Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Reversibility of Cisternal Stack Formation During Hypoxic Hypoxia and Subsequent Reoxygenation in Cerebellar Purkinje Cells

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca²⁺ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.     

Effect of dexmedetomidine on the release of [3H]-noradrenaline from rat kidney cortex slices: characterization of alpha2-adrenoceptor

The presynaptic modulation of [3H]-noradrenaline (NA) release from rat kidney cortex slices, a method used for the first time, was investigated. Rat kidney cortex slices were loaded with [3H]-NA and the release of radioactivity at rest and in response to field stimulation was determined. The alpha(2)-adrenoceptor agonist, dexmedetomidine inhibited the stimulation-evoked release of NA from kidney slices in a concentration-dependent manner, whereas alpha(2)-adrenoceptor antagonist CH-38083 (7,8-methyenedioxy-14-alpha-hydroxyalloberbane HCl), an alpha(2)-adrenoceptor antagonists, enhanced it. When dexmedetomidine and BRL-44408, a selective alpha(2A) antagonist, were added together, the effect of dexmedetomidine was significantly antagonized. In contrast, ARC-239 (2-(2,4-(o-piperazine-1-yl)-ethyl-4,4-dimethyl-1,3-(2H, 4H)disoguinolinedione chloride), a selective alpha(2B)-antagonist, had no effect on the release and failed to prevent the effect of dexmedetomidine. Prazosin, an alpha(1)- and alpha(2B/C)-adrenoceptor antagonist enhanced the release evoked by field stimulation. It is therefore suggested that there is a negative feedback modulation of NA release at the sympathetic innervation of kidney cortex, and dexmedetomidine, a clinically used anesthetic adjunct inhibits the release via activation of alpha(2C)-adrenoceptors.     

Effect of Lanthanum on the Release of Acetylcholine from the Myenteric Plexus and on Its Activation by Ouabain and Electrical Stimulation

The effect of lanthanum ions (La3+) on the release of acetylcholine (ACh) from longitudinal muscle strips of the guinea pig ileum with the myenteric plexus attached was investigated. After an exposure of the tissue to 2 mM LaCl3 for 18 min the rate of ACh release was increased approximately eightfold and the increased release lasted for more than 100 min. The augmented release of ACh was accompanied by enhanced synthesis. At the end of the experiments (102 min after LaCl3 had been removed), when the release of ACh was still more than six times higher than in controls, the content of ACh was the same in La3+-treated and untreated tissues. Electrical field stimulation failed to cause a further increase in the release of ACh from La3+-pretreated preparations whereas ouabain released considerable more ACh when compared to controls. It is concluded from this difference that electrical stimulation and ouabain release ACh from different pools.     

Presynaptic modulation by noradrenaline and an opioid of the substance P-induced release of [3H]acetylcholine from the myenteric plexus

Substance P (7.5-750 nM) applied in superfusion dose-dependently released 3H from isolated strips of myenteric plexus-longitudinal muscle of the guinea-pig ileum loaded with [3H]choline. Separation of the [3H]acetylcholine and [3H]choline components of the released radioactivity revealed that in response to substance P (SP) administration only the release of [3H]acetylcholine increased above resting level. A slowly developing tachyphylaxis to the effect of SP was observed. Evidence has been obtained that the slow tachyphylaxis developed to the acetylcholine-releasing effect of SP was not due to the exhaustion of releasable acetylcholine pool. Release of acetylcholine by 150 nM SP was completely prevented by tetrodotoxin or in a Ca2+-free medium and greatly reduced in the presence of noradrenaline or the opioid receptor agonist (D-Met2,Pro5)-enkephalinamide. The effect of noradrenaline and the opioid peptide was apparently prevented by yohimbine and naloxone, respectively.     

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.     

The role of catecholamines in the prolactin release induced by salsolinol

Salsolinol (1,2,3,4-tetrahydro-6,7-dihydroxy-1-methylisoquinoline) is an endogenous prolactin releasing agent. Its action can be inhibited by another isoquinoline, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), which has a strong norepinephrine releasing activity. Salsolinol does not alter the dopamine release in median eminence in vitro, providing evidence for the lack of interaction with presynaptic D2 dopamine receptors. At the same time, lack of norepinephrine transporter abolishes salsolinol's action. Salsolinol decreases tissue level of dopamine and increases norepinephrine to dopamine ratio in organs innervated by the sympathetic nervous system indicating a possible decrease of norepinephrine release. Enzymes of catecholamine synthesis and metabolism are probably also not the site of action of salsolinol. In summary, based upon all of these observations a physiologically relevant interplay might exist between the sympatho-neuronal system and the regulation of prolactin release.     

Neurochemical evidence for two types of presynaptic alpha2-adrenoceptors

Neurochemical and pharmacological evidence has been obtained that noradrenergic varicosities (in mouse and rat vas deferens) and cholinergic varicosities (in the Auerbach's plexus) contain heterogenous alpha₂-adrenoceptors through which the release of [³H]noradrenaline and [³H]acetylcholine can be modulated. The quantitative data also support the hypothesis that different noradrenaline and xylazine sensitive alpha₂-adrenoceptors are present prejunctionally in the vas deferens and Auerbach's plexus preparations. Prazosin, although it has a presynaptic inhibitory effect on alpha₂-adrenoceptors of noradrenergic axon terminals, has no effect on cholinergic axon terminals. These data suggest that there are two different types of alpha₂-adrenoceptors at the presynaptic axon terminals.Special Issue Dedicated to Dr. Abel Lajtha