Enhancement of noradrenaline release by 12-O-tetradecanoyl phorbol-13-acetate, an activator of protein kinase C

12-O-Tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C (PKC), enhanced the electrically evoked overflow of [3H]noradrenaline in a concentration-dependent manner in rabbit hippocampal slices. 4-O-Methyl-TPA, which lacks the ability to activate PKC had no effect on the evoked tritium overflow. The enhancement of noradrenaline release by TPA was affected by neither the alpha 2-adrenoceptor antagonist yohimbine nor the alpha 2-adrenoceptor agonist clonidine. It is concluded from these results that PKC is involved in the mechanism of stimulus-secretion coupling in noradrenergic nerve terminals of the rabbit hippocampus.     

3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampus: role of Na+ entry on Ca2+ pools and of protein kinase C.

Slices of rat hippocampus, preincubated with [3H]noradrenaline [(3H]NA), were superfused continuously and stimulated by addition of 3,4-diaminopyridine (3,4-DAP; 100 microM) for 10 min to the superfusion medium. An overflow of 3H evoked by 3,4-DAP (representing [3H]NA release) was measurable not only in the presence but also in the absence of extracellular Ca2+. Both the protein kinase C (PKC) activator 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) and the PKC inhibitor polymyxin B, affected mainly the evoked release in the absence of extracellular Ca2+ in a facilitatory or inhibitory manner, respectively. Moreover, in the absence of extracellular Ca2+, both the 3,4-DAP-evoked [3H]NA release and the facilitatory effect of 4 beta-PDB were abolished in the presence of tetrodotoxin or in the absence of Na+ in the superfusion medium. Ruthenium red, a blocker of mitochondrial Ca2+ reuptake, potently increased 3,4-DAP-evoked [3H]NA release in Ca(2+)-free EGTA-containing medium. The facilitatory effects of ruthenium red and 4 beta-PDB were additive. From these and earlier observations we conclude (1) that the mechanism of 3,4-DAP-evoked [3H]NA release involves both Ca2+ influx into the nerve terminals and mobilization of intraneuronal Ca2+ pools. Most probably Ca2+ release from cytoplasmic Ca2+ stores (e.g. endoplasmic reticular pools or mitochondria) is induced by Na+ ions entering the nerve endings during 3,4-DAP-evoked repetitive action potentials. (2) The facilitatory effect of phorbol ester on 3,4-DAP-evoked NA release appears to be mediated not by changes in Ca2+ influx, but by enhancement of intraneuronal events distal to Na+ ion entry and increased intracellular Ca2+ availability.     

The structural requirements for phorbol esters to enhance noradrenaline and dopamine release from rat brain cortex.

1. The effects of various protein kinase C (PKC) activators on the stimulation-induced (S-I) release of noradrenaline and dopamine was studied in rat cortical slices pre-incubated with [3H]-noradrenaline or [3H]-dopamine. The aim was to investigate a possible structure-activity relationship for these agents on transmitter release. 2. 4 beta-Phorbol 12,13-dibutyrate (4 beta PDB, 0.1-3.0 microM), enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner whereas the structurally related inactive isomer 4 alpha-phorbol 12, 13-dibutyrate (4 alpha PDB, 0.1-3.0 microM) and phorbol 13-acetate (PA, 0.1-3.0microM) were without effect on noradrednaline release. Another group of phorbol 12, 13-diesters containing a common 13-ester substituent (phorbol 12, 13-diacetate, PDA, 0.1-3.0 microM; phorbol 12-myristate 13-acetate, PMA, 0.1-3.0 microM; phorbol 12-methylaminobenzoate 13-acetate, PMBA, 0.03-3.0 microM) also enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner with PMA being the least potent. 3. The 12-deoxyphorbol 13-substituted monoesters, 12-deoxyphorbol 13-acetate (dPA, 0.1-3.0 microM), 12-deoxyphorbol 13-angelate (dPAng, 0.1-3.0 microM), 12-deoxyphorbol 13-isobutyrate (dPiB, 0.03-3.0 microM) and 12-deoxyphorbol 13-phenylacetate (dPPhen, 0.1-3.0 microM) enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner. In contrast, 12-deoxyphorbol 13-tetradecanoate (dPT, 0.1-3.0 microM) was without effect. 4. The involvement of PKC in mediating the effects of the various phorbol esters was further investigated. PKC was down-regulated by 20 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta PDB and dPA was abolished whilst that of dPAng was significantly attenuated. This indicates that these agents were acting selectively at PKC. In support of this the PKC inhibitors, polymyxin B (21 microM) and bisindolylmaleimide I (3 microM), attenuated the facilitatory effect of 4 beta PDB and dPAng although that of dPA was not significantly altered. 5. The effects of these agents on transmitter release were not correlated with their in vitro affinity and isozyme selectivity for PKC. Short chain substituted mono- and diesters of phorbol were more potent enhancers of action-potential evoked noradrenaline and dopamine release than the long chain esters. Interestingly, these former agents are the least potent or non effective (e.g. dPA, PDA) tumour promoters. We suggest that the reason for the poor effects of lipophilic long chain phorbol esters (PMA, dPT) on transmitter release is that they are sequestered in the plasmalemma and do not access the cell cytoplasm where the PKC may be located.     

