Anococcygeus Muscle Research Articles

Effect of butanolic fraction of Desmodium adscendens on the anococcygeus of the rat.

The chemical composition of plants can vary according to factors such as soil and time of collection. Desmodium adscendens (Sw.) D.C. var. adscendens (Papillionaceae) is a plant employed in the treatment of asthma in Ghana, Africa. Studies have shown that butanolic extract inhibits contraction of the ileum and trachea in guinea pigs. In Mato Grosso, this plant is used only in the treatment of ovarian inflammation. The objective of this work was to verify if the plant found in Mato Grosso also relaxes smooth muscle and to understand better its action. The cumulative application of the butanolic fraction relaxed the contraction maintained in the isolated anococcygeus of a rat, induced by high potassium, but not that induced by phenylephrine. Relaxation was not altered by methylene blue. The butanolic fraction reduced in a concentration-dependent way the maximum response of concentration-response curve to calcium in the anococcygeus muscle. The results suggest that the butanolic fraction acts, at least partly, through the blockade of voltage-sensitive Ca+2 channels.

Characterization of the rat isolated retractor penis muscle as a model for the study of nitrergic transmission

Introduction: The anococcygeus and retractor penis muscles are part of the erectile machinery in male rodents. The rat anococcygeus muscle is a widely used smooth muscle preparation for the study of the effects of test substances on adrenergic, nitrergic, and cholinergic transmission. There is, however, little information available on the process of autonomic transmission in the rat retractor penis muscle, although its autonomic innervation has generally been assumed to be similar to that of the anococcygeus muscle because of the contiguous nature of the two muscles. The present study investigated the involvement of nitrergic transmission in mediating relaxant responses of the rat retractor penis muscle to electrical field stimulation. Methods: The retractor penis muscle was isolated from Sprague–Dawley rats and mounted in Krebs solution. Phentolamine (5 μM) was added to the bath to block the adrenergic responses of the muscle, which was then precontracted with carbachol (10 μM). Results: Electrical field stimulation (20–30 V, 1 ms pulse width, at 0.5–20 Hz for 10 s) of the carbachol precontracted muscle elicited frequency-dependent relaxant responses (0.9–68%). Tetrodotoxin (1 μM), N G-nitro- l-arginine ( l-NOARG) (50 μM), N G-nitro- l-arginine methylester ( l-NAME) (100 μM), and haemoglobin (100 μM) inhibited these relaxant responses by 99.3%, 93.9%, 86.9%, and 77.5%, respectively. l-Arginine (250 μM) (but not its d-isomer) reversed the blockade produced by l-NOARG (72.7%) and l-NAME (81.5%). Discussion: Our results provide clear evidence that the inhibitory (relaxant) responses of the rat retractor penis muscle to electrical field stimulation are mediated by nitric oxide involving the l-arginine–nitric oxide synthase–nitric oxide pathway. The rat retractor penis muscle is a versatile preparation that can replace the cumbersome preparations from the pig, ox, and horse, hitherto used as pharmacological models for the study of the retractor penis muscle.

Biochemical and pharmacological characterization of the venom of the black scorpion Heterometrus spinifer

The sting of the black scorpion Heterometrus spinifer, which can cause intense localized pain, has not been reported to produce lethal cardiovascular complications, which are well known to result from scorpion envenomation as a consequence of a massive release of catecholamines. Therefore, we have undertaken a biochemical and pharmacological characterization of the venom of H. spinifer. Pharmacologically, the venom (0.125 μL/mL) produced a marked, reversible contracture in the chick biventer cervicis muscle that was blocked by d-tubocurarine (2 μM) but not by tetrodotoxin (5 μM) and ω-conotoxin GVIA (3 μM). The anticholinesterase neostigmine (1 μM) potentiated the contracture by 5.3-fold. An ultra-filtrate fraction of MW < 3000 (F3K) of the venom produced a similar contracture in the biventer muscle, whereas the retentate of MW > 3000 did not. In the rat anococcygeus muscle, the venom produced a contractile response that was partially (37.4 ± 1.6%) blocked by atropine (5 μM); phentolamine (5 μM) blocked the remaining response. Tetrodotoxin (5 μM) did not block the contractile response of the venom on the anococcygeus muscle. Electrospray ionization-mass spectrometry/mass spectrometry confirmed the presence of high concentrations of acetylcholine (79.8 ± 1.7 μM) and norepinephrine (146.7 ± 19.8 μM) in H. spinifer venom, which can fully account for the observed cholinergic and adrenergic effects. In contrast to scorpion venoms that selectively target neuronal ion channels in mediating transmitter release, our data show that H. spinifer venom does not possess such activity, which likely explains the apparent lack of lethality of black scorpion envenomation.

