Characterisation of excitatory and inhibitory transmitter systems in prostate glands of rats, guinea pigs, rabbits and pigs

Excitatory and inhibitory transmitter systems were investigated in strips of prostate glands from rats, guinea pigs, pigs and rabbits. In strips from all species, electrical field stimulation (1 ms pulses at 1-30 Hz for 10 s) produced frequency-dependent contractions which were abolished by tetrodotoxin (1 microM). In strips from rats, guinea pigs and rabbits, contractions were reduced by prazosin (1 microM), guanethidine (10 microM) and atropine (2 microM), indicating the presence of noradrenergic and cholinergic mechanisms. However, the smooth muscle in the pig prostate appears to have a non-(nor)adrenergic non-cholinergic (NANC) excitatory innervation for which the transmitter was not identified. When noradrenergic and cholinergic mechanisms were blocked by guanethidine and atropine, respectively, and tone was raised with noradrenaline or methoxamine, field stimulation produced relaxations only in strips of rabbit prostate, and these were greatly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), providing functional evidence for a nitrergic relaxant innervation. In accord with this, nitric oxide (NO) synthase activity was considerably higher in rabbit than in rat or pig prostates.     

Mosaicism in trisomy 8: phenotype differences according to tissular repartition of normal and trisomic clones

The functional role of non-adrenergic non-cholinergic (NANC) nerves in the autonomic control of the male mini-pig bladder neck was investigated in the present study. Electrical stimulation of muscle strips from male mini-pig bladder neck showed biphasic response with initial phasic contraction followed by post-contractile relaxation. Electrical stimulation in the presence of four autonomic blockers (atropine 10(-6) M, propanolol 10(-6) M, phentolamine 10(-6) M) showed suppression of 68 +/- 15% of the contractile response (P < 0.05, n = 8) but no significant change in the relaxation response. Alpha-chymotrypsin 2 U/ml, L-NG-monomethyl-L-arginine acetate (a nitric oxide synthetase inhibitor) 10(-4) M, 8-phenylthlophylline (a P1-purinoceptor antagonist) 10(-6) M, and pyridoxal-phosphate-6-azophenyl-2', 4'-disulphoric acid tetrasodium salt (a P2Y-purinoceptor antagonist) 3 x 10(-5) M did not alter the NANC response significantly. On the other hand, reactive blue-2 (a P2Y-purinoceptor antagonist) 3 x 10(-5) significantly reduced the relaxation by 79 +/- 9%. The result suggested that the P2Y-purinoceptor is involved in the electrically induced NANC post-contractile relaxation of the mini-pig bladder neck smooth muscle.     

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM). 6. Atropine-resistant contractions to EFS were enhanced by indomethacin (10 microM) and reduced or abolished by the non-selective NK1/NK2 tachykinin receptor antagonist D-Pro2, D-Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 microM).7. Exogenous tachykinins mimicked non-cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A>neurokinin B>substance P, suggesting a predominance of the NK2 sub-type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.8. These results suggest that: (i) in previous studies, non-cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non-cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as 'rebound' excitation; (iii) one or more tachykinins, possibly,acting via NK2 receptors, may mediate non-cholinergic excitatory responses.     

Placental tissue from human miscarriages expresses class II HLA-DR antigens

The role of peptides in mediating the nonadrenergic, noncholinergic (NANC) response of the rat urinary bladder was studied. Electrical stimulation of muscle strips from 3-month-old female Wistar rat urinary bladders in the presence of autonomic blockers (atropine 10(-6) mol/l, propanolol 10(-6) mol/l, phentolamine 10(-6) mol/l, and guanethidine 10(-6) mol/l) showed NANC contraction accounting for 60% of the maximum contractile responses at 40 Hz. Frequency-response studies showed that in the presence of alpha-chymotrypsin (2 U/ml, 30-min incubation), the NANC contractile responses to electrical stimulation at lower frequencies (3-10 Hz) were enhanced (p < 0.05; n = 9). However, no significant differences were observed at higher frequencies (20-40 Hz). With repetitive 4-Hz stimulation, alpha chymotrypsin caused a 19% increase in the NANC contractile response (p < 0.05; n = 8). It is postulated that the NANC response of the rat bladder smooth muscle is composed of an excitatory (contractile) and an inhibitory (relaxant) component. Some peptide(s) is/are responsible for mediating the inhibitory response.     