Experimental conditions required for the enhancement by alpha-adrenoceptor antagonists of noradrenaline release in the rabbit ear artery.

1 Segments of the rabbit ear artery were preincubated with (-)-[3H]-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of [3H]-noradrenaline, separated from its metabolites by column chromatography, was determined. 2 Tetrodotoxin abolished, cocaine increased, and clonidine reduced the overflow of [3H]-noradrenaline elicited by 10 shocks at 0.2 Hz, 10 shocks at 2 Hz or 100 shocks at 2 Hz. 3 The effects of yohimbine (0.1 or 1 microM), phentolamine (1 microM) and piperoxan (1 or 10 microM) depended on the stimulation conditions. No antagonist increased the overflow of [3H]-noradrenaline evoked by 10 pulses at 0.2 Hz, but all markedly increased the overflow evoked by 100 pulses at 2 Hz. Only piperoxan (10 microM) slightly enhanced the overflow at 10 pulses, 2 Hz. The effects of yohimbine and piperoxan were similar in arteries not exposed to cocaine and in those that were perfused/superfused with medium containing cocaine (10 microM) after preincubation. 4 It is concluded that yohimbine, phentolamine and piperoxan increase the release of noradrenaline only when the concentration of noradrenaline in the biophase of the ear artery is sufficiently high. The effect is, hence, an anti-noradrenaline effect and due to the blockade of presynaptic alpha-adrenoceptors. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train, indicating that the alpha-adrenergic auto-inhibition develops relatively slowly.     

The adenosine receptor-mediated inhibition of noradrenaline release possibly involves an N-protein and is increased by alpha 2-autoreceptor blockade.

The stimulation-evoked overflow of [3H]-noradrenaline from slices of the rabbit hippocampus is inhibited by alpha 2-autoreceptors as well as by adenosine (A1)-receptors. Slices of rabbit hippocampus were labelled with [3H]-noradrenaline, superfused continuously and stimulated twice electrically (rectangular pulses; 2 ms, 3 Hz, 24 mA, 5 V cm-1). Treatment of hippocampal slices with N-ethylmaleimide (NEM, 30 microM; 30 min), which functionally disturbs certain N-proteins, decreased the inhibitory action of adenosine receptor agonists like (-)-N6-(R-phenylisopropyl)-adenosine ((-)-PIA) and adenosine on noradrenaline release. Release inhibition caused by (-)-PIA (0.03-1 microM) was antagonized by NEM in a non-competitive manner in the absence and in the presence of the alpha 2-adrenoceptor antagonist yohimbine. The adenosine receptor antagonist 8-phenyltheophylline significantly increased the evoked noradrenaline release by about 15% in control slices by diminishing the inhibitory action of endogenous adenosine. In NEM-treated slices this effect of 8-phenyltheophylline was not seen. In the presence of (-)-PIA (0.1 microM), i.e. under conditions of an increased inhibitory tone, release facilitation by 8-phenyltheophylline was decreased by NEM compared to that in the respective controls. Occupation of the A1-receptor with (-)-PIA prior to and during the NEM treatment did not protect the A1-receptor-coupled signal transduction system from being affected by NEM. In the presence of the alpha 2-adrenoceptor antagonist yohimbine, the inhibitory action of (-)-PIA was strongly increased. The above results suggest the involvement of a regulatory N-protein in the A1-receptor-mediated inhibition of noradrenaline release and an interaction between the alpha 2-autoreceptor and the A1-receptor-coupled signal transduction system, possibly at the level of a N-protein.     