Calcium-Antagonistic Activity of Sumatriptan in the Rat Anococcygeus Muscle

The relaxant effect of sumatriptan, a 5-HT_1B/1D receptor agonist, on the rat anococcygeus muscle was investigated. Sumatriptan induced concentration-dependent relaxations of the phenylephrine-precontracted rat anococcygeus muscle. N^G-nitro-L-arginine, methylene blue, glibenclamide, tetrodotoxin, indomethacin, GR 113808, GR 55562, methysergide, ketanserin, ICS 205930, clozapine, methiothepin, metergoline, mesulergine, and ritanserin did not inhibit the relaxations induced by sumatriptan. Sumatriptan converted calcium-induced contractions into relaxations in the preparations depolarized by a calcium-free, high-potassium solution. In membrane preparations obtained from the rat anococcygeus muscle, the basal rate of cAMP production by adenylate cyclase was 4.77 ± 0.02 pmol/mg protein/min (n = 15). The enzyme activity was increased to 104 ± 51.7 pmol/mg protein/min by forskolin (10^–4 mol/l), but did not change in the presence of sumatriptan. The mRNA expression of 5-HT_1B, 5-HT_1D, and 5-HT₇ receptors was not observed in the rat anococcygeus muscle. The results indicate that sumatriptan causes relaxation of the precontracted rat anococcygeus muscle by a calcium-antagonistic activity.

Pharmacological characterization of mikatoxin, an α-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka

Symptoms of envenomation by the New-Guinean small-eyed snake Micropechis ikaheka (Elapidae) include peripheral neurotoxicity and myotoxicity. We have now purified to homogeneity a long-chain neurotoxin, mikatoxin, from M. ikaheka venom by successive gel filtration and reverse-phase chromatography. Electrospray ionization mass spectrometry showed mikatoxin to be a homogenous peptide of MW 7775.6. Mikatoxin was devoid of any phospholipase A2 activity associated with the crude venom and did not exhibit any intrinsic anticholinesterase activity. In the chick biventer cervicis muscle, it produced an irreversible, concentration-dependent block of responses to exogenously applied acetylcholine and carbachol as well as twitches evoked by nerve, but not by direct muscle stimulation. Moreover, mikatoxin, like α-bungarotoxin and erabutoxin-b, did not show significant fade response to train-of-four stimulation of the mouse phrenic nerve-hemi diaphragm muscle. It also failed to block ganglionic transmission in the guinea pig ileum and muscarinic responses in the rat anococcygeus muscle. Our study provides strong evidence for the presence of a neurotoxin (mikatoxin) in M. ikaheka venom that produces neuromuscular blockade in skeletal muscle attributable to selective and irreversible antagonism of postsynaptic nicotinic acetylcholine receptors of the neuromuscular junction and likely contributes to the peripheral neurotoxicity observed in M. ikaheka envenomation.

Effects of agents that inactivate free radical NO (NO*) on nitroxyl anion-mediated relaxations, and on the detection of NO* released from the nitroxyl anion donor Angeli's salt.