Development of cholinergic and inhibitory non-adrenergic non-cholinergic responses in the rat gastric funds

1. Cholinergic contractions and inhibitory non-adrenergic non-cholinergic (NANC) relaxations were studied in longitudinal muscle strips of the gastric funds of 2, 4 and 8 week old rats. 2. Contractions induced by electrical stimulation of the cholinergic neurones and by administration of acetylcholine decreased during development. The potentiating effect of physostigmine was similar in the 3 age groups. 3. Short train stimulation in NANC conditions induced fast relaxations, which were more pronounced in 4 and 8 week than in 2 week old rats. These relaxations were almost completely inhibited by NG-nitro-L-arginine methyl ester (L-NAME) in the 3 age groups. The nitric oxide-induced relaxations did not change during development. 4. Sustained electrical stimulation in NANC conditions induced an initial relaxation, which was almost totally blocked by L-NAME, followed by an almost complete recovery of tone at lower frequencies of stimulation. At higher frequencies of stimulation, the recovery of tone was incomplete or absent. This sustained relaxation was only partially reduced by L-NAME and almost abolished by L-NAME plus alpha-chymotrypsin. The initial relaxations increased during development, while the sustained relaxations remained similar during this period. Vasoactive intestinal polypeptide-induced relaxations were also similar in the 3 age groups. 5. These results show that the sensitivity of the gastric fundus to acetylcholine decreases from 2 weeks to 8 weeks postnatally, while the importance of the nitrergic innervation increases during this period.     

Effects of noise stress on EFS-mediated cholinergic and inhibitory NANC responses in tracheae from normal and sensitized guinea-pigs

1 The aim of the present research was to study the cholinergic and inhibitory non-adrenergic-non-cholinergic (NANC) responses obtained with electrical field stimulation (EFS) of tracheal tissues from sham- and noise-exposed guinea-pigs. A comparison was also made between normal and ovalbumin (OA)-sensitized animals. 2 In proximal tracheae pretreated with indomethacin (3 microM), propranolol (1 microM), alpha-chymotrypsin (2 U ml-1) and L-NAME (0.1 mM), frequency-dependent responses to EFS (0.1 ms width; 20 V, 0.1-100 Hz, 15 s train duration) were obtained, both contractile and relaxing in nature. The contractile responses were abolished by atropine (1 microM), and did not vary significantly between sham- and noise-exposed guinea-pigs, or between normal and sensitized animals. The NANC relaxing responses, present in spite of the pre-treatment of the tissues with L-NAME and alpha-chymotrypsin, and almost completely abolished by tetrodotoxin (TTX) treatment (10 microM), appeared to be enhanced in noise-exposed guinea-pigs, with respect to sham-exposed animals, but only when the animals were not OA-sensitized. 3 In distal tracheae contracted with histamine (10 microM), the study of the whole inhibitory NANC response (pre-treatment with propranolol, but not with alpha-chymotrypsin and L-NAME), which was mainly TTX-sensitive, revealed a statistically non-significant difference between sham- and noise-exposed guinea-pigs, both normal and OA-sensitized. When distal tracheae were preincubated with alpha-chymotrypsin (2 U ml-1) and L-NAME (0.1 mM), in addition to propranolol, a significant residual inhibitory NANC response to EFS was observed. Surprisingly, in this case, similarly to the evidence obtained in proximal tracheae, a significantly enhanced response was revealed in noise-exposed guinea-pigs with respect to sham-exposed animals. 4 The noise-induced enhancement of the relaxant response disappeared when the tissues were pretreated with the A2 purinergic antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 1 microM), while it persisted in the presence of the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM). 5 The above data indicate that, while not modifying the cholinergic and the whole inhibitory NANC response to EFS, noise stress selectively influences an inhibitory component of the NANC system in guinea-pig trachea with a mechanism probably involving an enhanced neurally mediated release of adenosine, which relaxes the smooth muscle via A2 receptors. This effect appears to be lacking or masked in sensitized guinea-pigs.     

