthesis of cis-PzACAla is reported, involving the hydrogenation of 4-aminophenylalanine on a rhodium catalyst to give the desired cis-isomer with a 53% yield.Abbreviations Cpa 3-(4-chlorophenyl)alanine - ILys N-isopropyllysine - Nal 3-(2-naphthyl)alanine - NicLys N-nicotinoyllysine - Pal 3-(3-pyridyl)alanine - PicLys N-picolinoyllysine - PzACAla 3-(4-pyrazinylcarbonylaminocyclohexyl)alanine - Qal 3-(3-quinolyl)alanine     

Behavioral sensitization and tolerance to cocaine and the occupation of dopamine receptors by dopamine

Data from the authors’ laboratory on the neural substrates of Pavlovian conditioning and behavioral sensitization to psychomotor stimulants are reviewed. The findings of a recent experiment on the role of occupation of dopamine receptors by dopamine and its association to behavioral sensitization are reported. Daily intermittent injections of cocaine produced behavioral sensitization to the locomotor response in rats, whereas continuous cocaine infusions produced behavioral tolerance. Behavioral sensitization to cocaine was blocked by coadministration of nimodipine, anL-type calcium channel blocker. The increases in locomotion produced by cocaine was associated with an increase in the occupation of striatal dopamine D₁ and D₂ receptors, measured as the density of receptors protected from denaturation byN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). This association was not observed when rats were given a challenge injection of cocaine 10 d after withdrawal from similar treatment regimens. Rats given a cocaine challenge after withdrawal from either intermittent or continuous cocaine treatment regimens exhibited increased occupations of striatal D₁ and D₂ receptors. This increase was similar in magnitude to that observed in rats without a history of cocaine treatments after a challenge injection of cocaine. This suggests tnat the differences in occupancy of striatal dopamine receptors by dopamine observed in the prewithdrawal condition are likely the result of differences in brain levels of cocaine achieved by the two treatment regimens. Occupancy of striatals dopamine D₁ and D₂ receptors does not appear to be related to the development of sensitization to the motor-stimulating effects of cocaine.     

NR2A-containing NMDA receptors depress glutamatergic synaptic transmission and evoked-dopamine release in the mouse striatum

NMDA receptors play essential roles in the physiology and pathophysiology of the striatum, a brain nucleus involved in motor control and reward-motivated behaviors. NMDA receptors are composed of NR1 and NR2A–D subunits. Functional properties of NMDA receptors are determined by the type of NR2 subunit they contain. In this study, we have examined the involvement of NR2B and NR2A in the modulatory effect of NMDA on glutamatergic and dopaminergic synaptic transmission in the striatum. We found that bath application of NMDA decreased the amplitude of the field excitatory post-synaptic potential/population spike (fEPSP/PS) measured in corticostriatal mouse brain slices. This depression was not affected by the NR2B-selective antagonists Ifenprodil and Ro 25-6981, but was abolished by the NR2A antagonist NVP-AAM077. Activation of corticostriatal neurons by NMDA did not contribute to synaptic depression because similar results were obtained in decorticated striatal slices. Synaptic depression was not dependent on GABA release because the GABA_A receptor antagonist bicuculline did not affect NMDA-induced decrease of the fEPSP/PS. NMDA also depressed evoked-dopamine release through NR2A- but not NR2B-containing NMDA receptors. Our results identify an important role for NR2A-containing NMDA receptors intrinsic to the striatum in regulating glutamatergic synaptic transmission and evoked-dopamine release.     

Decomposition and nutrient release by green manures in a tropical hillside agroecosystem

The decomposition and nutrient release of 12 plant materials were assessed in a 20-week litterbag field study in hillsides from Cauca, Colombia. Leaves of Tithonia diversifolia (TTH) and Indigofera constricta (IND) decomposed quickly (k=0.035±0.002 d^–1), while those of Cratylia argentea (CRA) and the stems evaluated decomposed slowly (k=0.007±0.002 d^–1). Potassium presented the highest release rates (k>0.085 d^–1). Rates of N and P release were high for all leaf materials evaluated (k>0.028 d^–1) with the exception of CRA (N and P), TTH and IND (P). While Mg release rates ranged from 0.013 to 0.122 d^–1, Ca release was generally slower (k=0.008–0.041 d^–1). Initial quality parameters that best correlated with decomposition (P>0.001) were neutral detergent fibre, NDF (r=–0.96) and in vitro dry matter digestibility, IVDMD (r=0.87). It is argued that NDF or IVDMD could be useful lab-based tests during screening of plant materials as green manures. Significant correlations (P>0.05) were also found for initial quality parameters and nutrient release, being most important the lignin/N ratio (r=–0.71) and (lignin+polyphenol)/N ratios (r=–0.70) for N release, the C/N (r=0.70) and N/P ratios (r=–0.66) for P release, the hemicellulose content (r=–0.75) for K release, the Ca content (r=0.82) for Ca release, and the C/P ratio (r=0.65) for Mg release. After 20 weeks, the leaves of Mucuna deerengianum released the highest amounts of N and P (144.5 and 11.4 kg ha^–1, respectively), while TTH released the highest amounts of K, Ca and Mg (129.3, 112.6 and 25.9 kg ha^–1, respectively). These results show the potential of some plant materials studied as sources of nutrients in tropical hillside agroecosystems.     

