The action of tetraethyl-ammonium chloride on the response of the rat anococcygeus muscle to motor and inhibitory nerve stimulation and to some drugs.

1 Tetraethylammonium chloride (TEA) 0.125 mM to 20 mM potentiates the response of the anococcygeus muscle to field stimulation of the motor adrenergic nerves without affecting the response to noradrenaline suggesting a pre-synaptic origin of potentiation. The potentiation is greatest at low, submaximal, frequencies (2 Hz) of stimulation and only slight at the higher frequency of 20 Hz. This difference is due to the restraint imposed on the demonstration of potentiation by maximal or near maximal motor responses since reduction of the mechanical response at 20 Hz by either phentolamine (post-synaptic block) or guanethidine (pre-synaptic block) resulted in a great increase in potentiation of the response at this frequency. 2 TEA in concentrations up to 1 mM similarly potentites the response to inhibitory nerve stimulation and again the greatest effect is at low frequencies. Higher concentrations (5-20 mM) progressively depress the inhibitory response. It is suggested that TEA may specifically antagonize the post-synaptic action of the inhibitory transmitter and that at higher concentrations of TEA this effect dominates the pre-synaptic action in increasing transmitter release. 3 TEA has no effect on the motor response to tyramine. 4 TEA (5-20 mM) causes a maintained rise in muscle tone. Part of this is abolished by phentolamine but part is resistant. A similar muscle stimulant action of TEA is observed in muscles from rats previously treated with 6-hydroxydopamine in which indirect sympathomimetic drugs and field stimulation could no longer produce a motor response. These results suggest that part of the motor effect of TEA is due to an increased spontaneous release of noradrenaline and part to a direct action on the muscle. 5 TEA 0.125 mM to 20 mM antagonize the stimulant action of carbachol. Dose-response curves show a parallel shift to the right with no change in the maximum response suggesting a competitive atropine-like action. Such an effect has previously been reported in amphibian tissue but not so far as we can determine in mammalian preparations. 6 The possible mode of action of TEA is discussed.     

The response of the cat anococcygeus muscle to nerve or drug stimulation and a comparison with the rat anococcygeus

1 The cat anococcygeus muscle is shown to possess a dual innervation similar to the rat anococcygeus, with a motor adrenergic innervation and an inhibitory innervation whose transmitter is unknown. The pharmacological properties of the cat muscle were investigated and compared with those of the rat muscle.2 The cat muscle contracts to noradrenaline, 5-hydroxytryptamine, tyramine, amphetamine, guanethidine, cocaine and lysergic acid diethylamide (LSD). The effects of noradrenaline and 5-hydroxytryptamine are blocked by phentolamine and methysergide respectively.3 The cat anococcygeus is relaxed by acetylcholine, carbachol, isoprenaline, ATP, prostaglandins E(1), E(2) and F(2alpha) and vasopressin, all of which contract the rat muscle. The effects of acetylcholine and carbachol are blocked by atropine and those of isoprenaline by propranolol.4 Field stimulation produces contraction of the cat anococcygeus, which is blocked by phentolamine and guanethidine but unaffected by hexamethonium, atropine or neostigmine.5 In the presence of guanethidine (10(-5)M), the tone of the muscle is raised and field stimulation produces relaxation of the muscle. These inhibitory responses are unaffected by phentolamine, hexamethonium, atropine or neostigmine.6 Neostigmine potentiates the effects of acetylcholine, but not of carbachol in relaxing the cat anococcygeus and in contracting the rat anococcygeus, but has no effect on either motor or inhibitory responses to field stimulation.7 Cold storage for up to eight days had little effect on either the motor response to noradrenaline or the motor or inhibitory response to field stimulation of the cat anococcygeus. Beyond eight days, the response to field stimulation diminishes more rapidly than the response to noradrenaline.     