The adrenergic, cholinergic and NANC nerve-mediated contractions of the female rabbit bladder neck and proximal, medial and distal urethra

1. The nerve-mediated contraction of the female rabbit bladder neck and different portions of the urethra (proximal, medial and distal) was studied in vitro by electrical stimulation (50 V, 30 Hz, 0.05 ms width, trains of 5 s every 5 min) by use of a superfusion system. 2. The amplitude (Emax) and the duration (Dmax) of the stimulated contraction were studied in the four tissues. The Emax value was significantly higher in distal urethra (2.07+/-0.15 g) compared to the bladder neck (1.08+/-0.10 g), proximal urethra (0.73+/-0.07 g) and medial urethra (0.87+/-0.07 g). In contrast, the Dmax value appeared slightly but significantly lower (P<0.05) in distal urethra (68.5+/-2.3 s) than in bladder neck (76.7+/-6.0 s), proximal urethra (84.5+/-5.0 s) and medial urethra (81.3+/-3.5 s). 3. Cocaine (1 microM) significantly increased the basal Emax values in medial and distal urethra and the basal Dmax values in the four tissues. 4. Prazosin (1 microM) significantly reduced E max value in proximal, medial and distal urethra and Dmax value in bladder neck and proximal urethra. Atropine (1 microM) also significantly reduced Emax values in bladder neck and proximal urethra and reduced Dmax value in bladder neck, but not in other tissues. Yohimbine (0.1 microM) was devoid of effect in the four tissues. 5. The association of prazosin (1 microM) and atropine (1 microM) did not modify the Emax and the Dmax values of the electrically-induced contractions, except in proximal urethra and in bladder neck where an additive inhibitory effect (on Emax only) was observed compared to prazosin and atropine alone. 6. The residual contractile response after combined treatment with prazosin and atropine was significantly diminished by tetrodotoxin (TTX; 1 microM) but not completely abolished. These NANC contractions were insensitive to P2X-purinoceptor desensitization by continuous tissue perfusion with alpha,beta-methylene ATP (30 microM). 7. These results demonstrate that bladder neck and proximal urethra are mainly innervated by the parasympathetic nervous system, whereas medial and distal urethras are to a greater extent under the control of the sympathetic innervation. The residual responses, insensitive to prazosin and atropine, may indicate a NANC innervation in the four tissues. However, the nature of the NANC neurotransmitter remains to be identified.     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.     

A re-appraisal of the nature of the atropine-resistant contraction to electrical field stimulation in the human isolated detrusor muscle

We investigated whether in human isolated detrusor strips the atropine-resistant contractile response to electrical field stimulation was mediated by ATP (or a related purine), as previously shown in the urinary bladder of other mammalian species. Electrical stimulation (1-50 Hz for 5 s at 1 min intervals, 0.1 ms pulse width, 60 V) elicited reproducible, frequency-dependent twitch contractions, which were markedly reduced by atropine (10 microM). Tetrodotoxin (TTX: 1 microM) inhibited contractile responses to a similar degree. When applied together, atropine and TTX caused an inhibition which was superimposable to that caused by either drug alone. The TTX-resistant contractions were totally unaffected by omega-conotoxin GVIA (omega-CTX: 0.1 microM). The atropine-resistant contractions were unaffected by the P2-purinoceptor antagonists suramin (300 microM) and PPADS (30 microM), at concentrations which virtually suppressed the contractile response induced by applied ATP (10 microM(-1) mM). As previously described, antagonism of the ATP-induced contractions by suramin (30, 100, 300 microM) and PPADS (3, 10, 30 microM) was insurmountable, with apparent 'pA2' values (calculated at the lowest antagonist concentrations) of 4.9 and 5.2, respectively. It is concluded that, under our experimental conditions, the non-cholinergic (atropine-resistant) component of the excitatory transmission in the human detrusor is not mediated by neural release of ATP, in spite of the presence of excitatory P2-purinoceptors on the effector cells. The TTX- and omega-CTX-resistant, non-cholinergic component might be related to the release of unknown transmitter(s) through a mechanism independent of both Na+- and N-type Ca2+-channels. More likely, the atropine-resistant component may reflect direct smooth muscle excitation since the human detrusor has a very short chronaxie (Sibley 1984).     

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.     

