Clemens Allgaier

Islet-activating protein (pertussis toxin) diminishes ?2-adrenoceptor mediated effects on noradrenaline release

SummaryThe effect of islet-activating protein (IAP) on α2-adrenoceptor mediated modulation of noradrenaline release in the rabbit hippocampus was studied. Slices of the hippocampus were incubated for 6 h with IAP, subsequently loaded with3H-noradrenaline and superfused continuously. IAP-pretreatment significantly enhanced the electrically evoked transmitter release and diminished the facilitatory effect of the α2-adrenoceptor antagonist yohimbine. In addition, the inhibitory effect of the α2-adrenoceptor agonist clonidine was reduced. These results provide circumstantial evidence that an inhibitory guanine-nucleotide-binding protein, most probably N1 of a presynaptically located adenylate cyclase, is involved in the α2-autoreceptor mediated modulation of noradrenaline release.

Co-release of noradrenaline and ATP from cultured sympathetic neurons

The vesicles of postganglionic sympathetic axons store both noradrenaline and ATP. The theory of noradrenaline-ATP co-transmission implies that both compounds are released by nerve action potentials and elicit postjunctional effects. Many properties of postjunctional responses support the theory. However, neural release of ATP has been difficult to detect biochemically: by far the major part of the overflow of ATP from intact tissues upon sympathetic nerve stimulation comes from non-neural elements, especially smooth muscle and endothelial cells. Here we describe a parallel electrically evoked overflow of [3H]noradrenaline and endogenous ATP from cultured chick sympathetic neurons. The overflow was abolished by tetrodotoxin, omega-conotoxin and withdrawal of Ca2+, was increased by tetraethylammonium and 4-aminopyridine, and was not changed by prazosin or suramin. The results demonstrate directly the action potential-evoked, Ca(2+)-dependent and presumably vesicular and exocytotic release of ATP from postganglionic sympathetic neurons. They support the co-transmitter theory and suggest that cultured sympathetic neurons are a preparation in which noradrenaline-ATP co-release can be examined free from postjunctional components.

The adenosine receptor‐mediated inhibition of noradrenaline release possibly involves a N‐protein and is increased by α2‐autoreceptor blockade

1 The stimulation‐evoked overflow of [3H]‐noradrenaline from slices of the rabbit hippocampus is inhibited by α2‐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). 2 Treatment of hippocampal slices with N‐ethylmaleimide (NEM, 30 μm; 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 μm) was antagonized by NEM in a non‐competitive manner in the absence and in the presence of the α2‐adrenoceptor antagonist yohimbine. 3 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‐phenytheophylline was not seen. In the presence of (‐)‐PIA (0.1 μm), 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. 4 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. 5 In the presence of the α2‐adrenoceptor antagonist yohimbine, the inhibitory action of (‐)‐PIA was strongly increased. 6 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 α2‐autoreceptor and the A1‐receptor‐coupled signal transduction system, possibly at the level of a N‐protein.

Polymyxin B, a selective inhibitor of protein kinase C, diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

SummarySlices of the rabbit hippocampus were labelled with 3H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 μmol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.

Presynaptic K-Opioid Receptors on Noradrenergic Nerve Terminals Couple to G Proteins and Interact with the α2-Adrenoceptors

Abstract: Stimulation‐induced noradrenaline (MA) release in rabbit hippocampus is inhibited by activation qf presynaptic α2‐adrenoceptors and k‐opioid receptors. The purpose of the present study was to investigate (a) an interference between the α2‐ and k‐mechanisms, and (b) a coupling of the opioid receptors to pertussis toxin (PT)‐sensitive guanine nucleotide‐binding proteins (G proteins), as has been previously shown for the α2‐receptors. [3H]NA release from hippocampal slices was evoked by electrical field stimulation (360 pulses/3 Hz). Inhibition of stimulation‐evoked NA release by the preferential K‐receptor agonist ethylketocyclazocine (EKC) was increased in the presence of the α2‐adrenoceptor antagonist yohimbine (0.1 or 1.0 μM). When autoinhibitionn was completely removed, EKC (1 μM) almost abolished transmitter release. Pretreatment of hippocampal tissue wiih either PT (8 μg/ml; 18 h) or N‐ethylmaleimide (NEM) (30 μM; 30 min), which has been shown to alkylate PT substrates, diminished the EKC‐produced inhibition of NA release. The K‐mecha‐nism was still impaired by these compounds when the α2‐ receptors were blocked with yohimbine. An effect of NEM on the active site of the K‐receptor seems to be unlikely, because NEM diminished the EKC‐induced inhibition of release irrespective of whether or not the opioid receptor was occupied by EKC during exposure to NEM. The present results suggest an interference of both α2‐ and k‐opioid receptor‐coupled signal transduction possibly through competition for a common pool of G proteins.