Phorbol ester-mediated enhancement of hippocampal noradrenaline release: which ion channels are involved?

Enhancement of neurotransmitter release following phorbol ester-induced activation of protein kinase C (PKC) may be mediated by changes in ion conductance through the presynaptic membrane. This question was studied with rabbit hippocampal slices preincubated with [3H]noradrenaline ([3H]NA). NA release was evoked by pulses of either high K+ or Ca2+ (in the presence of high K+), or by electrical field stimulation. 4 beta-Phorbol 12,13-dibutyrate (PDB) increased and polymyxin B (PMB) reduced the K+-evoked NA release independent of the K+ concentration used for depolarization. The effects of PDB and PMB were not reduced by tetrodotoxin. PDB still enhanced the NA release triggered by short Ca2+ pulses in depolarized, axon terminal membranes (30 mM K+ and no Ca2+). The electrically evoked NA release was markedly enhanced by PDB even in the absence of Cl- in the medium or in the presence of the K+ channel blockers, tetraethylammonium, 4-amino- and 3,4-diaminopyridine. The inhibitory effect of the Ca2+ channel blocker, Cd2+, remained almost unchanged in the presence of PDB. It is concluded that PKC activation facilitates NA release in the hippocampus but not via presynaptic changes in Na+, K+ or Cl- currents. Whether phorbol ester mediates an increased intracellular Ca2+ availability, or whether a triggering 'normal' Ca2+ influx simply initiates, and synergistically supports, the PKC-mediated reactions leading to enhanced exocytosis, cannot be decided from the results of the present experiments.     

3H]noradrenaline release evoked by selegiline ((-)-deprenyl) in the isolated main pulmonary artery of the rabbit

High concentrations of selegiline[-)-deprenyl) (greater than 10(-5) M) enhanced the nerve stimulation (2 Hz)-evoked release of [3H]noradrenaline from the isolated main pulmonary artery of the rabbit. This facilitation of stimulation-evoked [3H]noradrenaline release by selegiline was reduced by exogenous (-)-noradrenaline, an agonist of presynaptic alpha 2-adrenoceptors. This inhibitory action of (-)-noradrenaline was partly antagonized by yohimbine, a selective alpha 2-adrenoceptor blocker. When the stimulation-evoked [3H]noradrenaline release had already been increased by inhibition of Na+-pump (K+-free solution), selegiline further enhanced the nerve-evoked release of labelled neurotransmitter.     

Alpha 2-adrenoceptor mediated inhibition of exocytotic noradrenaline release in the absence of extracellular Ca2+.

The effect of the alpha 2-adrenoceptor agonist clonidine on 3,4-diaminopyridine (3,4-DAP)-evoked [3H]noradrenaline ([32H]NA) release in rat hippocampus slices was studied in the presence or absence (+1 mM EGTA) of extracellular Ca2+. 3H overflow (consisting mainly of unmetabolized [3H]NA) was evoked by addition of 100 microM 3,4-DAP for 10 min to the medium, which always contained 1 microM desipramine. Ligands for L-type voltage-sensitive Ca2+ channels (VSCC) did not affect the evoked [3H]NA release, whereas the preferential N-type VSCC antagonist omega-conotoxin was inhibitory, both in the presence and even more potently in the absence of Ca2+, suggesting an involvement of N-type VSCC in the mechanism of 3,4-DAP-evoked [3H]NA release. In the absence of extracellular Ca2+ the initial Na+ influx, which has been previously proposed to liberate Ca2+ from intracellular stores for the exocytotic process, most probably occurs via N-type VSCC. Clonidine inhibited the 3,4-DAP-evoked [3H]NA release in a concentration-dependent manner, both in the presence and even more potently in the absence of Ca2+; its effects were antagonized by yohimbine. In the presence of extracellular Ca2+ the clonidine effect was not changed by addition of omega-conotoxin. Similar effects of clonidine were found in slices from the rabbit hippocampus. Since the availability of Ca2+ from intracellular stores seems to predominate in the present model, our results lend some support to the suggestion that alpha 2-adrenoceptor activation might affect intracellular mechanisms of Ca2+ homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of 5-HT2 receptors inhibits the evoked release of [3H]noradrenaline in the rat spinal cord.

