Pharmacological sensitivities of two afterhyperpolarizations in rat optic nerve

Optic nerves were removed from 4-8-week-old rats and studied in a modified sucrose gap chamber in order to examine the pharmacological sensitivities of two afterhyperpolarizations (AHPs), an early and a late one. The peak latency of the early AHP, which occurred immediately after the action potential, was 6-12 ms, and its duration was 44-75 ms. The early AHP was present at resting potential in about 75% of recorded nerves. In the nerves in which an early AHP was not present at resting potential, an AHP was present during delivery of constant current depolarization through the sucrose gap. The early AHP was increased in amplitude by depolarization of the nerve, decreased in amplitude or reversed in polarity by hyperpolarization, was not affected by tetraethylammonium (TEA) or Co2+, and was obliterated by 4-aminopyridine (4-AP) concomitant with action potential broadening. The late AHP followed a broadened action potential in the presence of the potassium channel blocker 4-AP. Its peak latency ranged from 112 to 254 ms and its duration from 336 to 710 ms. It was increased in amplitude with repetitive stimulation, was reversibly obliterated by TEA, but was not significantly altered by Co2+, Cd2+, Ca2+ removal, charybdotoxin or apamin. The results demonstrate the presence of two AHPs mediated by pharmacologically distinct potassium conductances in rat optic nerve, neither of which is calcium-dependent.     

Apamin, a blocker of the calcium-activated potassium channel, induces neurodegeneration of Purkinje cells exclusively

Following acute intracerebroventricular injections of 1 ng of apamin and chronic apamin infusion (0.4 ng/μl, 0.5 μl/h, 14 days), the rat brains exhibited bilateral damage only in the cerebellum. The argyrophilic cells were Purkinje cells in copula pyramis, flocculus, paraflocculus, and paramedian lobules. These data demonstrate that the inactivation of small conductance Ca²⁺-activated K⁺ channels by apamin induces a non-limbic neurodegeneration.     

Spinale Wirkungen von Apamin

Zusammenfassung Die spinalen Wirkungen von Apamin, einem neurotoxischen Polypeptid aus Bienengift, wurden an spinalisierten Katzen untersucht. 15 min nach i.v. Injektion von 0,5–1,0 mg/kg war der Mittelwert und die Streuung der Amplitudenmaxima der monosynaptischen Extensorreflexpotentiale signifikant gewachsen. Die Mittelwerte der Amplitudenmaxima bei den monosynaptischen Flexorreflexpotentialen verhielten sich unter Apamin uneinheitlich, jedoch nahm die Streuung der Einzelwerte ebenfalls stark zu. Die polysynaptischen Komponenten der Reflexpotentiale nach Reizung von Flexorreflexafferenzen wurden stark vergrößert, das Dorsalwurzelpotential und der Dorsalwurzelreflex nahmen mäßig zu. Die Effektivität der polysynaptischen Hemmung eines Gastrocnemiusreflexes durch konditionierende Reizung des N. biceps post. + semitendinosus nahm jedoch ab. Mit der Konditionierungstechnik ließ sich eine Änderung der direkten Hemmung, der rekurrenten Hemmung sowie der räumlichen und zeitlichen Förderung durch Apamin nicht nachweisen. Aus den Versuchen wird geschlossen, daß Apamin vornehmlich polysynaptische Reflexbögen erregt und daß hierbei excitatorische Mechanismen stärker gefördert werden als inhibitorische.Herrn Prof. Dr. L. Lendle zum 70. Geburtstag gewidmet.     

Calcium-activated hyperpolarizations in neurons of the mediolateral part of the lateral septum: intracellular studies from guinea pig brain slices

Intracellular recordings were used to study the afterpotentials that followed a single spike and trains of spikes in class A neurons (n=85) of the mediolateral part of the lateral septum (LSml) of the guinea pig in in vitro slices. Following a single spike, LSml neurons (n=56) developed a slow afterhyperpolarization (sAHP), called early sAHP. These sAHP did not sum; other LSml neurons (n=8) showed a depolarizing afterpotential (DAP) that summed. Twenty-one neurons did not exhibit an afterpotential. Following a train of spikes, LSml neurons (n=79) developed a long-lasting sAHP, called late sAHP; these sAHP summed. Both the DAP and the early and late sAHP were markedly suppressed in amplitude by addition of Co²⁺ but persisted in the presence of tetrodotoxin (TTX). Increase in external K⁺ markedly depressed the early and late sAHP. Apamin and d-tubocurarine selectively blocked early sAHP, with no effect on late sAHP. These results indicate that the early and late sAHP are mainly generated by an activation of two types of Ca²⁺-dependent K⁺ conductances, with different time courses and pharmacological properties. In LSml neurons, late sAHP mediates the long-term adaptation of repetitive firing.     