Whitemere, a lake that defies some conventions about nutrients

1. Whitemere, a kettle-hole lake in north-west England (z_m,14 m, area 22.5 ha) has extremely high maximal concentrations (around 1 mg L⁻¹) of total phosphorus (TP) and soluble reactive phosphorus (SRP), and comparatively low maximal concentrations of dissolved inorganic nitrogen (<0.5 mg N L⁻¹). Bioassays indicate that its phytoplankton is nitrogen limited, and it has surface blue-green algal blooms in summer. Palaeoecological investigations have shown that high populations of cyanophytes occurred more than 6000 years ago, even before human settlement of the catchment.
2. The reasons for this combination of features are not immediately clear. This paper attempts to distinguish between two hypotheses: that the high phosphorus concentrations arise from input of phosphate-rich groundwater; and that the high phosphorus concentrations arise from concentration and recycling mechanisms within the lake.
3. The lake is entirely fed by ground water, direct rainfall and surface sheet flow. The concentrations of phosphorus in the ground water are much lower than in the lake water. The lake is stratified and has high phosphorus concentrations in the anaerobic hypolimnion in summer as a result of substantial release from the sediment. Vertical turbulence in the relatively weakly stratified water column may transfer substantial amounts to the epilimnion.There is also substantial phosphorus release from the sediments under the aerobic epilimnion.
4. There is little outflow of water and phosphorus. Most of the phosphorus is returned to the sediment under winter isothermal conditions, to be released again the following summer. The lake has probably maintained these mechanisms for millennia. Recent eutrophication is likely to have been driven by nitrogen inputs from greater intensification of agriculture in the catchment.     

Real-Time Effects of N-Methyl-d-Aspartic Acid on Dopamine Release in Slices of Rat Caudate Putamen: A Study Using Fast Cyclic Voltammetry

Abstract: The functional role of N-methyl-d -aspartic acid (NMDA) glutamate receptors in the real-time regulation of single electrical pulse (1 p)-stimulated endogenous dopamine release was investigated in slices of rat caudate putamen using fast cyclic voltammetry at a carbon fibre electrode. In the presence of Mg²⁺, 20 µM NMDA had a weak effect on background signals but did not affect 1 p-stimulated dopamine release. Removal of Mg²⁺ increased the background and doubled 1 p-stimulated dopamine release. In the absence of Mg²⁺, 20 µM NMDA caused a transient “release” of dopamine and decreased the background signal. The 1 p-stimulated dopamine release was subsequently reduced. In the presence of 1 µM (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), superfusion with 20 µM NMDA did not cause a transient “release” of dopamine, and 1 p-stimulated dopamine release was not subsequently attenuated. In the presence of 1 µM tetrodotoxin, 1 p-stimulated dopamine release was abolished, but 20 µM NMDA still caused a transient “release” of dopamine. Removal of Ca²⁺ from the artificial CSF abolished 1 p-stimulated dopamine release and resulted in a decline in the baseline but did not affect dopamine “release” when 20 µM NMDA was added. The dopamine release-inducing effect of 20 µM NMDA was less pronounced in sites in the caudate putamen where dopamine release increased with frequency of electrical stimulation (hot spots) than in sites where there was little frequency-dependent dopamine release (cold spots). Subsequent 1 p-stimulated dopamine release was less attenuated in cold spots than in hot spots. We conclude that in the absence of Mg²⁺, NMDA induces release of dopamine by acting at CPP-sensitive NMDA receptors in a Ca²⁺-independent manner. This transient release depletes dopamine from a storage site from which dopamine is released by 1 p electrical stimulation. These real-time observations of the effects of NMDA on electrical stimulus-independent and -dependent dopamine release may explain the apparently conflicting observations of the effects of NMDA on dopamine release made in previous studies. They also indicate that dopamine release and storage are heterogeneous at different sites in the rat caudate putamen.     

Comparison of the Effects of a Convulsant Barbiturate on the Release of Endogenous and Radiolabeled Amino Acids from Slices of Mouse Hippocampus

The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) stimulates the spontaneous release of endogenous and radiolabeled acetylcholine (ACh) from mouse hippocampal slices in vitro. In order to determine if the ability of CHEB to release ACh was unique to this neurotransmitter, we have studied the action of this drug in vitro on the release of both radiolabeled and endogenous putative neurotransmitter and non-transmitter amino acids in the hippocampus. Although CHEB stimulated the spontaneous release of both [3H]gamma-n-aminobutyric acid (GABA) and endogenous GABA, CHEB had different effects on the spontaneous release of radiolabeled and endogenous L-glutamate and L-aspartate: L-[3H]glutamate release was inhibited by CHEB, but endogenous L-glutamate release was unaffected by CHEB, but endogenous L-aspartate release was stimulated. The spontaneous release of the amino acids L-alanine and glycine (not thought to be neurotransmitters in the hippocampus) was not affected by CHEB. The results of this study indicate that CHEB does not always stimulate the release of all putative neurotransmitters. The ability of this drug to release ACh, GABA, and L-aspartate may be the result of some specific interaction of CHEB with nerves using these neurotransmitters in the hippocampus. In addition, the results suggest some problems that may be encountered when radiolabeled substances are used to study neurotransmitter release.