Investigation of the role of adrenergic and non-nitrergic, non-adrenergic neurotransmission in the sheep isolated internal anal sphincter.

Nitric oxide is widely established as an important neurotransmitter in the control of anal sphincter tone; although, a number of other transmitters have also been tentatively implicated. Whilst alpha-adrenoceptor antagonists reduce anal sphincter pressure in man, the role of noradrenaline as a possible transmitter is poorly characterised. We have investigated the contribution of these transmitters to neurogenic relaxations, and evaluated the possible role of a non-nitrergic, non-adrenergic transmitter. The magnitude and duration of neurogenic responses were examined by measuring responses to electrical field stimulation (EFS) in segments of sheep internal anal sphincter following the development of spontaneous myogenic tone. Neurogenic relaxations induced by EFS were significantly reduced in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) suggesting major involvement of nitric oxide as a neurotransmitter. The duration of neurogenic relaxations was inversely related to the frequency of EFS, with contractile responses often manifest at higher frequencies. The duration of relaxations at high frequencies of EFS was increased by bretylium (adrenergic neurone blocker) and prazosin (alpha(1)-adrenoceptor antagonist). At higher frequencies of EFS, 60% of preparations also produced a residual non-nitrergic, non-adrenergic, apamin-sensitive relaxation which was unaffected by vasoactive intestinal polypeptide (VIP) and inhibitors of purinergic responses [suramin, pyridoxal-phosphate-6-azophenyl 2',4' disulfonic acid (PPADS) and alpha,beta-methylene adenosine triphosphate (ATP)]. However, MRS2179 (P2Y(1) receptor antagonist) showed a modest inhibitory effect. We conclude that endogenous noradrenaline acts via postjunctional alpha(1)-adrenoceptors to antagonize neurogenic relaxations that are largely mediated by nitric oxide. Our results indicate the involvement of a non-nitrergic, non-adrenergic, apamin-sensitive transmitter which is inhibited by MRS2179, suggesting a possible role for purines.

Vitamin D insufficiency and the blunted PTH response in established osteoporosis: the role of magnesium deficiency.

INTRODUCTION: Vitamin D insufficiency is common, however within individuals, not all manifest the biochemical effects of PTH excess. This further extends to patients with established osteoporosis. The mechanism underlying the blunted PTH response is unclear but may be related to magnesium (Mg) deficiency. The aims of this study were to compare in patients with established osteoporosis and differing degrees of vitamin D and PTH status : (1) the presence of Mg deficiency using the standard Mg loading test (2) evaluate the effects of Mg loading on the calcium-PTH endocrine axis (3) determine the effects of oral, short term Mg supplementation on the calcium-PTH endocrine axis and bone turnover. METHODS: 30 patients (10 women in 3 groups) were evaluated prospectively measuring calcium, PTH, Mg retention (Mg loading test), dietary nutrient intake (calcium, vitamin D, Mg) and bone turnover markers (serum CTX & P1CP). Multivariate analysis controlling for potential confounding baseline variable was undertaken for the measured outcomes. RESULTS: All subjects, within the low vitamin D and low PTH group following the magnesium loading test had evidence of Mg depletion [mean(SD) retention 70.3%(12.5)] and showed an increase in calcium 0.06(0.01) mmol/l [95% CI 0.03, 0.09, p=0.007], together with a rise in PTH 13.3 ng/l (4.5) [95% CI 3.2, 23.4, p=0.016] compared to baseline. Following oral supplementation bone turnover increased: CTX 0.16 (0.06) mcg/l [95%CI 0.01, 0.32 p=0.047]; P1CP 13.1 (5.7) mcg/l [95% CI 0.29, 26.6 p=0.049]. In subjects with a low vitamin D and raised PTH mean retention was 55.9%(14.8) and in the vitamin replete group 36.1%(14.4), with little change in both acute markers of calcium homeostasis and bone turnover markers following both the loading test and oral supplementation. CONCLUSIONS: This study confirms that in patients with established osteoporosis, there is also a distinct group with a low vitamin D and a blunted PTH level and that Mg deficiency (as measured by the Mg loading test) is an important contributing factor.

Comparison of the effects of nitric oxide donors and calcium channel blockers on the intrinsic myogenic tone of sheep isolated internal anal sphincter.

BACKGROUND: Chronic anal fissure is associated with considerable pain and anal hypertonia. Numerous clinical studies attest to the effectiveness of individual nitro-containing drugs and organic calcium channel blockers in this condition but there are few comparative studies. METHODS: Isolated segments of sheep internal anal sphincter were prepared for isometric tension recording. The effect of various drugs on myogenic tone was examined in the absence or presence of sodium orthovanadate (SOV), an agent used to mimic anal hypertonia by increasing myogenic tone. RESULTS: All the drugs tested produced concentration-dependent inhibition of myogenic tone, with the maximum effect ranging from 66.4 per cent (verapamil) to 100 per cent (sodium nitroprusside). Sodium nitroprusside and diltiazem were the most potent, followed by glyceryl trinitrate (GTN), nifedipine and verapamil, which had similar potency, and finally nicorandil. The potency of GTN and diltiazem was reduced threefold in the presence of 1 mmol/l SOV. The combined effect of GTN and diltiazem was greater than the effect of either agent alone, even in the presence of 3 mmol/l SOV. CONCLUSION: Nitro-containing drugs and organic calcium channel blockers are potent inhibitors of anal sphincter myogenic tone that may be used in combination to treat chronic anal fissure.