The role of tachykinin NK1 and NK2 receptors in atropine-resistant colonic propulsion in anaesthetized guinea-pigs

1. The role of endogenous tachykinins on guinea-pig colonic propulsion was investigated by using potent and selective tachykinin NK₁ and NK₂ receptor antagonists. Colonic propulsion and contractions were determined by means of a balloon-catheter device, inserted into the rectum of guanethidine (68 μmol kg⁻¹, s.c., 18 and 2 h before)-pretreated, urethane-anaesthetized guinea-pigs. Propulsion of the device (dynamic model) was determined by measuring the length of the catheter expelled during 60 min filling of the balloon (flow rate 5 μl  min⁻¹).
2. In control conditions the tachykinin NK₁ receptor antagonist SR 140333 (1 μmol kg⁻¹, i.v.) did not affect either colonic propulsion or the amplitude of contractions. The tachykinin NK₂ receptor antagonists MEN 10627 and MEN 11420 (1 μmol kg⁻¹, i.v.) increased colonic propulsion at 10 min (+120% and 150%, respectively) but at 60 min the effect was significant only for MEN 10627 (+84%). SR 48968 (1 μmol kg⁻¹, i.v.) did not significantly enhance the colonic propulsion. None of these tachykinin NK₂ receptor antagonists modified the amplitude of colonic contractions. In contrast, both atropine (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹) and hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished propulsion (81% and 87% inhibition, respectively) and decreased the amplitude of contractions (68% inhibition for either treatment).
3. In atropine-treated animals (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹), apamin (30 nmol kg⁻¹, i.v.) restored colonic propulsion (+416%) and increased the amplitude of contractions (+367% as compared to atropine alone). Hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished the apamin-induced, atropine-resistant colonic propulsion (97% inhibition) and reduced the amplitude of the atropine-resistant contractions (52% inhibition).
4. The apamin-induced, atropine-resistant colonic propulsion was inhibited by SR 140333 (−69% at 1 μmol kg⁻¹), SR 48968 (−78% at 1 μmol kg⁻¹), MEN 11420 (−59% at 1 μmol kg⁻¹) and MEN 10627 (−50% at 1 μmol kg⁻¹), although the latter effect was not statistically significant. The combined administration of SR 140,333 and MEN 10,627 (1 μmol kg⁻¹ for each antagonist) almost completely abolished colonic propulsion (90% inhibition). The amplitude of colonic contractions was also reduced by SR 140333 (−42%), SR 48968 (−29%), MEN 11420 (−45%) but not by MEN 10627 (−16%). The combined administration of SR 140333 and MEN 10,627 reduced the amplitude of contractions by 47%. SR 140603 (1 μmol kg⁻¹, i.v.), the less potent enantiomer of SR 140333, was inactive.
5. In control animals, apamin (30 nmol kg⁻¹, i.v.) enhanced colonic propulsion (+84%) and increased the amplitude of contractions (+68%), as compared to the vehicle. Hexamethonium (55 μmol kg⁻¹, i.v. plus infusion of 17 μmol h⁻¹) inhibited propulsion (86% inhibition) and decreased the amplitude of contractions (49% inhibition). SR 140333, SR 48968, MEN 11420, MEN 10627, or the coadministration of SR 140333 and MEN 10627 had no effect.
6. In a separate series of experiments, the mean amplitude of colonic contractions was also recorded under isovolumetric conditions through the balloon-catheter device kept in place at 75 mm from the anal sphincter (static model). In control conditions, neither SR 140333 nor MEN 11420 modified the amplitude of contractions. In atropine-pretreated guinea-pigs, SR 140333 and MEN 11420 (0.1–1 μmol kg⁻¹) dose-dependently decreased the amplitude of contractions. In apamin- and atropine-pretreated animals, only the highest (1 μmol kg⁻¹) dose of SR 140333 or MEN 11420 significantly decreased the amplitude of contractions. The inhibitory potency of atropine (0.3–1 μmol kg⁻¹) was similar in apamin-pretreated animals and in controls.
7. It was concluded that, in anaesthetized guinea-pigs, endogenous tachykinins, acting through both NK₁ and NK₂ receptors, act as non-cholinergic excitatory neurotransmitters in promoting an apamin-evoked reflex propulsive activity of the distal colon.