1. The participation of 5-HT2 receptors in the modulation of the evoked release of [3H]noradrenaline from rat spinal cord slices has been examined. 2. In rat spinal cord slices preincubated with [3H]noradrenaline, the alpha 2-receptor agonist clonidine (10(-6) mol/l) decreased the release of tritium evoked by field stimulation (600 pulses at 5 Hz, 20 mA, 2 msec), while the alpha 2-antagonist yohimbine (10(-6) mol/l) increased it. 3. The 5-HT2/5-HT1c agonist DOI (3 x 10(-7) mol/l) decreased the evoked release of tritium, an effect which was prevented by ketanserin (10(-7) mol/l). 4. It is suggested that in addition to presynaptic alpha 2-adrenoceptors, there are 5-HT2 receptors which modulate the release of noradrenaline in the rat spinal cord.     

Presynaptic alpha 2-adrenoceptors modulate the release of [3H]noradrenaline from rat spinal cord dorsal horn neurones

Slices of the dorsal half of the rat spinal cord were used to investigate the existence of a noradrenergic feedback modulation of noradrenaline release. After crude preparation of the vertebral column, the spinal cord was ejected by hydraulic pressure and transverse slices were cut. These were preincubated with [3H]noradrenaline during 0.1 Hz electrical stimulation and then superfused and stimulated electrically for two periods. The stimulation-evoked release of [3H]noradrenaline was Ca2+-dependent and tetrodotoxin-sensitive. Pretreatment of the animals with the noradrenergic neurotoxin, DSP-4, reduced the tritium content in the slices and the stimulation-evoked release to less than 10% of the controls. Clonidine (0.01-1 microM) inhibited the evoked overflow by 60% maximally and yohimbine (0.1-1 microM) enhanced it by 160% maximally. The effects of clonidine were antagonized by yohimbine. These results provide evidence that noradrenaline release from spinal cord slices is controlled by an alpha 2-adrenoceptor-mediated, negative feedback mechanism.     

Stimulation-evoked release of [3H]-noradrenaline by 1, 10 or 100 pulses and its modification through presynaptic alpha 2-adrenoceptors

1 Mice isolated vasa deferentia were loaded with 1-[7,8-3H]-noradrenaline and subsequently field stimulated with 1, 10 or 100 pulses (2 ms pulse width, 1 Hz). The tritium overflow was separated into [3H]-noradrenaline and its 3H-metabolites. 2 The resting release of tritium contained about 7% [3H]-noradrenaline, 33% [3H]-3, 4-dihydroxyphenylglycol ([3H]-DOPEG) and 60% 3H-non-catechols with usually less than 1% [3H]-dihydroxymandelic acid ([3H]-DOMA). The proportion of the tritium as [3H]-noradrenaline increased with stimulation train length to 35% with 100 pulses; this increase in [3H]-noradrenaline was associated with falls in [3H]-DOPEG and 3H-non-catechols. Generally the proportional increase in [3H]-noradrenaline on stimulation was about 10 x total tritium when compared with the resting release. 3 The fractional release of [3H]-noradrenaline per pulse was independent of train length, averaging about 6 x 10(-6). This was reduced by the alpha 2-adrenoceptor agonist clonidine (0.3 - 30 nM) with an IC50 of 4.8 nM (10 pulses at 1 Hz). 4 The alpha 2-adrenoceptor antagonist, yohimbine (10 - 100 nM), did not alter the fractional release of [3H]-noradrenaline elicited by 1 pulse. The antagonist did not change the amount or composition of the resting or evoked tritium overflow. However, yohimbine (1 - 100 nM) increased the fractional release of [3H]-noradrenaline per pulse for trains of 10 or 100 pulses (1 Hz) in a concentration-dependent fashion. An increase above controls was significant only with 100 pulses and yohimbine, 30 nM. 5 The results show that the release of noradrenaline during trains of pulses in the mouse vas deferens can be regulated through presynaptic alpha 2-adrenoceptors. There was no evidence of inhibition by noradrenaline of its own release following a single pulse.     

