Anesthetic management in epidermolysis bullosa: review of 129 anesthetic episodes in 32 patients

Non-adrenergic non-cholinergic (NANC) inhibitory nerves have been described in all regions of the gastrointestinal tract, but have not been shown previously in the human gall bladder. Electrical field stimulation was used in the presence of various agonists and antagonists to show NANC inhibitory innervation in strips of human gall bladder muscle. Gall bladder strips were set up isometrically in an organ bath containing oxygenated Krebs's solution. Electrical field stimulation was applied at 10 Hz, pulse width 0.3 ms and supramaximal voltage at intervals of 3 to 5 minutes. Of 60 strips that contracted in response to electrical field stimulation, 30 showed relaxation on electrical field stimulation in the presence of either carbachol (5-10 microM) or else atropine (0.5-2 microM) plus cholecystokinin octapeptide (0.01-0.1 microM) or caerulein (0.1 nM) or histamine (5-10 microM). In 22 strips this relaxation was not abolished by guanethidine (2-5 microM) showing the NANC nature of this response. The NANC relaxation was abolished by L-nitroarginine (100 microM) and this effect was partly reversible by L arginine (200 microM). All responses to electrical field stimulation were abolished by tetrodotoxin (0.2-2 microM). These results show for the first time a NANC inhibitory innervation in human gall bladder muscle. The probable neurotransmitter is nitric oxide.     

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.     

Influence of a selective guanylate cyclase inhibitor, and of the contraction level, on nitrergic relaxations in the gastric fundus

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus. 2. ODQ (10(-6) and 10(-5) M) concentration-dependently inhibited the short-lasting relaxations by NO (2 x 10(-6) M-10(-4) M) administered as a bolus without influencing the relaxation by 3 x 10(-8) M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10(-6) and 10(-5) M ODQ. 3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5-16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10(-6) and 10(-5) M ODQ, and by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M). 4. ODQ (10(-6) M) and L-NAME (3 x 10(-4) M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz). 5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F2alpha (PGF2alpha), the inhibitory effect of 3 x 10(-4) M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3 x 10(-7) M PGF2alpha. 6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.     

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.     

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.     

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.     

Transmural care. A new approach in the care for terminal cancer patients: its effects on re-hospitalization and quality of life

The pharmacological properties of non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission were investigated in the rabbit choledocho-duodenal junction (CDJ), using the microelectrode and tension recording methods. L-NAME (10(-4) M) and apamin (5 X 10 (-6) M) suppressed NANC relaxation evoked by electrical field stimulation (EFS) in the presence of atropine and guanethidine (each 10(-6) M) to a similar extent (to about 40% of the initial control). However, combined application of L-NAME (10(-4) M) and apamin (5 X 10(-6) M) did not abolish it. EFS also evoked biphasic inhibitory junction potentials (IJPs) consisting of initial fast and slow sustained components in the presence of atropine and guanethidine (each 10(-6) M). Apamin (5 X 10(-8)-5 X 10(-6) M) dose-dependently suppressed the initial fast component by about 70%. In contrast, L-NAME (10(-4) M) did not affect either the amplitude of IJP or the resting membrane potential. PACAP-38 (> 10(-8) M) dose-dependently hyperpolarized the smooth muscle membrane of rabbit CDJ followed by a slow repolarization to the original level. After pretreatment with apamin (5 X 10(-7) M), PACAP-38 (10(-6) M) failed to evoke membrane hyperpolarization. During repolarization in the continued presence of PACAP-38, the amplitude of initial fast component of IJP was reduced to about 40-60% of control value, while that of the slow one was unaffected. A similar suppression of initial fast component of IJP (about 40% of the control value) also occurred after application of PACAP (6-38), a PACAP antagonist, or prolonged treatment with monoclonal antibodies to PACAP-27 or PACAP-38. Furthermore, the substantial part of residual fast IJP in the presence of PACAP (6-38) was suppressed by desensitization to alpha,beta-methylene ATP (10(-3) M). These results indicate that in rabbit CDJ NANC relaxation consists mainly of apamin- and L-NAME-sensitive components, which occur in a membrane potential dependent (through membrane hyperpolarization) and independent fashion, respectively. It has further been suggested that PACAP, together with a smaller contribution of ATP, may be involved as the principal apamin-sensitive transmitter in NANC relaxation of this muscle.     