The effect of immunosympathectomy and of 6-hydroxydopamine on the responses of the rat anococcygeus to nerve stimulation and to some drugs

1. The rat anococcygeus muscle possesses a dense motor adrenergic innervation and also a powerful inhibitory innervation whose transmitter is unknown. The possibility that the adrenergic nerves released both noradrenaline and the unknown inhibitory transmitter was investigated by destroying the adrenergic nerves either by immunosympathectomy or by 6-hydroxydopamine.2. Immunosympathectomy was only partially effective in destroying the adrenergic neurones to the anococcygeus. 6-Hydroxydopamine destroyed the terminal adrenergic varicosities abolishing almost completely the motor response. Neither treatment affected the inhibitory response.3. 6-Hydroxydopamine induced a specific hypersensitivity of the muscle to noradrenaline which was not shared with carbachol.4. The maximum tension response of the anococcygeus muscle was greater in the male than in the female probably because the muscle is bigger.     

The effects of L-arginine and NG-monomethyl L-arginine on the response of the rat anococcygeus muscle to NANC nerve stimulation.

The effect of the competitive inhibitor of L-arginine, NG-monomethyl L-arginine (L-NMMA) on the response of the rat anococcygeus muscle to non-adrenergic, non-cholinergic (NANC) inhibitory nerve stimulation has been examined. L-NMMA causes a rise in muscle tone and inhibition of the response to nerve stimulation. The stereoisomer D-NMMA is without effect. The rise in tone and inhibition of the nerve response is reversed by L-arginine. Another analogue, L-canavanine, which is effective against L-arginine utilization in the macrophage, was without effect on the rat anococcygeus. These results provide indirect evidence for nitric oxide (NO) or a substance releasing NO as the transmitter of the NANC nerves in this tissue.     

Effect of serum albumin upon the response of the rat isolated superfused anococcygeus muscle to catecholamines and to nerve stimulation

The presence of bovine serum albumin in the superfusing medium reduced the response of the rat isolated anococcygeus muscle to catecholamines and to nerve stimulation. It is suggested that this is a consequence of the binding of catecholamines to serum albumin. When the binding was prevented, either by decreasing the pH or by the addition of a drug such as oxytetracycline, then the inhibitory effects of serum albumin were much reduced.     

Endotoxin impairs the response of rat anococcygeus muscle to electrical field stimulation.

Rat anococcygeus muscles were isolated 4 h after treatment with either E. coli endotoxin (20 mg kg-1 i.p.) or saline. The contractile responses of the muscle to electrical field stimulation, and to the alpha 1-adrenoceptor agonist cirazoline were impaired by endotoxin treatment. Inhibition of the L-arginine pathway with L-NG-nitro arginine methylester (3 x 10(-5) M) failed to restore responsiveness to either electrical field stimulation or to cirazoline. Endotoxin impaired inhibitory responses to electrical field stimulation in preparations precontracted with cirazoline. Responsiveness to sodium nitroprusside was also impaired by endotoxin under these conditions. These results show an impairment by endotoxin of both contractile and inhibitory responses of the rat anococcygeus muscle to electrical field stimulation. The impairment appears to be post-junctional. In contrast to the findings in vascular smooth muscle, the endotoxin-induced impairment of contractile responses does not appear to involve the L-arginine pathway.     

The postsynaptic effects of antidepressant drugs in the rat anococcygeus muscle

The effects of antidepressants of tricyclic (amitriptyline, nortriptyline, protriptyline, doxepin, imipramine, and desipramine) and atypical (maprotiline, nomifensine, tandamine, viloxazine, CGP 6085A, and YM-08054-1) structures on contractile responses to exogenously applied acetylcholine and (?)-noradrenaline were studied in rat isolated anococcygeus muscle previously incubated with 6-hydroxydopamine. Atropine, amitriptyline, protriptyline, doxepin, imipramine, maprotiline and nortriptyline inhibited contractile responses to acetylcholine whereas desipramine, nomifensine, tandamine, viloxazine, CGP 6085A and YM 08054-1 did not. The contractile responses to (?)-noradrenaline were inhibited by low concentrations of tricyclic antidepressants and by higher concentrations of the atypical agents. These results illustrate that, in the preparation, the order of potency of antidepressants as muscarinic and as postsynaptic α-adrenoceptor antagonists is similar. The ability of tricyclic, but not atypical agents, to increase the concentration of noradrenaline bound to postsynaptic α-adrenoceptors may be severely limited by the antagonistic effect these agents have at this receptor.     