1. The effects of agents that inactivate free radical nitric oxide (carboxy-PTIO, hydroxocobalamin and pyrogallol) were tested on relaxations produced by the nitroxyl anion (NO(-)) donor Angeli's salt in rat aortic rings and anococcygeus muscles. The amount of NO(*) generated from Angeli's salt in the presence of these agents was measured using a NO(*)-selective electrode sensor. 2. Carboxy-PTIO (100, 300 microM), hydroxocobalamin (30, 100 microM) and pyrogallol (10, 30 microM) significantly reduced relaxations produced by Angeli's salt (0.3 microM) in aortic rings but not in anococcygeus muscles. 3. NO(*) generated from Angeli's salt (0.1 - 10 microM), as detected by the sensor electrode, was less than 0.5% of the amount of Angeli's salt added. Carboxy-PTIO (100 microM) and hydroxocobalamin (30 microM), but not pyrogallol significantly increased the amount of NO(*) detected. 4. In the presence of an oxidizing agent copper [II] (as CuSO(4) 100 microM), the amount of NO(*) detected from 0.3 microM of Angeli's salt increased from an undetectable level of 142.7+/-15.7 nM (equivalent to 47.6% of Angeli's salt added). Under these conditions, carboxy-PTIO, hydroxocobalamin and pyrogallol significantly reduced the amount of NO(*) detected from Angeli's salt as well as the signal generated by an equivalent amount of authentic NO (0.33 microM). 5. The difference in effects of these agents on relaxations to Angeli's salt in the aorta and the anococcygeus muscle may be explained by the ready conversion of NO(-) to NO(*) in the aorta through an unidentified mechanism, which makes NO(-) susceptible to inactivation by these agents. Furthermore, in addition to inactivating NO(*), carboxy-PTIO and hydroxocobalamin may themselves oxidize NO(-) to NO(*), albeit slightly.

Functional site of bukatoxin, an α-type sodium channel neurotoxin from the Chinese scorpion ( Buthus martensi Karsch) venom: probable role of the 52PDKVP 56 loop

α-Toxins from scorpion venoms prolong the action potential of excitable cells by blocking sodium channel inactivation. We have purified bukatoxin, an α-toxin from scorpion ( Buthus martensi Karsch) venom, to homogeneity. Bukatoxin produced marked relaxant responses in the carbachol-precontracted rat anococcygeus muscle (ACM), which were mediated through the L-arginine–nitric oxide synthase–nitric oxide pathway, consequent to a neuronal release of nitric oxide. Based on the presence of proline residues in the flanking segments of protein–protein interaction sites, we predicted the site between 52PP 56 to be the potential interaction site of bukatoxin. A homology model of bukatoxin indicated the presence of this site on the surface. Buka11, a synthetic peptide designed based on this predicted site, produced a concentration-dependent nitric oxide-mediated relaxant response in ACM. Using alanine-substituted peptides, we have shown the importance 53DKV 55 flanked by proline residues in the functional site of bukatoxin.

Peroxynitrite produces relaxations on the rat anococcygeus muscle

Effects of peroxynitrite (ONOO −), its stable product 3-nitrotyrosine (NT) and sodium nitroprusside (SNP) on isolated rat anococcygeus muscle were investigated. Administration of 0.1–1.0 mM ONOO − or 0.01–100.0 μM SNP produced concentration-dependent relaxations on the phenylephrine (2 μM)-precontracted muscle. Time courses of these relaxations to ONOO − and SNP were not similar and decomposed ONOO − caused a biphasic response composed of an initial contraction followed by a relaxation. NT (0.01–0.1 mM) either incubated for 20 min prior to precontraction or given during precontraction plateau did not attenuate precontraction or ONOO −-induced relaxations. Results of the present study demonstrate that ONOO − relaxes rat anococcygeus muscle specifically while its stable metabolite NT has no effect.

Characterization of the ORL(1) receptor on adrenergic nerves in the rat anococcygeus muscle.

1. Nociceptin, the endogenous ORL(1) receptor agonist inhibited the motor response to electrical-field stimulation in the rat anococcygeus muscle. This effect was characterized using the peptide ligands acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH(2) (Ac-RYYRWK-NH(2)), acetyl-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)) and [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) ([F/G]NC(1-13)NH(2)), and the non-selective opioid antagonist naloxone benzoylhydrazone (NalBzOH). 2. Nociceptin produced a concentration-dependent inhibition of the adrenergic motor response to electrical-field stimulation (EC(50) 19 nM, pEC(50) 7.7+/-0.1, n=8), but the response to exogenous noradrenaline (0.2 - 1 microM) was unaffected. The inhibitory nerve response was not affected by up to 1 microM nociceptin. 3. After inhibition of nitric oxide synthase (N(omega)-nitro-L-arginine 100 microM), and in the presence of peptidase inhibitors, nociceptin produced full inhibition of the pure adrenergic motor response (EC(50) 4 nM; pEC(50) 8.4+/-0.1, E(max) 98.3+/-1.2%, n=12). Ac-RYYRWK-NH(2) was a potent partial-agonist (pEC(50) 9.0+/-0.1, E(max) 66.4+/-5.2; n=11) but the efficacy of Ac-RYYRIK-NH(2) (pEC(50) 8.0+/-0.2, E(max) 36.7+/-3.5; n=12) was lower and the peptide could be tested as an antagonist (pA(2) 9.01). 4. [F/G]NC(1-13)NH(2) and NalBzOH had little or no efficacy and were competitive antagonists with pK(B) values of 7.4 (95% c.l. 7.1, 7.7) and 6.9 (95% c.l. 6.7, 7.1) respectively. Both increased the response to field stimulation at high concentrations, suggesting the release of an endogenous agonist for the ORL(1) receptor during stimulation. 5. Rat nocistatin did not affect the response to electrical-field stimulation, nor did it modify the inhibitory action of nociceptin. 6. Our findings suggest there is a significant endowment of ORL(1) receptors on sympathetic terminals of the rat anococcygeus, where nociceptin mediates a powerful inhibitory effect on adrenergic neuromuscular transmission.