Evidence supporting a role for ATP as non-adrenergic non-cholinergic inhibitory transmitter in the porcine ileum

The aim of this study was to investigate the nature of the non-adrenergic non-cholinergic (NANC) inhibitory transmitter of the circular muscle of the porcine ileum. For this purpose, the effects of putative NANC mediators i.e. NO, vasoactive intestinal polypeptide (VIP) and ATP were measured in isolated organ bath experiments (in basal conditions and after incubation with neostigmine 3 x 10[-5] M) and using the microelectrode technique. The NO donor sodium nitroprusside (NaNP) up to 10(-4) M, VIP up to 10(-7) M and ATP up to 10(-4) M failed to cause significant relaxation in the basal state. However, all of them induced marked relaxations when the tissue had been preincubated with neostigmine (3 x 10[-5] M) which was added to increase basal mechanical activity. The resting membrane potential (RMP) was unaffected by NaNP(up to 10(-4) M and VIP up to 10(-7) M whereas ATP (up to 10[-4] M) induced a transient hyperpolarization. The inhibitory junction potentials (IJPs) induced by electrical field stimulation (EFS) were not affected by N omega-nitro-L-arginine (L-NNA) (10[-4] M) whereas suramin, a purinoceptor antagonist, decreased (10[-4] M) or abolished (10[-3] M) the IJPs. Relaxations induced by ATP in neostigmine preincubated tissue were resistant to 10(-6) M tetrodotoxin, an axonal blocker, and inhibited by suramin. Apamin (10[-6] M, a small conductance calcium activated potassium channel blocker, completely abolished the IJP (n=5) and significantly decreased the relaxation induced by ATP (n=5). The present data provide support to the hypothesis that ATP is the NANC inhibitory transmitter in the porcine ileum acting on P2 muscular receptors. Nevertheless, VIP and NaNP do also cause relaxation of preparations preincubated with neostigmine.     

Nervous regulation of the tone of the retractor penis muscle in the goat

The nervous control of the retractor penis muscle (rp) was investigated in the anaesthetized goat. Also, isolated field stimulated strips of the muscle were studied. The noradrenaline (NA) and acetylcholine (ACh) content of the rp was determined, and histochemistry for adrenergic and acetylcholinesterase (AChE) positive nerves was performed. The muscle exhibited spontaneous activity that persisted after section of all nerves. There was, however, also a tendency of the activity to follow the general vasomotor tone, which disappeared after section of the sympathetic chains. The excitatory adrenergic nerves which innervate the muscle come from the sympathetic chains and run along the pudendal, the hypogastric and the pelvic nerves. The rp has a dense network of adrenergic fibres and is very sensitive to excitatory adrenergic stimulation. It has a fairly large NA content, which is higher in old goats (5.95 +/- 0.42 micrograms g-1) than in young goats (2.87 +/- 0.78 micrograms g-1). Inhibitory non-adrenergic non-cholinergic (NANC) innervation reaches it via the pelvic and the hypogastric nerves. The maximum inhibitory response is reached at low frequencies (2-4 Hz). Cholinergic prejunctional inhibition of the excitatory response to sympathetic chain stimulation was effected by simultaneous stimulation of the hypogastric nerves. In vitro experiments confirmed the presence of endogenous cholinergic muscarinic suppression of the excitatory adrenergic neurotransmission. Significant amounts of ACh (0.81 +/- 0.18 micrograms g-1) are present in the muscle, and it contains strongly AChE positive nerve fibres and nerve cell bodies. It is concluded that the goat rp is innervated by sympathetic adrenergic excitatory nerves and parasympathetic NANC inhibitory nerves. It further has a direct sympathetic inhibitory NANC innervation, and an indirect inhibitory cholinergic innervation which at least in part is sympathetic.     

Comparison of the pharmacological profile of S-nitrosothiols, nitric oxide and the nitrergic neurotransmitter in the canine ileocolonic junction