Release of [3H]-noradrenaline from the sympathetic nerves to bovine mesenteric lymphatic vessels and its modification by alpha-agonists and antagonists.

1. Isolated segments of bovine mesenteric lymphatic vessels were loaded with [3H]-noradrenaline and its efflux in response to field stimulation examined. Vessels were attached to an isometric force transducer for the simultaneous recording of mechanical activity. 2. Field stimulation at 1, 4 and 8 Hz (0.3 ms pulses, 1 min train) increased spontaneous contraction rate and evoked 3H release up to a maximum of 4.5% of total tissue 3H at 8 Hz. Output per pulse was maximal at 4 Hz. 3. Tetrodotoxin (3 x 10(-6) M) blocked the release of 3H in response to field stimulation although the drug did not attenuate release evoked by high K+ (65 mM) solution. Field-evoked release of 3H was also absent in Ca2+ -free solution containing EGTA (1 mM). 4. When vessels were preincubated with labelled transmitter plus cocaine (5 x 10(-5) M) evoked release of 3H was absent. After preloading with [3H]-noradrenaline, cocaine (10(-6) M) potentiated both the mechanical response to field stimulation and evoked 3H release. 5. The relatively non selective alpha-adrenoceptor antagonist phentolamine (3 x 10(-6) M) and the alpha 2-antagonists yohimbine (10(-8) M) and rauwolscine (10(-6) M) significantly increased evoked 3H release at both of the frequencies examined (1 and 4 Hz). In contrast, the selective alpha 1-antagonist prazosin (10(-6) M) failed to alter 3H release to 4 Hz stimulation although release at 1 Hz was potentiated in the presence of the drug. 6. The postsynaptic excitatory response to field stimulation remained in the presence of prazosin (10(-6) M), but was converted to an inhibitory effect in the presence of phentolamine (3 x 10(-6) M), yohimbine (10(-6) M) or rauwolscine (10(-6) M). 7. Evoked 3H efflux was significantly reduced by clonidine (10(-6) M), xylazine (10(-6) M) and exogenous noradrenaline (5 x 10(-7) M), although phenylephrine (10(-6) M) reduced release only at the lower of the two frequencies tested (1 Hz). 8. These findings suggest that release of 3H by field stimulation reflects endogenous transmitter release and that this is subject to autoinhibition via feedback onto inhibitory prejunctional alpha 2-adrenoceptors. The postjunctional excitatory response is mediated via postjunctional alpha 2-adrenoceptors.     

Presynaptic alpha 2-adrenoceptor antagonism by verapamil but not by diltiazem in rabbit hypothalamic slices.

1 Rabbit hypothalamic slices prelabelled with [3H]-noradrenaline and superfused with Krebs solution were stimulated electrically at a frequency of 5 Hz. Exposure to verapamil (0.1 to 10 microM) significantly increased, in a concentration-dependent manner, the electrically-evoked overflow of tritium, without affecting the spontaneous outflow of radioactivity. 2 Exposure to diltiazem in concentrations up to 100 microM had no effect on the electrically evoked release of [3H]-noradrenaline, but increased the basal outflow of radioactivity at 10 and 100 microM. 3 The preferential alpha 2-adrenoceptor antagonist, yohimbine (0.1 microM) significantly antagonized the inhibitory effect of clonidine or adrenaline on [3H]-noradrenaline overflow elicited by electrical stimulation. Verapamil (3 microM) also antagonized this inhibitory effect of the alpha 2-adrenoceptor agonists on [3H]-noradrenaline release. In contrast to these results, exposure to diltiazem (10 microM) was ineffective in blocking the action of the alpha 2-adrenoceptor agonist. 4 These results suggest that the two Ca2+-antagonists verapamil and diltiazem differ in their ability to affect central noradrenergic neurotransmission. While verapamil is a relatively potent alpha 2-adrenoceptor antagonist, diltiazem is devoid of presynaptic alpha 2-adrenoceptor antagonist properties.     

Effects of 5-HT receptor agonists on depolarization-induced [3H]-noradrenaline release in rabbit hippocampus and human neocortex.