Flowering-time genes modulate the response to LEAFY activity

Angiotensin 1-7 (Ang 1-7) has been reported to induce relaxation which is partially blocked by a kinin receptor antagonist. We investigated the relationship between kinins and angiotensin peptides with use of preconstricted isolated pig coronary arteries. Ang 1-7 alone (up to 10(-5) M) had no relaxant effect. Bradykinin (BK) (10(-10)-10(-7) M) induced transient relaxation, returning to basal tone, although BK remained in the bath. In these BK-stimulated rings, Ang 1-7 but not BK (both 5 x 10(-6) M) again relaxed the rings by approximately 50%. This relaxation was blocked by a BK B2 antagonist, a kininase, and a nitric oxide synthase inhibitor. Ang 1-7 inhibited purified angiotensin-converting enzyme (ACE) by 30 +/- 3.5% (n = 4) at 10(-6) M. However, in BK-pretreated rings, the ACE inhibitor ramiprilat did not induce relaxation, nor did it affect the relaxant response to Ang 1-7, which suggests that the effect of Ang 1-7 was not caused by ACE inhibition. Ang 1-7-induced vasodilation was reduced by 69.9 +/- 6.2% by an AT2 receptor blocker, PD-123319, and 29.3 +/- 7.3% by an AT1 antagonist, losartan. Neither the nonselective AT1/AT2 receptor antagonist sarthran nor saralasin inhibited the response to Ang 1-7. Ang II did not elicit relaxation either alone or in the presence of losartan, which suggests that activation of AT2 receptors does not cause relaxation. Thus, in the presence of bradykinin, Ang 1-7 relaxes pig coronary arteries via a PD-123319-sensitive mechanism involving nitric oxide, kinins and the BK B2 receptor. The kallikrein-kinin and renin-angiotensin systems may be linked through the interaction of Ang 1-7 and BK.     

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.     

Tumor necrosis factor-alpha induces emphysema-like pulmonary tissue rebuilding. Changes in type II alveolar epithelial cells

This study has investigated the relative involvement of cholinergic, adrenergic, nitric oxide and tachykininergic transmission in extrinsic neural influences on the lower oesophageal sphincter (LOS) in urethane anaesthetized ferrets. A micromanometric assembly (OD 1.75 mm) incorporating a sleeve sensor was used for high-fidelity oesophageal, LOS and gastric pressure measurement at low perfusion rates (< 0.1 ml/min). The LOS response to vagal and splanchnic nerve stimulation (0.5 ms pulse width, 10 s duration) was frequency- and voltage-dependent. LOS responses to stimulation at 20 V, 10 Hz were investigated in separate groups of animals with either L-NAME (100 mg/kg), hexamethonium (15 mg/kg), guanethidine (5 mg/kg), CP96,345 (NK-1 antagonist, 4 mg/kg), atropine (0.4 mg/kg) or propranolol (1 mg/kg). Propranolol treatment was followed by yohimbine (1 mg/kg) and prazosin (0.25 mg/kg). Vagal stimulation caused an immediate decrease in LOS pressure, followed by increase on cessation of stimulation, followed by a prolonged decrease (77 +/- 2%) for up to 5 min. L-NAME did not affect inhibition, but increased excitation 4-fold (p < 0.001). Guanethidine and CP96,345 had no major effect. Hexamethonium decreased the inhibitory (p < 0.05) and excitatory (p < 0.01) responses. Atropine reduced the excitatory response (p < 0.05). Some inhibition still remained if all treatments were combined. Splanchnic stimulation reduced LOS pressure by 70 +/- 6% for 101 +/- 17 s. L-NAME, guanethidine, hexamethonium and CP96,345 all independently significantly reduced inhibition. The combination of guanethidine and CP96,345 usually abolished splanchnic-induced inhibition. Atropine was without effect. Propranolol (1 mg/kg) changed the splanchnic-induced response from mainly inhibition to excitation (100 +/- 44% increase). LOS responses to noradrenaline (1-10 micrograms close IA) showed similar features to responses to splanchnic stimulation. We conclude that vagal stimulation evokes LOS relaxation via activation of established cholinergic and NANC mechanisms and other, unidentified mechanisms. Splanchnic stimulation activates adrenergic neurones probably via nicotinic and non-nicotinic ganglionic mechanisms, which in turn elicit beta adrenergic inhibitory effects on the LOS. Splanchnic stimulation also antidromically activates spinal afferent fibres. These may release substance P from peripheral myenteric plexus and prevertebral ganglionic endings causing activation of myenteric NANC inhibitory neurones and sympathetic neurones, respectively.     