The postsynaptic effects of antidepressant drugs in the rat anococcygeus muscle

The effects of antidepressants of tricyclic (amitriptyline, nortriptyline, protriptyline, doxepin, imipramine, and desipramine) and atypical (maprotiline, nomifensine, tandamine, viloxazine, CGP 6085A, and YM-08054-1) structures on contractile responses to exogenously applied acetylcholine and (-) noradrenaline were studied in rat isolated anococcygeus muscle previously incubated with 6-hydroxydopamine. Atropine, amitriptyline, protriptyline, doxepin, imipramine, maprotiline and nortriptyline inhibited contractile responses to acetylcholine whereas desipramine, nomifensine, tandamine, viloxazine, CGP 6085A and YM 08054-1 did not. The contractile responses to (-)-noradrenaline were inhibited by low concentrations of tricyclic antidepressants and by higher concentrations of the atypical agents. These results illustrate that, in the preparation, the order of potency of antidepressants as muscarinic and as postsynaptic alpha-adrenoceptor antagonists is similar. The ability of tricyclic, but not atypical agents, to increase the concentration of noradrenaline bound to postsynaptic alpha-adrenoceptors may be severely limited by the antagonistic effect these agents have at this receptor.     

Neostigmine augments responses of the rat anococcygeus muscle to field stimulation.

1 The effects of neostigmine on noradrenergic transmission have been studied in the field stimulated, isolated anococcygeus muscle of the rat. 2 In those muscles where the excitatory response to field stimulation was not completely inhibited by guanethidine (5 X 10(-6) to 10(-5) M) or phentolamine (10(-6) M), atropine (5 X 10(-8) M) gave no further inhibition of the response. 3 The shape of the response to field stimulation was altered in a dose-dependent manner by neostigmine (5 X 10(-7) to 5 X 10(-6) M), such that a 'shoulder' appeared during the relaxation phase. The 'shoulder', present at all stimulation frequencies tested between 3 and 40 Hz, was abolished by atropine (5 X 10(-8) M) and unaffected by tubocurarine (10(-6) M). 4 Neostigmine (2.5 X 10(-6) M), whether alone or in the presence of atropine (5 X 10(-8) M), had no effect on the uptake or stimulation-induced release of [3H]-noradrenaline. 5 Using electron microscopy, small Schwann/axon bundles close to smooth muscle cells rarely showed cholinesterase staining, whereas larger bundles at the outer serosal aspects of the muscle exhibited dense staining. 6 It is concluded that the observed effects of neostigmine are not due to a presynaptic effect on noradrenergic transmission.     

The effects of drugs on the sensitivity of the rat anococcygeus muscle to agonists

1. The effects of cocaine, 6-hydroxydopamine (6-OHDA), reserpine and 17-beta-oestradiol on the sensitivity of the rat anococcygeus muscle to noradrenaline (NA), acetylcholine (ACh) and KCl were investigated.2. Cocaine (10(-5)M) increased the sensitivity of the anococcygeus to NA (100-fold) but not to ACh or KCl.3. 6-OHDA treatment, whether discontinuous over 6 days or continuous for 12 days, also produced a specific increase in sensitivity to NA (50-fold).4. Reserpine ((1 mg/kg)/day) produced a small increase in sensitivity of the muscle to NA and ACh but not to KCl, after 6 days and 12 days treatment.5. 17-beta-Oestradiol (10(-5)M) had no effect on the sensitivity of the anococcygeus to NA or ACh, but reduced the sensitivity to KCl and the possible mechanism of this effect is discussed.6. Cocaine increased, while reserpine, 6-OHDA and 17-beta-oestradiol decreased, the maximum response of the muscle to KCl. The response to KCl was shown to consist of at least three components; a direct action on the muscle, an effect due to release of NA, and an inhibitory action, probably due to release of the unknown inhibitory transmitter.     

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.     

Development of motor response to intramural nerve stimulation and to drugs in rat small intestine.