1. The present study attempted to determine whether noradrenaline (NA) release in rabbit hippocampus and human neocortex is modulated by presynaptic 5-hydroxytryptamine (5-HT) receptors. 2. Slices of rabbit hippocampus and human neocortex, loaded with [3H]-noradrenaline ([3H]-NA) were superfused and the effects of 5-hydroxytryptamine (5-HT) receptor ligands on electrically evoked [3H]-NA release were investigated. 3. In rabbit hippocampus, 5-HT, 5-carboxamidotryptamine (5-CT; 32 microM) and 2-CH3-5-HT (32 microM) increased [3H]-NA release elicited with 360 pulses/3 Hz. Facilitation of transmitter release was not influenced by the 5-HT3 receptor antagonist, tropisetron but was prevented by the alpha 2-adrenoceptor antagonist, rauwolscine. When autoinhibition was avoided by stimulating the tissue with 4 pulses/100 Hz (pseudo-one pulse-(POP) stimulation), 2-CH3-5-HT decreased evoked transmitter release, whereas 5-HT and 5-CT had no effect. Inhibition caused by 2-CH3-5-HT was not affected by tropisetron but counteracted by the alpha 2-adrenoceptor ligands, clonidine and rauwolscine. Inhibition caused by clonidine was diminished in the presence of 5-CT or 2-CH3-5-HT. 4. In human neocortex, [3H]-NA release elicited with 360 pulses/3 Hz was increased by 10 microM 5-HT and 32 microM 5-CT, whereas 2-CH3-5-HT was ineffective. [3H]-NA release evoked with a modified POP stimulation (2 bursts of 4 pulses/100 Hz, 3.5 min apart) was not affected by 2-CH3-5-HT or 5-CT. 5. The present results indicate that 5-HT, 2-CH3-5-HT and 5-CT can act on presynaptic alpha 2-autoreceptors as partial agonists (2-CH3-5-HT; in rabbit hippocampal tissue) or antagonists (5-HT and 5-CT; in tissue of rabbit hippocampus and human neocortex). Furthermore the existence of autoinhibition dictates whether these drugs cause facilitation of release, inhibition or have no effect.     

Protein kinase C involvement in maintenance and modulation of noradrenaline release in the mouse brain cortex.

1. The role of protein kinase C in the modulation of noradrenaline release was investigated in mouse cortical slices which were pre-incubated with [3H]-noradrenaline. The aim was to investigate the hypothesis that protein kinase C is activated during high levels of transmitter release to maintain transmitter output. 2. The protein kinase C activators, phorbol myristate acetate (0.01-0.3 microM) and to a greater extent 4 beta-phorbol 12,13-dibutyrate (0.01-0.3 microM) significantly enhanced stimulation-induced noradrenaline release whereas 4 alpha-phorbol 12,13-dibutyrate (0.1 microM) which does not activate protein kinase C was without effect. The effect of the protein kinase C activator, phorbol myristate acetate, on noradrenaline release was attenuated by the protein kinase C inhibitor, polymyxin B (21 microM) which by itself inhibited stimulation-induced noradrenaline release. 3. Protein kinase C was down-regulated by 10 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta-phorbol 12,13-dibutyrate (0.1 microM) on noradrenaline release was abolished as was the inhibitory effect produced by polymyxin B. This indicates that polymyxin B was acting selectively at protein kinase C. 4. The inhibitory effect of polymyxin B on noradrenaline release, when expressed as a percentage of the appropriate frequency control, was constant at 1, 5 and 10 Hz. Furthermore, the ratio of release at 5 Hz to that at 10 Hz was not altered by protein kinase C down-regulation, indicating that there is no additional effect of protein kinase C at higher stimulation frequencies. 5. When transmitter release was elevated by blocking alpha 2-adrenoceptor auto-inhibition with idazoxan (0.1 microM) or K+ channels with tetraethylammonium (300 microM), the elevation in transmitter release was significantly attenuated by protein kinase C down-regulation, suggesting an involvement of protein kinase C. 6. We conclude that protein kinase C is involved in the modulation of noradrenaline release over a wide range of stimulation frequencies, in addition to a role when noradrenaline release is elevated by presynaptic mechanisms.     

The structural requirements for phorbol esters to enhance serotonin and acetylcholine release from rat brain cortex.