Neurogenic contraction and relaxation of human penile deep dorsal vein

1. The aim of the present study was to characterize neurogenic and pharmacological responses of human penile deep dorsal vein and to determine whether the responses are mediated by nitric oxide from neural or endothelial origin. 2. Ring segments of human penile deep dorsal vein were obtained from 22 multiorgan donors during procurement of organs for transplantation. The rings were suspended in organ bath chambers for isometric recording of tension. We then studied the contractile and relaxant responses to electrical field stimulation and to vasoactive agents. 3. Electrical field stimulation (0.5-2 Hz) and noradrenaline (3 x 10(-10)-3 x 10(-5) M) caused frequency- and concentration-dependent contractions that were of greater magnitude in veins denuded of endothelium. The inhibitor of nitric oxide synthesis NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, l0(-4) M) increased the adrenergic responses only in rings with endothelium. 4. In preparations contracted with noradrenaline in the presence of guanethidine (10(-6) M) and atropine (10(-6) M), electrical stimulation induced frequency-dependent relaxations. This neurogenic relaxation was prevented by L-NAME, methylene blue (3 x 10(-5) M) and tetrodotoxin (10(-6) M), but was unaffected by removal of endothelium. 5. Acetylcholine (10(-8)-3 x 10(-5) M) and substance P (3 x 10(-11) -3 x 10(-7) M) induced endothelium-dependent relaxations. In contrast, sodium nitroprusside (10(-9)-3 x 10(-5) M) and papaverine (10(-8) 3 x 10(-5) M) caused endothelium-independent relaxations. 6. The results provide functional evidence that the human penile deep dorsal vein is an active component of the penile vascular resistance through the release of nitric oxide from both neural and endothelial origin. Dysfunction in any of these sources of nitric oxide should be considered in some forms of impotence.     

Effect of omega-agatoxin-IVA on autonomic neurotransmission

omega-Agatoxin-IVA, a peptide from the venom of the funnel-web spider Agelenopsis aperta and a P type Ca2+ channel inhibitor, was examined for effects on responses to nerve stimulation in isolated autonomic neuroeffector preparations from the rabbit, guinea-pig and rat. Ca(2+)-dependent, tetrodotoxin sensitive, noradrenergic excitatory responses of rabbit pulmonary artery, rat vas deferens, and anococcygeus muscles, and cholinergic guinea-pig myenteric plexus preparations (all highly sensitive to the N type Ca2+ channel inhibitor omega-conotoxin-GVIA) were unaffected by omega-agatoxin-IVA (100 nM). Similarly, the neurogenic response of rat bladder, which has cholinergic, and non-adrenergic non-cholinergic (NANC) excitatory components, and the NANC inhibitory response of rat jejunum (atropine 0.5 microM- and guanethidine 5.0 microM-treated), which are partially sensitive and insensitive to omega-conotoxin-GVIA, respectively, were unaffected by omega-agatoxin-IVA (100 nM). Neurogenic NANC inhibitory responses of the guinea-pig taenia caecum, and rat anococcygeus muscles (atropine- and guanethidine-treated, and tone raised with prostaglandin F2 alpha), were also insensitive to omega-agatoxin-IVA. These results suggest that P type Ca2+ channels, if present, play an insignificant role in supplying the Ca2+ necessary for neurotransmitter release in the peripheral autonomic nervous system.     