     

5-HT-induced neurogenic relaxations of the guinea-pig proximal colon: investigation into the role of ATP and VIP in addition to nitric oxide

In the guinea-pig proximal colon, 5-hydroxytryptamine (5-HT) relaxes the longitudinal muscle by stimulating neuronal 5-HT receptors, which induces the release of nitric oxide (NO). It was investigated whether the inhibitory neurotransmitters adenosine 5-triphosphate (ATP) and/or vasoactive intestinal polypeptide (VIP) could be involved as well.Antagonists to block the contractile response to 5-HT via 5-HT₂, 5-HT₃ or 5-HT₄ receptors were present throughout the experiments and methacholine was administered to precontract the strips. ATP, VIP and 5-HT induced concentration-dependent relaxations, in the case of 5-HT yielding a non-monophasic concentration-response curve. Tetrodotoxin (TTX; 300 nM), N^G-nitro-l-arginine (l-NNA, 100 M) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 M) or 0.3–3 M ATP but reduced those by 10 M ATP. Suramin (300 M) strongly inhibited the relaxations to ATP and VIP. l-NNA and suramin also inhibited the relaxations to 5-HT. In the presence of l-NNA (100 M), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 M), demonstrating its specificity. Apamin (30 nM) inhibited both the relaxations to ATP (by 70–100%) and to 5-HT; relaxations to isoprenaline were partially inhibited, indicating a non-specific component in the inhibitory action of apamin. However, relaxations to exogenous VIP (up to 0.3 M), NO (1 ,M) and to nitroglycerin were not inhibit ed. In the presence of l-NNA (100 M), apamin inhibited the relaxations to 5-HT only at 30 M. ,\-methylene-ATP (,-Me-ATP; 100 M) did not desensitize the responses to ATP. Reactive blue 2 affected the relaxations to isoprenaline at concentrations necessary to significantly inhibit the relaxations to ATP (i.e. from 10 M onwards). Thus, it was not possible to test either ,-Me-ATP or reactive blue 2 against the relaxations to 5-HT. -Chymotrypsin (0.015 mg·ml^–1) and trypsin (0.005 mg·ml^–1) almost abolished the relaxations to VIP, but did not affect those to isoprenaline and 5-HT. The VIP receptor antagonists [p-Cl-d-Phe⁶, Leu¹⁷]VIP (1 M) and VIP_10–28 (1 and 3 M) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 M) had no effect on the relaxations to VIP or 5-HT.It can be concluded that in the guinea-pig colon longitudinal muscle, VIP and ATP induce relaxation via a direct effect on the smooth muscle, not involving NO. 5-HT-induced relaxations are mediated by NO as well as by a substance which is sensitive to inhibition by suramin but not apamin. It is suggested that this substance is ATP and not a peptide.     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

Intestinal Adaptation in Atrophic Rat Ileum is Accompanied by Supersensitivity to Vasoactive Intestinal Peptide,Pituitary Adenylate Cyclase-activating Peptide and Nitric Oxide

Background: Intestinal inactivity leads to atrophic changes and concomitant alterations in the expression of neurotransmitters in the enteric nervous system. In atrophic rat ileum neurones expressing vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) decrease in number while nitric oxide synthase (NOS) expressing neurones increase. Since little is known about functional changes accompanying intestinal atrophy the aim of the present study was to investigate relaxatory responses to VIP, PACAP-27 and nitric oxide (NO) in longitudinal smooth muscle from atrophic rat ileum. Methods: To create a dysfunctional (atrophic) intestine, the distal 10 cm of rat ileum was surgically bypassed. In vitro experiments were carried out on longitudinal muscle strips from rat ileum having been sham-operated, one week or four weeks bypassed. Results: The amplitudes of the relaxatory responses to PACAP-27, VIP and the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), but not forskolin, were significantly increased in the one-week bypassed ileum. In the four-weeks bypassed ileum the VIP, PACAP-27, SNAP and forskolin evoked relaxations were of the same magnitude as those of the shamoperated. The augmented responses to both VIP and PACAP-27 could be blocked by pre-treatment with apamin while N_G-nitro-L-arginine methyl ester (L-NAME) and tetrodotoxin were ineffective. In contrast to sham-operated and four-weeks bypassed ileum, cross-desensitization between VIP and PACAP-27 was noted after one week of bypass. Conclusion: Intestinal adaptation after bypassing the distal ileum of the rat includes a transient supersensitivity of the longitudinal muscle to the NO donor SNAP, VIP and PACAP27. These augmented relaxatory responses may contribute to the hypomotility noted in inactive intestine.     

Effects of apamin on the discharge properties of putative dopamine-containing neurons in vitro

An in vitro brain slice preparation was used to evaluate the effects of apamin, a selective inhibitor of calcium-activated potassium channels, on the discharge characteristics of presumed dopamine-containing neurons within the substantia nigra. Apamin administration (1 μM) was associated with an increase in neuronal excitability as evidenced by the emergence of both sustained irregular and intermittent bursting activity similar to that seen spontaneously in vivo. These data suggest that the characteristic regular activity observed among putative dopamine-containing neurons in vitro is mediated, in part, by a calcium-activated potassium conductance.