1. In organ bath experiments, hydroquinone (30-100 microM) and hydroxocobalamin (30-100 microM) concentration-dependently inhibited the relaxations induced by NO (0.3-30 microM) but not those by nitroglycerin (GTN, 1 microM) in the canine ileocolonic junction (ICJ). Hydroxocobalamin reduced the relaxation to low frequency (2 Hz) stimulation of the non-adrenergic, non-cholinergic (NANC) nerves, whereas hydroquinone only reduced the NANC nerve-mediated relaxations to electrical stimulation at 16 Hz, 0.5 ms. 2. Relaxations to S-nitroso-L-cysteine (CysNO, 1-30 microM), or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1-30 microM) were not inhibited by hydroquinone (30-100 microM), hydroxocobalamin (30-100 microM), pyrogallol (30-100 microM) or L-cysteine (1-3 microM). Hydroquinone (100 microM) only reduced the relaxation to 10 microM CysNO. Hydroxocobalamin, but not hydroquinone, pyrogallol or L-cysteine, potentiated the relaxations to the lowest concentration (1 microM) of S-nitrosoglutathione (GSNO, 1-30 microM). 3. In the superfusion bioassay, hydroquinone (100 microM) and hydroxocobalamin (1 microM) concentration-dependently inhibited the biological activity of authentic NO (1-4 pmol) to the same extent as that of the transferable nitrergic factor, released from the canine ICJ in response to NANC nerve stimulation (8-16 Hz, 2 ms). Responses to GTN (10 pmol) or adenosine 5'-triphosphate (10 nmol) were not affected. 4. In conclusion, the nitrosothiols CysNO, SNAP and GSNO relax the canine ileocolonic junction, but these relaxations, pharmacologically, behave differently from the NANC nerve-mediated relaxations. From the bioassay experiments, we conclude that the nitrergic factor, released in response to NANCnerve stimulation of the canine ICJ, behaves pharmacologically like NO but not like a nitrosothiol.Therefore, we suggest NO, and not CysNO, SNAP or GSNO as the inhibitory NANC neurotransmitter in the canine ICJ.     

Nitrergic modulation of cholinergic responses in the opossum lower oesophageal sphincter

1. Electrical field stimulation (EFS) of the superfused lower oesophageal sphincter from opossum (Monodelphis domestica) elicited biphasic responses. The first phase (relaxation) was strictly dependent on the duration of the EFS. The second phase (contraction) started following termination of the EFS (< or = 15 Hz). EFS at frequencies above 15 Hz led only to contraction, which started immediately upon initiation of the stimulation. 2. In the presence of NG-nitro-L-arginine (L-NOARG; 0.1-300 microM), the relaxation phase was abolished and the contractile response started with the initiation of EFS (at all frequencies) and was greater in magnitude. The contractile response to EFS was completely blocked with scopolamine (10 microM). 3. Exogenous acetylcholine (1-100 microM) elicited concentration-dependent contractions of the sphincter in the presence of botulinum toxin. These contractions were abolished when EFS was applied during administration of acetylcholine. This inhibitory effect of EFS was completely reversed when the tissue was treated with L-NOARG (100 microM). 4. These results suggest that the cholinergic response in the opossum lower oesophageal sphincter is under nitrergic control.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.     

Further studies concerning possible transmitters from NANC nerves in the circular muscle of the rat stomach fundus

We examined the characteristics of the non-adrenergic, non-cholinergic (NANC) inhibitory response of the circular muscle of the rat stomach fundus to transmural nerve stimulation or high K+. Treatments with isotonic high K+ (20 mM), nitric oxide (NO) and sodium nitroprusside (SNP) all elevated cyclic GMP levels in the rat stomach fundus in the presence of atropine and guanethidine. Isotonic high K+-induced formation of cyclic GMP was completely inhibited by tetrodotoxin (TTX) or NG-nitro-L-arginine (L NNA). The K+ also increased cyclic AMP levels and this response was completely inhibited by TTX. Dose-dependent relaxation of the fundus in response to SNP was shifted to the right by a prior incubation with high concentration of SNP (10(-4) M) for 2 hrs. Incubating the fundus with SNP for 2 hrs significantly inhibited NO induced cyclic GMP formation. Relaxation responses to transmural stimulation (1 Hz or 30 Hz), isotonic high K+ and NO were significantly reduced by a prior incubation with SNP. Isotonic high K+ (20 mM)-induced relaxation of circular muscle strips was not completely inhibited by combined treatment with 10(-5)M L-NNA, 5 x 10(-5)M oxyhemoglobin and anti VIP (1:200). These results suggest that NO as well as VIP is possible transmitter from NANC nerves in the circular muscle of the rat stomach fundus and there should be one or more inhibitory mediators other than VIP and NO.     