M3-type muscarinic receptors predominantly mediate neurogenic quick contraction of bovine ciliary muscle

1. The present experiments were designed to investigate which subtypes of muscarinic receptors are involved in the neurogenic quick contraction of bovine ciliary muscle in connection to quick eye focal accommodation. 2. Transmural electrical stimulation (TES) produced a transient contraction, which was abolished in the presence of 3 x 10(-7) M tetrodotoxin and 10(-6) M atropine, but greatly augmented by 3 x 10(-7) M physostigmine. 3. The exogenously applied acetylcholine (ACh: 10(-9) to 3 x 10(-6) M) produced a concentration-dependent contraction, which was competitively antagonized by 10(-6) M atropine and augmented by 3 x 10(-7) M physostigmine, but unaffected by 3 x 10(-7) M tetrodotoxin. 4. The magnitude and time to peak of the maximal contraction produced by TES were significantly greater (1267.5 +/- 86.0 mg, P < 0.005) and shorter (9.0 +/- 0.2 sec, P < 0.005) than corresponding values (97.0 +/- 9.9 mg and 20.3 +/- 2.1 sec, respectively) of the phasic contraction caused by exogenously applied 10(-5) M ACh, at which concentration the agonist caused the maximal contraction. The velocity (140.6 +/- 7.8 mg/sec) of the transient contraction caused by TES was approximately 28-fold greater than that of the phasic contraction caused by ACh (5.1 +/- 0.9 mg/sec). 5. The contractions produced by TES were greatly attenuated by 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) as an M3 antagonist and slightly by pirenzepine as an M1 antagonist (20.2 +/- 7.9% inhibition at the highest concentration), but not by methoctramine (MET) as an M2 antagonist. The IC50 value (-log M) for 4-DAMP was determined to be 7.17 +/- 0.14. 6. Scatchard plot analysis of [3H]-quinuclidinylbenzilate (QNB) binding revealed that the binding sites constituted a single population with a Kd of 31.2 +/- 0.8 pM and a Bmax of 895.5 +/- 93.2 fmol/mg protein. The activity in inhibiting [3H]-QNB binding was most potent with 4-DAMP (-log Ki = 7.98 +/- 0.02), but less potent with pirenzepine (-log Ki = 6.43 +/- 0.04) and MET (-log Ki = 7.32 +/- 0.16). 4-DAMP was approximately 35- and 5-fold more potent than pirenzepine and MET in terms of -log Ki values, respectively, suggesting the predominant localization of M3 receptor subtypes in the bovine ciliary muscle membrane. 7. These results suggest that TES produces a neurogenic quick contraction of the bovine ciliary muscle, which would be mediated mainly by ACh released from the intramural nerve terminals and subsequent excitation of M3 receptor subtypes localized on the ciliary muscle cells, and that neurogenic quick contraction of the ciliary muscle is possibly involved in part in eye focal accommodation.     

Potentiating action of MKC-242, a selective 5-HT1A receptor agonist, on the photic entrainment of the circadian activity rhythm in hamsters

1. Purinergic and cholinergic components of parasympathetic neurotransmission and contractile responses to exogenous alpha,beta-methylene ATP, acetylcholine, substance K, substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and capsaicin have been investigated in the urinary bladder of hibernating hamsters (4 weeks), cold exposed (4 weeks) and age-matched controls. 2. Electrical field stimulation (EFS) evoked increased frequency-dependent contractions in the detrusor strips from hibernating hamsters compared with those obtained from cold-exposed and age-matched animals. Tetrodotoxin (10(-6) M) completely blocked the frequency-dependent contractions in all groups. 3. The purinergic component of the parasympathetic neurotransmission was not affected in hibernating and cold-exposed animals while the cholinergic component was increased with respect to age-matched animals. The neurogenic response to EFS, still present after incubation with atropine (10(-6) M) and suramin (10(-4) M), was attenuated by indomethacin (10(-6) M) and blocked by tetrodotoxin (10(-6) M). 4. Exogenous administration of alpha,beta-methylene ATP elicited a significantly reduced contraction in strips from hibernating and cold-exposed hamsters relative to age-matched animals. The contractile response to exogenous acetylcholine was greater in the detrusors from hibernating hamsters than in cold-exposed and age-matched animals. Substance K elicited reduced contractions in preparations from hibernating animals compared with cold-exposed and control animals. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P and capsaicin did not elicit any relaxant or contractile response either at resting tone or in carbachol (5 x 10(-7) M)-precontracted tissues. 5. In summary, our findings indicate that 4 weeks of hibernation can significantly increase neurogenic responses in the hamster urinary bladder. This appears to be due to an increase in postjunctional responses to acetylcholine. In contrast, there was a decrease of the postjunctional responses to the parasympathetic cotransmitter ATP and also to the sensory-motor neurotransmitter substance K.     