1. The onset and development of functional innervation and transmitter reactivity in the small intestine, isolated from foetal and neonatal rat, was examined in relation to the development of two muscle layers. 2. About half of the preparations tested at embryonic day 15 responded to electrical field stimulation, although both acetylcholine (ACh, 1 micro M) and excess K (50 mM) evoked a response in all preparations. The response to these stimuli was the extension of the preparation longitudinally. All six preparations examined at embryonic day 16 extended in response to electrical field stimulation. 3. The extension response to electrical field stimulation was observed up to embryonic day 19. Then, the response changed to a biphasic one and finally to shortening after 4-days postnatal. The responses to electrical field stimulation were blocked by tetrodotoxin (TTX, 0.3 micro M) or atropine (0.3 micro M). The response to ACh and excess K similarly changed and became only a shortening at embryonic day 21 and 6-days postnatal, respectively. 4. The extension and biphasic responses produced by these stimuli were invariably converted to shortening after the preparation had been opened longitudinally. 5. The pD2 value for ACh was 6.74-7.37 during the period embryonic day 15-6-days postnatal. 6. Theses results suggest that in the rat intestine, functional cholinergic innervation is established at least by embryonic day 16. In the early stages, the development of the circular muscle layer precedes the preparation in response to stimuli. The reverse takes place following the development of the longitudinal muscle layer.     

The effects of pyrogallol and hydroquinone on the response to NANC nerve stimulation in the rat anococcygeus and the bovine retractor penis muscles.

1. The effects of pyrogallol and hydroquinone on the bovine retractor penis (BRP) and rat anococcygeus muscles to non-adrenergic, non-cholinergic (NANC) nerve stimulation have been examined. Both drugs at a concentration of 10(-4) M significantly reduced the response in the rat anococcygeus muscle but had no effect in the BRP muscle. 2. The inhibition of the NANC response in the rat anococcygeus muscle by pyrogallol was completely reversed by superoxide dismutase suggesting it was due to the generation of superoxide anions. 3. Pyrogallol inhibited the response to nitric oxide (NO) in the rat anococcygeus muscle but not that to 3-isobutyl-1-methyl-xanthine (IBMX) which confirmed a selective action. 4. These results suggest that the NANC neurotransmitter in the rat anococcygeus muscle is susceptible to superoxide anions and may be NO or a substance that can liberate NO.     

The effects of neostigmine on the response of the rat anococcygeus muscle to field stimulation are not a consequence of cholinesterase inhibition

Neostigmine (5 X 10(-7) to 5 X 10(-6) M) and physostigmine (10(-5) M) each augment the responses of the rat anococcygeus muscle to field stimulation whereas iso-OMPA (5 X 10(-6) to 5 X 10(-4) M) or BW 62c47 (5 X 10(-7) to 5 X 10(-5) M) do not. At a concentration of 10(-5) M, neostigmine. BW 62c47 and iso-OMPA respectively produced a 48, 50 and 68% inhibition of cholinesterase activity in homogenates of anococcygeus muscle. The ED50 for cholinesterase inhibition by neostigmine (1.4 X 10(-5) M) is approximately 15 fold greater than the ED50 for the augmentation of the response to field stimulation (9.5 X 10(-7) M). It is concluded that the action of neostigmine in augmenting the response of the rat anococcygeus muscle to field stimulation is not a consequence of cholinesterase inhibition even though stimulation of muscarinic receptors is implicated.     

The effect of pithing and of nerve stimulation on the depletion of noradrenaline by reserpine in the rat anococcygeus muscle and vas deferens

1 The depletion of noradrenaline (NA) in the rat anococcygeus muscle and vas deferens by reserpine and the effect on this of the abolition of nerve activity by pithing and reinforcement of nerve activity by stimulation of the spinal cord outflows has been studied.2 NA depletion of the anococcygeus and vas deferens measured 24 h after reserpine was similar and was related to dose. The heart was depleted faster than the two smooth muscle tissues.3 In the absence of reserpine neither abolition of nerve activity by pithing nor its reinforcement by nerve stimulation had a detectable influence on NA content of the anococcygeus or vas deferens.4 In rats given reserpine (200 mug/kg), increasing nerve activity by spinal stimulation significantly increased NA depletion in both the anococcygeus and the vas deferens when compared with animals pithed but not stimulated. These results confirm that nerve impulse traffic can be an important factor in determining the rate of depletion of NA by reserpine.5 The mechanical response to nerve stimulation in both the vas deferens and anococcygeus was resistant to quite severe depletion of their NA content, with the exception of the initial fast component of the response in the vas. The implications of these results for motor transmission in the vas deferens are discussed.