Does serotonin relax the rat anococcygeus muscle via 5-HT7 receptors?

The mechanisms of serotonin (5-HT)-induced contraction and relaxation were studied in the rat anococcygeus muscle. In the presence of prazosin (1 nM) and ketanserin (10 nM), concentration/response curves to 5-HT were shifted to the right and the maximum effects were not affected (pKB values 9.09+/-0.29 and 8.66+/-0.06 for prazosin and ketanserin, respectively). On contrary, guanethidine (10 microM) antagonised the 5-HT-induced contractions non-competitively. In the presence of guanethidine, prazosin or ketanserin further inhibited the responses to 5-HT at lower concentrations. The serotonergic receptor agonists 5-carboxamidotrypamine, metoclopramide and sumatriptan did not produce any effect in tissues under baseline conditions. 5-HT caused concentration-dependent relaxation in phenylephrine (1 microM) -precontracted preparations in the presence of guanethidine (10 microM). Tetrodotoxin (1 microM), N(G)-nitro-L-arginine (100 microM) and capsaicin (1 microM) were ineffective in antagonising the relaxation induced by 5-HT. The serotonergic receptor antagonists: ketanserin (0.1 microM), ICS 205930 (10 microM), GR 113808 (10 microM), GR 55562 (10 microM), methiothepin (10 nM), mesulergine (10 microM), ritanserin (0.1 microM) and spiperon (0.1 microM) did not antagonise 5-HT-induced relaxation. On the other hand, clozapine (0.01-0.1 microM) and metergoline (0.1-1 microM) attenuated the relaxation induced by 5-HT. These results demonstrate that 5-HT contracts rat anococcygeus muscle directly by an action on alpha1-adrenergic receptors and indirectly by releasing noradrenaline from adrenergic nerve endings. None of the 5-HT receptors plays a role in the 5-HT-induced contractions. Moreover 5-HT induces concentration-dependent relaxation in the precontracted preparations which apparently are not mediated through known 5-HT receptor types.

Sildenafil citrate on nitrergic transmission in anococcygeus muscles from the urogenital system of male and female mice

The effects of sildenafil on nitrergic relaxations were compared in anococcygeus muscles from male and female mice. In muscles from both sexes, sildenafil (10–300 nM) produced a weak, direct relaxation of carbachol-induced tone, and increased both the amplitude and duration of nitrergic relaxations. The most marked effect was on nitrergic duration (300–400% increase with 300 nM sildenafil); no differences in potency were observed between male (EC 50, 30 nM) and female (EC 50, 25 nM). The rate of onset for potentiation of nitrergic duration was similar in both sexes; but, on washout, the effects of sildenafil declined more slowly in the male muscle. Relaxations to both nitric oxide (NO) and sodium nitroprusside were also increased in amplitude and duration by 50 nM sildenafil, while those to forskolin and papaverine were unaffected. The results demonstrate that sildenafil causes a similar, potent and selective potentiation of nitrergic transmission in urogenital smooth muscle from both male and female mice.

Thapsigargin-induced tone and capacitative calcium influx in mouse anococcygeus smooth muscle cells.