The effects of various phorbol-based protein kinase C (PKC) activators on the electrical stimulation-induced (S-I) release of serotonin and acetylcholine was studied in rat brain cortical slices pre-incubated with [3H]-serotonin or [3H]-choline to investigate possible structure-activity relationships. 4beta-phorbol 12,13-dibutyrate (4betaPDB, 0.1-3.0 microM), enhanced S-I release of serotonin in a concentration-dependent manner whereas the structurally related inactive isomer 4alpha-phorbol 12, 13-dibutyrate (4alphaPDB) and phorbol 13-acetate (PA) were without effect. Another group of phorbol esters containing a common 13-ester substituent (phorbol 12,13-diacetate, PDA; phorbol 12-myristate 13-acetate, PMA; phorbol 12-methylaminobenzoate 13-acetate, PMBA) also enhanced S-I serotonin release with PMA being least potent. The deoxyphorbol monoesters, 12-deoxyphorbol 13-acetate (dPA), 12-deoxyphorbol 13-angelate (dPAng), 12-deoxyphorbol 13-phenylacetate (dPPhen) and 12-deoxyphorbol 13-isobutyrate (dPiB) enhanced S-I serotonin release but 12-deoxyphorbol 13-tetradecanoate (dPT) was without effect. The 20-acetate derivatives of dPPhen and dPAng were less effective in enhancing S-I serotonin release compared to the parent compounds. With acetylcholine release all phorbol esters tested had a far lesser effect when compared to their facilitatory action on serotonin release with only 4betaPDB, PDA, dPA, dPAng and dPiB having significant effects. The effects of the phorbol esters on serotonin release were not correlated with their reported in vitro affinity and isozyme selectivity for PKC. A comparison across three transmitter systems (noradrenaline, dopamine, serotonin) suggests basic similarities in the structural requirements of phorbol esters to enhance transmitter release with short chain substituted mono- and diesters of phorbol being more potent facilitators of release than the long chain esters. Some compounds notably PDA, PMBA, dPPhen, dPPhenA had different potencies across noradrenaline, dopamine and serotonin.     

Different alpha-adrenoceptors modulate the release of 5-hydroxytryptamine and noradrenaline in rat cortex.

1 The potassium-evoked release of [3H]-noradrenaline from slices of rat occipital cortex and the potassium-evoked release of [3H]-5-hydroxytryptamine from slices of rat frontal cortex were measured using a superfusion system. 2 The rank order of potency for a number of alpha-adrenoceptor agonists was different for the two neuronal systems, clonidine and azepexole being the most potent inhibitors of noradrenaline release and methoxamine and phenylephrine being the most potent against 5-hydroxytryptamine release. 3 The rank order of potency for a series of alpha-adrenoceptor antagonists in reversing the inhibition of noradrenaline release produced by clonidine was: phentolamine greater than rauwolscine = yohimbine = corynanthine much greater than WB4101, whereas against methoxamine-inhibition of 5-hydroxytryptamine release the rank order of potency was: WB4101 greater than phentolamine greater than corynanthine greater than yohimbine greater than rauwolscine. 4 The results suggest that the alpha-adrenoceptors which modulate potassium-evoked 5-hydroxytryptamine release are not identical with the alpha 2-adrenoceptors which modulate potassium-evoked 5-hydroxytryptamine release are not identical with the alpha 2-adrenoceptors located on noradrenergic nerve terminals and may more closely resemble alpha 1-than alpha 2-adrenoceptors.     

Differential abilities of phorbol esters in inducing protein kinase C (PKC) down-regulation in noradrenergic neurones