Long duration pressor responses following activation of subfornical organ neurons in rats are the result of increased circulating vasopressin

Electrical stimulation in the subfornical organ (SFO) of male Sprague-Dawley rats resulted in biphasic increases in blood pressure (BP) without a change in heart rate. The initial short duration (0-10 s) increase in BP lasted throughout the 10 s stimulation period (area under the curve (AUC) = 104.3+/-15.26 mmHg/s, (mean+/-SEM) P < 0.001). Upon termination of the electrical stimulus, the BP remained elevated for approximately 55 s (long duration response, AUC = 327.5+/-48.22 mmHg/s, P < 0.001). This long duration BP response was determined to be the result of an increase in circulating vasopressin (VP) as administration of a V1 receptor antagonist abolished this response (AUC = -210.7 +/- 42.38 mmHg/s, P < 0.01). The results of the present study demonstrate that the long duration component of the biphasic increase in BP observed on response to electrical stimulation of the SFO is the result of increased concentrations of circulating VP.     

Acetylcholine-induced endothelium-independent relaxations in monkey isolated superior and inferior caval veins

1. We examined the effects of acetylcholine (ACh), isoprenaline (Isop) and Ca-ionophore, A23187 on monkey isolated superior (SCV) and inferior caval veins (ICV) with and without intact endothelium, which had been partially contracted by 2 x 10(-6)-5 x 10(-6) M prostaglandin F2 alpha (PGF2 alpha). 2. Low concentrations of ACh (10(-10)-10(-9) M) produced a dose-dependent relaxation in the precontracted venous segments with endothelium. ACh at concentrations more than 10(-7) M elicited a transient contraction followed by a relaxation in these segments. 3. An addition of 5 x 10(-7) M A 23187 induced about 60% of maximum relaxation produced by 10(-5) M sodium nitroprusside (SNP) in each venous segment with endothelium. 4. Isop (10(-10)-10(-5) M) caused a dose-related relaxation in the precontracted caval veins with intact endothelium. 5. Removal of endothelium caused no significant effect on the ACh-induced dual responses but a significant inhibition of the A23187-induced relaxation. 6. Pretreatment with atropine antagonized competitively the ACh-induced relaxations in the endothelium-intact and endothelium-denuded caval veins. The Schild plot analysis showed that the pA2 values of the segments with and without endothelium were 9.72 +/- 0.14 (n = 5) and 10.01 +/- 0.23 (n = 6) in the ICV; and 9.95 +/- 0.20 (n = 5) and 9.70 +/- 0.10 (n = 5) in the SCV, respectively. 7. Pretreatment with 5 x 10-5M aspirin, 3 x 10-5M N0-nitro-L-arginine methylester, 1 mM tetraethylammonium,or 3 x 10-6 M glibenclamide caused no significant effect on the basal tone, ACh induced transient contraction, and ACh;.induced relaxation in the precontracted venous segments with and without endothelium.8. Pretreatment with 10-5 M methylene blue produced a significant reduction of the ACh- and SNP induced relaxations in the precontracted venous segments with and without endothelium. The pretreatment with the same concentration of methylene blue, however, caused no significant effect on the Isop-induced relaxation in venous segments with endothelium.9. The results suggest that ACh acts directly on the venous smooth muscle cells via a high-affinity muscarinic receptor subtype to accumulate cellular cyclic GMP producing endothelium-independent relaxation in the monkey caval veins.     

Force and intracellular Ca2+ during NANC-mediated relaxation of rat anococcygeus muscle and the effects of cyclopiazonic acid

1. Simultaneous recordings of tension and [Ca2+]i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca2+]i. 3. The nitric oxide synthase inhibitor, NG-nitro-L-Arginine (NOLA, 100 mumol/L) completely abolished the decreases in the [Ca2+]i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca2+]i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 mumol/L), partially inhibited the force and [Ca2+]i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, cyclopiazonic acid, (10 mumol/L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca2+ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca2+]i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca2+ sequestration into the sarcoplasmic reticulum. However, other Ca2+ lowering mechanisms and possible Ca2+ independent mechanisms may also contribute to the NANC relaxation response.     

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.