Changes in cholinergic and purinergic neurotransmission in pathologic bladder of chronic spinal rabbit

PURPOSE: We evaluated the changes in cholinergic and purinergic neurotransmission in pathologic bladder of chronic spinal rabbits. MATERIAL AND METHODS: Detrusor muscle strips were obtained from normal rabbits and chronic spinal rabbits with detrusor hyperreflexia and detrusor sphincter dyssynergia (DSD). Muscle strips were mounted in an organ bath, and transmural nerve electrical field stimulation (EFS: supamaximal voltage, 0.5 msec. duration, 10 second trains) was performed. The responses to EFS and agonists were determined by recording the isometric tension of muscle strips. RESULTS: Both normal and pathologic detrusor strips contracted in a frequency dependent fashion in response to transmural electrical nerve stimulation. At each frequency, atropine reduced the nerve-mediated contraction in a dose-dependent fashion and left an atropine-resistant response at a concentration of 1 microM. The atropine-resistant contraction was abolished by desensitization of P2X-purinoceptors with repeated exposure to alpha, beta-methylene ATP (10 microM). The atropine sensitive (cholinergic) and resistant (purinergic) contractions increased with an increase in frequency and reached maximum at 20 Hz. The relative contribution of cholinergic and purinergic transmission to the nerve-mediated contraction was determined at this frequency. In normal detrusor, the cholinergic and purinergic components were approximately 40% and 60%. In pathologic detrusor, the cholinergic component increased to 75% whereas the purinergic component decreased to 25%. Exogenously administered acetylcholine and ATP produced dose-dependent contractions of detrusor strips. The concentration-response curves for each agonist did not show significant differences between normal and pathologic detrusor. CONCLUSION: These results suggest that neurotransmission is shifted to a cholinergic dominance in pathologic rabbit bladder affected by detrusor hyperreflexia and DSD.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

Role of nitric oxide in the colon of patients with ulcerative colitis

The cause of impaired motility, such as diarrhea and toxic megacolon, in patients with ulcerative colitis (UC) is unknown. Nitric oxide (NO) has been shown to be a neurotransmitter in the nonadrenergic noncholinergic (NANC) inhibitory nerves in the human gut. To assess the physiologic significance of NO in the colon of patients with UC, we investigated enteric nerve responses on lesional and normal bowel segments derived from patients with ulcerative colitis (n = 6) and patients who underwent colon resection for colonic cancers (n = 10). A mechanographic technique was used to evaluate in vitro muscle responses to electrical field stimulation (EFS) of adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers, including NG-nitro-L-arginine (L-NNA) and L-arginine. The results showed that (1) NANC inhibitory nerves were found to act on both normal colon and UC colon; (2) the colon with UC was more strongly innervated by NANC inhibitory nerves than the normal colon; (3) L-NNA concentration-dependently inhibited the relaxation in response to EFS in the colon of both normal and UC colon; and (4) this inhibitory effect in the colon of both normal and UC patients was reversed by L-arginine; (5) NO acts more strongly in the UC colon than the normal colon. These findings suggest that NANC inhibitory nerves play an important role in the impaired motility observed in patients with UC and that NO plays an important role as a neurotransmitter in NANC inhibitory nerves of human colon.     

A reporter gene assay for fungal sterol biosynthesis inhibitors

We evaluated the effect of estrogen on nitric oxide (NO)-mediated urethral relaxation in rabbits. Female New Zealand white rabbits, 4-5 weeks old, were treated with 5 mg/kg estradiol dipropionate (estrogen group) or saline (control group) injected intramuscularly weekly for 2 weeks. Electrical field stimulation (supramaximum voltage, 2 ms pulse duration, 0.3-15 Hz and 3 s train) caused frequency-dependent relaxation of urethral strips in both groups, which was inhibited by Nomega-nitro-L-arginine (L.-NNA). This inhibition was overcome by addition of L-arginine. The relaxation induced by nitrergic nerve stimulation was significantly lower in the estrogen group than in the control group. There was no significant difference in sodium nitroprusside-induced urethral relaxation between the two groups. The production of NO in urethral strips during nitrergic nerve stimulation was evaluated by measuring nitrite/nitrate (NO2-/NO3-) levels in both groups, using microdialysis. The NO2-/NO3- production during electrical field stimulation in the estrogen group was significantly less than that in the control group. The NADPH diaphorase-positive reaction in the control group was greater than that in the estrogen group. The results suggest that estrogen treatment may reduce NO synthase activity, and inhibit the relaxation induced by nitrergic nerve stimulation in rabbit urethral smooth muscle.