Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

Binding of kynurenine to catecholamine

Summary Papiliochrome II is a pale yellow pigment of butterflies and consists of one molecule each ofL-kynurenine and N--alanyldopamine (NBAD). The aromatic amino nitrogen of kynurenine is bonded to the-carbon of NBAD. There are isomers IIa and IIb which show opposite circular dichroism. The-alanine contents of IIa and IIb were determined and the molar ratio of IIa to IIb has proved to be 1.17. The IIa and IIb were decomposed toL-kynurenine and N--alanylnorepinephrine (NBANE) by being heated in water at 80°C for 30 min. In both IIa and IIb, circular dichroism of the NBANE showed the same positive peak at 280 nm. The NBANE were further decomposed to-alanine and norepinephrine (NE) by being heated in 1 N HC1 at 100°C for 2 hr. The NE was submitted to enantioseparation and has proved to be a racemic mixture in both cases of IIa and IIb. These results are discussed in the light of the enzymic synthesis of IIa and IIb.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Increased 5-Hydroxytryptamine and Norepinephrine Release from Rat Brain Slices by the Red Sea Flatfish

The effect of the Red Sea flatfish toxin pardaxin was examined on K+-evoked and on basal release of either [3H]norepinephrine or [3H]5-hydroxytryptamine from preloaded rat cortical slices. The K+-induced release of the neurotransmitters was stimulated in a dose-related manner at concentrations ranging from 0.5 to 4 micrograms/ml. Basal release of the two transmitters was elevated to a lesser extent. Although the stimulation of evoked release was approximately equivalent for the two neurotransmitters, the response to 5-hydroxytryptamine was reversible whereas that of norepinephrine was not washed by 20 min of superfusion. The mechanisms involved in producing these actions of pardaxin are not known; however, they may be mediated by changes in electrolyte fluxes across the neuronal membranes.     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

Effects of p,p''-DDT on the Rat Brain Concentrations of Biogenic Amine and Amino Acid Neurotransmitters and Their Association with p,p''-DDT-Induced Tremor and Hyperthermia

Male, Fischer strain 344 adult rats were given various doses (25-100 mg/kg) of p,p'-DDT by oral gavage, and levels of biogenic amines, their metabolites, and amino acid neurotransmitters, tremor activity, and rectal temperature were measured at several intervals (2, 5, 12, and 24 h) after dosing. Dose-related increases in rectal temperature and in tremor activity were observed at 50-100 mg/kg 12 h after dosing. Tremorigenic doses of DDT increased the 5-hydroxyindoleacetic acid (5-HIAA) level in hypothalamus, brainstem, and striatum, whereas doses of 75 and 100 mg/kg increased the 3-methoxy-4-hydroxyphenylglycol (MHPG) level in hypothalamus and brainstem and the 3,4-dihydroxyphenylacetic acid level in striatum. Six amino acids were assayed in the brainstem, hypothalamus, and striatum; aspartate and glutamate levels were increased only in brainstem at 25-100 mg/kg. No consistent changes in concentrations of taurine, glutamine, glycine, or gamma-aminobutyric acid were observed in any of the regions assayed. Time-related increases in rectal temperature were seen 2-12 h after dosing, and the presence of tremor was observed 5-12 h after dosing; for both the time of peak effect was at 12 h. The DDT-induced hyperthermia and tremor were associated with dose- and time-related increases in levels of 5-HIAA, MHPG, aspartate, and glutamate. It is suggested that an increase in the turnover rate of 5-hydroxytryptamine (5-HT) may be responsible for the DDT-induced hyperthermia, whereas increases in the metabolism of 5-HT and norepinephrine may be involved in the tremor.     

Desipramine Binding: Relationship to Central and Sympathetic Noradrenergic Activity

We examined the effects of treatments affecting norepinephrine release on the number of norepinephrine reuptake recognition sites as reflected by desipramine binding. To do this, we used manipulations having similar presynaptic but contrasting postsynaptic effects. Presynaptic inhibition by 6-hydroxydopamine lesion or by clonidine, and postsynaptic receptor stimulation by isoproterenol, reduced desipramine binding. Presynaptic stimulation by d-amphetamine and postsynaptic receptor blockade by prazosin increased desipramine binding. Similar effects and binding properties were seen in cerebral cortex, heart, and soleus muscle. After unilateral noradrenergic lesions, reduction in desipramine binding correlated with reduction in norepinephrine uptake. These results show that norepinephrine reuptake appears to be regulated by transmitter release regardless of effects on postsynaptic transmission, and that this regulation is analogous in the central and sympathetic nervous systems.     

ACTH 1-24-induced potentiation of norepinephrine contractile responses in aortic strips from spontaneously hypertensive (SH) and normotensive (WKY) rats

Norepinephrine (NE)-induced contractile responses were less in aortic strips from SH compared to WKY rats. ACTH 1-24 potentiated NE responses in both SH and WKY aortic strips. This effect was more potent in SH aortic strips. NE-induced contractions in SH aortic strips were less sensitive to changes in external Ca2+ levels than were those of WKY aortic strips. ACTH 1-24 did not potentiate NE responses under low external Ca2+ conditions in SH aortic strips or under high external Ca2+ conditions in WKY aortic strips. The greater sensitivity of NE responses following ACTH 1-24 in SH aortic strips may imply that this peptide is modulating a mechanism related to an impaired contractility and that Ca2+ plays a key role in the observed effects.