3,4-diaminopyridine-induced noradrenaline release from CNS tissue as a model for action potential-evoked transmitter release: effects of phorbol ester

We used rabbit hippocampus slices preincubated with [3H]noradrenaline (NA) and applied short pulses of 3,4-diaminopyridine (3,4-DAP) during superfusion to investigate the mechanism underlying the 3H overflow evoked by 3,4-DAP and the effects of the protein kinase C (PKC) activator, 4 beta-phorbol 12,13-dibutyrate (PDB), in this model. The 3H overflow evoked by 200 microM 3,4-DAP (about 4-5% of tissue-tritium) was largely Ca2+-dependent, tetrodotoxin-sensitive and markedly reduced by clonidine, but it was enhanced by yohimbine. We also demonstrated that the response could be inhibited via presynaptic adenosine (A1-) and opioid (kappa-) receptors. PDB (1 microM) markedly increased the 3,4-DAP-evoked 3H overflow, its effect being almost unchanged following activation of presynaptic alpha 2-, A1- or kappa-receptors. Inhibitors of PKC (polymyxin B, staurosporine) almost abolished the 3,4-DAP-evoked 3H overflow and antagonized the effects of PDB. It is concluded that application of 3,4-DAP (200 microM for 2 min) to brain slices leads to depolarization of the neuronal membrane, Na+ current-carried action potentials, Ca2+ influx and the exocytotic release of NA, which in many aspects resembles the release evoked by electrical field stimulation. The findings with phorbol ester further support the involvement of PKC in transmitter release. Activation of PKC apparently does not directly interfere with signal transduction mechanisms of presynaptic inhibitory receptors on noradrenergic nerve terminals.

Possible involvement of protein kinase C (PKC) in the regulation of electrically evoked serotonin (5-HT) release from rabbit hippocampal slices

Protein kinase C (PKC)-activating phorbol esters enhanced the electrically evoked 5-HT release from rabbit hippocampal slices preincubated with [3H]5-HT. The release was diminished by polymyxin B, an inhibitor of PKC. These results are compatible with a stimulatory effect of PKC on the 5-HT release induced by action potentials. The mutual effects of PKC affecting drugs on 5-HT release suggest a functional but not a competitive interaction. The attenuation or the enhancement of effects of 5-HT autoreceptor ligands at various 5-HT biophase concentrations found after PKC-affecting drugs are in line with the view that autoreceptor-mediated events are not directly influenced by the enzyme PKC.

Postnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system: I. Axon terminal region: hippocampus

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the rat hippocampus. To this end, hippocampal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the cholinergic ingrowth. In hippocampal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.11 (P3) to 0.81% of tissue 3H (P16), the latter value being still much lower than that of hippocampal slices from adult rats (2.89% of tissue 3H). Already at P3 the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release. The muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in hippocampal slices with increasing effectivity from P4 to P16; no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine and the muscarinic antagonist atropine (1 microM, each) exhibited significant inhibitory and facilitatory effects, respectively, only at P15-16. The specific activities of both hippocampal HACU (pmoles/mg protein/min) and ChAT (nmoles/mg protein/min) continuously increased from P3 to P16. It is concluded (1) that cholinergic nerve terminals arriving at the hippocampal formation during postnatal ingrowth are already endowed with the apparatus for action potential-induced, Ca(2+)-sensitive (exocytotic) ACh release; (2) that, in contrast, the expression of presynaptic muscarinic autoreceptors on these cholinergic axon terminals is delayed; and (3) that autoinhibition due to endogenous ACh develops even later, probably when the density of presynaptic terminals in the hippocampus and hence, the concentration of released ACh has reached a suprathreshold value.