The sarcoplasmic reticulum Ca-ATPase inhibitor thapsigargin (Tg; 0.4-100 nM) produced concentration-related, strong and sustained contractions of the mouse-isolated anococcygeus muscle; these contractions were dependent on extracellular calcium but were only partially reduced (by about 50%) in the presence of verapamil (10 and 100 microM). The verapamil-resistant component of the Tg-induced contraction was relaxed by the general calcium entry blockers SKF96365 (0.4-40 microM) and cadmium (50-300 microM), and by the tyrosine kinase inhibitor genistein (10-180 microM). In single smooth muscle cells loaded with Fura-2, addition of Tg (100 nM) to calcium-free medium produced a small, transient increase in fluorescence; subsequent addition of calcium (2.5 mM) produced a larger and sustained increase which was abolished on return to calcium-free conditions, but was only partially reduced by verapamil (10 microM; by about 30%). Manganese quenching of Fura-2 was enhanced in cells treated with Tg. The verapamil-resistant calcium influx was reduced by SKF96365 (20 microM) and to a lesser extent by genistein (40 microM); cadmium (200 microM) produced an initial decrease in fluorescence followed by a marked increase. These results demonstrate that, in the mouse anococcygeus, Tg can cause sustained contractions and elevations of calcium influx in the presence of verapamil; the time-course, calcium dependence and, although to a lesser extent, pharmacology of these effects generally support the proposal that excitation-contraction coupling in this tonic smooth muscle involves sustained capacitative calcium influx.

Insulin Reversal of Impaired Nitrergic Transmission in the Anococcygeus Muscle from Diabetic Rats

The aim of this study was to investigate whether insulin could reverse the impairment in nitrergic neurotransmission in the anococcygeus muscle from streptozotocin-induced diabetic rats. Relaxations to nitrergic nerve stimulation and sodium nitroprusside were significantly reduced in precontracted muscles from 8- and 4-week diabetic rats compared to the corresponding control rats. Treatment of diabetic rats with Lente insulin (1–12 units/day s.c.) for the final 4 weeks of the 8-week diabetes duration reversed the reductions, but treatment of diabetic rats with insulin for the last week only did not. The impairment of relaxations was also not altered by in vitro exposure of muscles from 8-week diabetic rats to soluble insulin (0.02 units/ml, 2 h). The findings suggest that in vivo insulin treatment can reverse the existing diabetes-induced impairment of nitrergic transmission in rat anococcygeus muscle. However, short-term treatment with insulin, either in vivo or in vitro, does not reverse the impairment.

Characterisation of nitrergic transmission in the isolated anococcygeus muscle of the female mouse

Field stimulation of anococcygeus muscles from female mice produced frequency-dependent relaxations of carbachol-induced tone, which were independent of the oestrus cycle but were abolished by the nitric oxide synthase (NOS) inhibitor l- N G-nitroarginine ( l-NOARG; 100 μM) and the soluble guanylyl cyclase inhibitor 1 H-[1,2,4]oxodiazolo[4,3- a]quinoxalin-1-one (ODQ; 5 μM); l-NOARG inhibition was reversed by l-, but not d-arginine. The selective phosphodiesterase V inhibitor zaprinast (1–130 μM) directly relaxed tone and enhanced both the amplitude and duration of field stimulation-induced relaxations; the effect on amplitude was greater at lower frequencies of stimulation, while increased duration dominated at higher frequencies. The duration, but not the amplitude, of relaxations to exogenous nitric oxide (NO; 15 μM) was also increased by zaprinast. The mouse anococcygeus provides a useful model for pharmacological investigation of nitrergic neurotransmission in female urogenital smooth muscle.

Impairment of epithelium-dependent relaxation in coaxial bioassay by reactive oxygen species

The purpose of the present study was to investigate the effects of reactive oxygen species on the activity of epithelium-derived relaxant factor (EpDRF) released by guinea-pig tracheal epithelium. Reactive oxygen species were generated by the electrolysis of the physiological buffer in which the tissues were bathed. Epithelium-dependent relaxation induced by acetylcholine in precontracted rat anococcygeus muscle that was placed in epithelium-intact guinea-pig trachea (coaxial bioassay system) was significantly attenuated when the tissues were exposed to electrolysis. Impairment of the acetylcholine response was prevented by incubation with free radical scavengers prior to electrolysis. In isolated rings of guinea-pig trachea, the contractile responses elicited by acetylcholine, histamine and 5-hydroxytryptamine were not altered after electrolysis of the bathing solution. The results of the present study suggested that exposure to reactive oxygen species impaired EpDRF release from guinea-pig trachea epithelium but did not alter the contractility of tracheal smooth muscle.