1. The ability of several phorbol ester protein kinase C (PKC) activators (phorbol 12, 13-dibutyrate, PDB; phorbol 12, 13-diacetate, PDA; and 12-deoxyphorbol 13-acetate, dPA) to down-regulate PKC was studied by assessing their effects on electrical stimulation-induced (S-I) noradrenaline release from rat brain cortical slices and phosphorylation of the PKC neural substrate B-50 in rat cortical synaptosomal membranes. 2. In cortical slices which were incubated for 20 h with vehicle, acute application of PDB, PDA and dPA (0.1 - 3.0 microM) enhanced the S-I noradrenaline release in a concentration-dependent manner to between 200 - 250% of control in each case. In slices incubated with PDB (1 microM for 20 h), subsequent acute application of PDB (0.1 - 3.0 microM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 microM) for 20 h, there was no reduction in the facilitatory effect of their respective phorbol esters or PDB (0.1 - 3.0 microM) when acutely applied, indicating that PKC was not down-regulated. This was confirmed using Western blot analysis which showed that PDB (1 microM for 20 h) but not PDA (3 microM for 20 h) caused a significant reduction in PKCalpha. 3. Incubation with PDB for 20 h, followed by acute application of PDB (3 microM) failed to increase phosphorylation of B-50 in synaptosomal membranes, indicating down-regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 microM) or PDB (3 microM) induced a significant increase in B-50 phosphorylation. 4. Acutely all three phorbol esters elevate noradrenaline release to about the same extent, yet PDA and dPA have lower affinities for PKC compared to PDB, suggesting unique neural effects for these agents. This inability to cause functional down-regulation of PKC extends their unusual neural properties. Their neural potency and lack of down-regulation may be related to their decreased lipophilicity compared to other phorbol esters. 5. We suggest that PKC down-regulation appears to be related to binding affinity, where agents with high affinity, irreversibly insert PKC into artificial membrane lipid and generate Ca(2+)-independent kinase activity which degrades and deplete PKC. We suggest that this mechanism may also underlie the ability of PDB to down-regulate PKC in nerve terminals, in contrast to PDA and dPA.     

Effects of botulinum A toxin on presynaptic modulation of evoked transmitter release

A possible influence of botulinum A toxin on the modulation of evoked neurotransmitter release was investigated in hippocampus tissue. Rabbit hippocampal slices prelabelled with [3H]noradrenaline ([3H]NA), [3H]5-hydroxytryptamine ([3H]5-HT) or [3H]choline were superfused with physiological medium and were stimulated electrically during superfusion. The evoked release of [3H]NA, [3H]5-HT and [3H]acetylcholine [( 3H]ACh) was inhibited by botulinum A toxin in a concentration- and time-dependent manner. Neither the inhibition of release of [3H]NA and [3H]5-HT by the alpha 2-adrenoceptor agonist clonidine nor facilitation of release in the presence of alpha 2-antagonists were influenced by pretreatment of the tissue with botulinum toxin. The toxin caused no [32P]ADP ribosylation of synaptosomal proteins of hippocampus. The facilitation of the stimulation-induced [3H]NA and [3H]5-HT release by the specific protein kinase C (PKC) activator 4 beta-phorbol-12,13-dibutyrate (PDB) was significantly diminished by botulinum A toxin. These results show that the evoked transmitter release is inhibited by botulinum A toxin by a mechanism which does not involve ADP ribosylation or an interaction with the alpha 2-adrenoceptor mechanism.     

Evidence that M1 muscarinic receptors enhance noradrenaline release in mouse atria by activating protein kinase C.

1. The M1 selective muscarinic agonist, McNeil A 343, enhanced the electrically evoked release of noradrenaline from postganglionic sympathetic nerves in mouse atria. This has been found previously to be due to activation of muscarinic receptors of the M1 subtype, probably located on sympathetic nerve terminals. The present study investigated the signal transduction mechanisms involved in the release-enhancing effects of McNeil A 343. The release of noradrenaline from mouse atria was assessed by measuring the electrically-induced (3 Hz, 60 s) outflow of radioactivity from atria which had been pre-incubated with [3H]-noradrenaline. 2. 8-Bromo cyclic AMP in the presence of IBMX was used to enhance maximally S-I noradrenaline release through cyclic AMP-dependent mechanisms. However, the facilitatory effect of McNeil A 343 (10 microM) was not different from the effect in the absence of these drugs, suggesting that McNeil A 343 enhances noradrenaline release independently of the cyclic AMP system. Furthermore, the release-enhancing effect of McNeil A 343 (10 microM) on noradrenaline release was also not altered by the 5-lipoxygenase inhibitor, BW A4C. 3. The facilitatory effect of McNeil A 343 was not altered in the presence of drugs (trifluoperazine, W7, and calmidazolium) which inhibit calmodulin-dependent processes, suggesting that the mechanisms of action of McNeil A 343 does not depend on calmodulin. 4. It was considered likely that the facilitatory effect of McNeil A 343 on noradrenaline release may be due to activation of protein kinase C, since activators of protein kinase C enhance noradrenaline release.(ABSTRACT TRUNCATED AT 250 WORDS)