Cultured chick sympathetic neurons: modulation of electrically evoked noradrenaline release by P2-purinoceptors

The present study investigates the pharmacological profile of P2-purinoceptors modulating noradrenaline release from cultured chick sympathetic neurons. ATP (30 μM-3 mM) and 2-methylthio-ATP (3–100 μM), but not α,β-methylene-ATP (up to 100 μM), caused a significant facilitation of electrically evoked [3H]-noradrenaline release when added 2 min before depolarization. The facilitation declined with time of exposure suggesting receptor desensitization. The facilitatory effect was markedly diminished by the P2-purinoceptor antagonists reactive blue 2 (3 μM) and suramin (300 μM), but not changed by mecamylamine (10 μM), a nicotinic receptor antagonist. At 1 mM and higher concentrations, ATP added for 12 min, inhibited noradrenaline release; release was virtually abolished by 6 mM ATP. The inhibitory effect of ATP was slightly diminished by suramin but not affected by reactive blue 2. Electrically evoked [3H]-noradrenaline release remained unaffected in the presence of the adenosine (P1)-receptor agonists R(−)N6-(2-phenylisopropyl)adenosine (R-PIA), 2-[p-(2-carboxyethyl)phenylethylamino]5′-N-ethylcarboxamidoadenosine (CGS-21680), 5′-N-ethylcarboxamidoadenosine (NECA), and N6-2-(4-aminophenyl)ethyladenosine (APNEA), used up to 1 μM.The present results confirm the existence of two P2-purinoceptors affecting noradrenaline release: 1) a facilitatory receptor which is activated by 2-methyl thio-ATP as well as ATP, and blocked by suramin as well as reactive blue 2, and 2) an inhibitory receptor which is activated by ATP, only slightly affected by suramin but not at all by reactive blue 2 and does not belong to the established P2-purinoceptor subtypes.

Studies on the interaction between presynaptic α2-adrenoceptors and adenosine A1 receptors located on noradrenergic nerve terminals

SummaryThe aim of the present study was to obtain a more detailed understanding of the interaction between presynaptic α2-adrenoceptors and A1 adenosine receptors mediating inhibition of noradrenaline release in the central nervous system. Slices of rabbit hippocampus, prelabelled with [3H]noradrenaline, were superfused in the presence of the re-uptake inhibitor (+)-oxaprotiline and electrically stimulated during superfusion. During stimulation with 36 pulses at 3 Hz the α2-adrenoceptor antagonist yohimbine induced a five-fold increase of noradrenaline release indicating a pronounced autoinhibition of approximately 80%. In these experiments the inhibition of release caused by R-PIA, a preferential A1 agonist, as well as its facilitation caused by DPCPX, a selective A1 antagonist, were smaller in comparison to the effects of these compounds on release virtually free of autoinhibition (i. e. by stimulating the tissue with 4 pulses at 100 Hz (POP-stimulation) or with 36 pulses at 3 Hz in presence of yohimbine). Clonidine, an α2-adrenoceptor agonist, was used to impose a distinct α2-adrenoceptor-mediated inhibition of release elicited by POP-stimulation. Only, however, in the presence of 30 nmol/l clonidine, causing maximum inhibition of approximately 80% of 3H-overflow, but not in the presence of 6 nmol/l clonidine, causing approximately 50% inhibition, a significant diminution of the inhibitory effect of R-PIA was seen. Similarly, the α2-adrenoceptor mechanism was affected only by 10 μmol/l R-PIA causing maximum inhibition of approximately 80%, but remained unchanged in the presence of 30 nmol/l R-PIA diminishing release by 50%. In addition, the α2-adrenoceptor-mediated inhibition remained unaffected in the presence of DPCPX, indicating that the concentration of endogenous adenosine attained was not sufficient to influence the α2-adrenoceptor mechanism. In conclusion, the present results suggest that activation of the presynaptic α2 and A1 receptors inhibits depolarization-induced noradrenaline release in an additive manner. Only extensive (near maximal or maximal) activation of one receptor mechanism impairs the inhibition due to activation of the other.