Effects of hydroxocobalamin and carboxy-PTIO on nitrergic transmission in porcine anococcygeus and retractor penis muscles.

The effects of carboxy-PTIO and hydroxocobalamin were studied on nitrergic transmission in anococcygeus and retractor penis muscles taken during post mortem examination from young male pigs. In both muscles under resting conditions, electrical field stimulation (EFS) caused contractions that were sensitive to tetrodotoxin (1 microM) and were greatly inhibited by prazosin (1 microM) and guanethidine (10-30 microM), but were not significantly affected by atropine (1 microM). In the anococcygeus muscle, but not in the retractor penis muscle, guanethidine produced a prolonged contraction. After tone was raised by guanethidine in the anococcygeus or by phenylephrine (1 microM) in the presence of guanethidine in the retractor penis, EFS caused tetrodotoxin-sensitive relaxations. The EFS-induced relaxations were abolished by the NO synthase inhibitor N(G)-L-nitro-arginine methyl ester (L-NAME; 100 microM) and its effect was partly overcome by L-arginine (1 mM), indicating it was mediated by nitrergic nerves. Carboxy-PTIO (0.1-1 mM) had no significant effect in reducing stimulation-induced nitrergic relaxations in either muscle. However, hydroxocobalamin (0.1-1 mM) caused concentration-dependent reductions of nitrergic relaxations in both muscles. Relaxations to exogenous nitric oxide (1 microM) in both muscles were abolished by carboxy-PTIO (0.3 mM) and hydroxocobalamin (0.1 mM). There were no differences in reactivity to carboxy-PTIO or hydroxocobalamin between anococcygeus and retractor penis muscles from the same species (pig). The finding also confirms earlier observations that the nitrergic transmitter is generally resistant to the NO-scavenger carboxy-PTIO.

Diabetes does not alter the activity and localisation of nitric oxide synthase in the rat anococcygeus muscle

Functional studies have revealed diabetes specifically impairs smooth muscle reactivity to nitric oxide in the rat anococcygeus muscle. The present study was conducted to examine whether concurrent prejunctional defects in nitrergic neurotransmission exist in anococcygeus muscles from diabetic rats. Nitric oxide synthase (NOS) activity was assessed by the conversion of 3 H - l-arginine to 3 H - l-citrulline in homogenates of anococcygeus muscles obtained from 8-week diabetic rats and control rats. NOS activity measured in all tissue samples was dependent on the presence of calcium (2 mM), NADPH (1 mM), tetrahydrobiopterin (100 μM) and flavin adenine dinucleotide (10 μM); however, removal of calmodulin (50 U/ml) did not reduce l-citrulline production. Both N G-nitro- l-arginine (100 μM) and N G-nitro- l-arginine methyl ester (100 μM) produced significant inhibition of enzyme activity. NOS activity measured in tissue samples from diabetic rats (369.6±75.9 fmol l-citrulline/mg protein) did not significantly differ from that measured in samples from control rats (423.9±110.6 fmol l-citrulline/mg protein). However, NOS activity measured after removal of the cofactor tetrahydrobiopterin, was significantly greater in samples from control rats than that from the diabetic group. NOS-immunoreactive and NADPH-diaphorase reactive nerve terminals were found to be sparsely distributed throughout longitudinal sections or whole mounts of anococcygeus muscles from both control and diabetic rats. Quantification of NADPH-diaphorase positive fibres intersecting transects of whole tissue mounts, revealed no significant difference in fibre number between the treatment groups. All NOS-immunoreactive fibres also showed vasoactive-intestinal-polypeptide immunoreactivity. In conclusion, the findings together provide no evidence to indicate that diabetes can induce prejunctional changes in NOS activity or localisation, concurrent with the reported postjunctional impairment in smooth muscle reactivity to nitric oxide, in the rat anococcygeus muscle.

Sympathetic neuroeffector transmission in the rat anococcygeus muscle.

1. When intracellular recordings were made from preparations of rat anococcygeus muscle, transmural nerve stimulation evoked noradrenergic excitatory junction potentials (EJPs) made up of two distinct components. Both components were abolished by either guanethidine or alpha-adrenoceptor antagonists, indicating that they resulted from the release of transmitter from sympathetic nerves and the subsequent activation of alpha-adrenoceptors. 2. The first component was associated with a transient increase in the intracellular concentration of calcium ions ([Ca2+]i) and a contraction. Although the second component was often associated with a long lasting increase in [Ca2+]i it was not associated with a contraction unless the second component initiated an action potential. 3. The increase in [Ca2+]i associated with the first component resulted from Ca2+ release from an intracellular store and from entry of Ca2+ through voltage-dependent Ca2+ channels. The increase in [Ca2+]i associated with the second component resulted only from the entry of Ca2+ through L-type Ca2+ channels (CaL channels). The depolarization associated with the initial increase in [Ca2+]i was abolished by reducing the external concentration of chloride ions ([Cl-]o), suggesting that it involved the activation of a Cl- conductance. 4. When the relationships between changes in [Ca2+]i, membrane depolarization and contraction produced by an increasing number of sympathetic nerve stimuli were determined in control, and caffeine- and nifedipine-containing solutions, it was found that an increase in [Ca2+]i recorded in nifedipine produced a larger contraction and larger membrane depolarization than did a similar increase in [Ca2+]i recorded in either control or caffeine-containing solutions. These observations indicate that Ca2+ released from stores more readily triggers contraction and membrane depolarization than does Ca2+ entry via CaL channels.

Inhibition of nitrergic relaxations by the M3-selective antagonist 4-DAMP

The inhibitory effect of the selective M3 musarinic acetylcholine receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP, 0.1–10 μM) on nicotine (100 μM)-induced nitrergic relaxation was investigated in comparison to d-tubocurarine (0.1–10 μM) and hexamethonium (0.1–10 μM) by using phenylephrine (1 μM)-precontracted rat anococcygeus muscles in vitro. Nicotine produced a 60.1± 2.4% (n = 40) inhibition of phenylephrine precontractions. But this relaxant response was at a significantly lower magnitude of 20.2± 4.6% (n = 18, P hexamethonium > d-tubocurarine with IC50 values being 0.47± 0.04 μM, 0.75± 0.06 μM, and 1.02± 0.05 μM, respectively. Therefore, it was concluded that the selective M3 muscarinic acetylcholine receptor antagonist 4-DAMP also possesses potent antagonistic action on nicotinic receptors of peripheral nitrergic neurons that innervate the rat anococcygeus muscle. Drug Dev. Res. 46:148–154, 1999. © 1999 Wiley-Liss, Inc.

Effects of pyrogallol, hydroquinone and duroquinone on responses to nitrergic nerve stimulation and NO in the rat anococcygeus muscle

1. The hypothesis that endogenous superoxide dismutase (SOD) protects the nitrergic transmitter from inactivation by superoxide and that this explains the lack of sensitivity of the transmitter to superoxide generators was tested in the rat isolated anococcygeus muscle. 2. Responses to nitrergic nerve stimulation or to NO were not significantly affected by exogenous SOD or by the Cu/Zn SOD inhibitor diethyldithiocarbamic acid (DETCA). 3. Hydroquinone produced a concentration-dependent reduction of responses to NO with an IC50 of 27 microM, and higher concentrations reduced relaxant responses to nitrergic nerve stimulation with an IC50 of 612 microM. The effects of hydroquinone were only slightly reversed by SOD, so it does not appear to be acting as a superoxide generator. 4. Pyrogallol produced a concentration-dependent reduction in responses to NO with an IC50 value of 39 microM and this effect was reversed by SOD (100-1000 u ml(-1)). Pyrogallol did not affect responses to nitrergic nerve stimulation. Treatment with DETCA did not alter the differentiating action of pyrogallol. 5. Duroquinone produced a concentration-dependent reduction of relaxations to NO with an IC50 value of 240 microM and 100 microM slightly decreased nitrergic relaxations. After treatment with DETCA, duroquinone produced greater reductions of relaxant responses to NO and to nitrergic stimulation, the IC50 values being 8.5 microM for NO and 40 microM for nitrergic nerve stimulation: these reductions were reversed by SOD. 6. The findings do not support the hypothesis that the presence of Cu/Zn SOD explains the greater susceptibility of NO than the nitrergic transmitter to the superoxide generator pyrogallol, but suggest that it may play a role in the effects of duroquinone.