The relationship between vitamin D and parathyroid hormone: calcium homeostasis, bone turnover, and bone mineral density in postmenopausal women with established osteoporosis.

It is evident from several studies that not all patients with hypovitaminosis D develop secondary hyperparathyroidism. What this means for bone biochemistry and bone mineral density (BMD) remains unclear. The aim of this study was to investigate the effects of hypovitaminosis D (defined as a 25OHD < or = 30 nmol/l) and patients with a blunted PTH response (defined arbitrarily as a PTH within the standard laboratory reference range in the presence of a 25OHD < or = 30 nmol/l) in comparison to patients with hypovitaminosis D and secondary hyperparathyroidism (defined arbitrarily as a PTH above the standard laboratory reference range in the presence of a 25OHD < or = 30 nmol/l) and vitamin D-replete subjects (25OHD > 30 nmol/l). Four hundred twenty-one postmenopausal women (mean age: 71.2 years) with established vertebral osteoporosis were evaluated by assessing mean serum calcium, 25OHD, 1,25(OH)2D, bone turnover markers, and BMD. The prevalence of hypovitaminosis D was 39%. Secondary hyperparathyroidism was found in only one-third of these patients who maintained calcium homeostasis at the expense of increased bone turnover relative to the vitamin D-replete subjects (bone ALP mean difference: 43.9 IU/l [95% CI: 24.8, 59.1], osteocalcin: 1.3 ng/ml [95% CI: 1.1, 2.5], free deoxypyridinoline mean difference: 2.6 nmol/nmol creatinine [95% CI: 2.5, 4.8]) and bone loss (total hip BMD mean difference: 0.11 g/cm2 [95% CI: 0.09, 0.12]). Patients with hypovitaminosis D and a blunted PTH response were characterized by a lower serum calcium (mean difference: 0.07 mmol/l [95% CI: 0.08, 0.2]), a reduction in bone turnover (bone ALP mean difference: 42.4 IU/l [95% CI: 27.8, 61.9], osteocalcin: 1.6 ng/ml [95% CI: 0.3, 3.1], free-deoxypyridinoline mean difference: 3.0 nmol/nmol creatinine [95% CI: 1.9, 5.9]), but protection in bone density (total hip BMD mean difference: 0.10 g/cm2, [95% CI: 0.08, 0.11]) as compared to those with hypovitaminosis D and secondary hyperparathyroidism. This study identifies a distinct group of patients with hypovitaminosis D and a blunted PTH response who show a disruption in calcium homeostasis but protected against PTH-mediated bone loss. This has clinical implications with respect to disease definition and may be important in deciding the optimal replacement therapy in patients with hypovitaminosis D but a blunted PTH response.

Pharmacological examination of contractile responses of the guinea-pig isolated ileum produced by mu-opioid receptor antagonists in the presence of, and following exposure to, morphine.

We have assessed the potential of several mu-opioid receptor antagonists to elicit a response in the guinea-pig isolated ileum in the presence of, and following overnight exposure to, morphine. Naloxone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), (-)-5, 9alpha-diethyl-2-(3-furyl-methyl)-2'-hydroxy-6,7-benzomorphan (MR2266), but not D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), produced a transient inhibition of electrically-evoked contractions of the guinea-pig ileum. The effect of 1 microM CTOP, but not that to MR2266, was inhibited by 1 microM somatostatin. Naloxone (0.3 microM), CTOP (3 microM), CTAP (3 microM) and MR2266 (0.3 microM) antagonized the inhibitory effect of morphine on electrically-evoked contractions of the guinea-pig to a similar degree and, following 60 min exposure to morphine, produced non-sustained contractions. The response to 3 microM CTOP was significantly smaller than that to 3 microM CTAP. None of the antagonists produced a response in the absence of morphine. Following overnight exposure of the ileum to 0.3 microM morphine (4 degrees C), and repeated washing to remove the agonist, all four antagonists elicited non-sustained contractions. However, the responses to 3 microM CTOP and 0.3 microM MR2266 were significantly smaller than those elicited by 0.3 microM naloxone and 3 microM CTAP. Somatostatin (1 microM) significantly reduced naloxone-induced contractions, but not those to CTAP. While all four mu-opioid antagonists elicited contractions in the presence of, and following prolonged exposure to, morphine, differences between them were noted which may be a consequence of non-opioid actions.

Pharmacological characterization of neurogenic responses of the sheep isolated internal anal sphincter.

The aim of the study was to establish the nature of the neurogenic responses of the sheep isolated anal sphincter. Isolated strips of sheep internal anal sphincter develop intrinsic contractile tone following the application of stretch tension. On transmural stimulation (1 - 20 Hz, 10 V pulse strength, 0.5 ms pulse width, 1 s every 180 s) transient relaxations were observed. The amplitude of the relaxations were frequency-dependent reaching a maximal response at 10 - 20 Hz and were inhibited by tetrodotoxin (0.3 microM). Neither atropine (0.3 microM) nor phentolamine (1 microM) affected control responses. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and the selective inhibitor of soluble guanylyl cyclase ODQ, (1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one) (1 microM) completely inhibited the neurogenic relaxations and uncovered contractions that were abolished by 1 microM phentolamine and 0.1 microM prazosin. The effect of L-NAME, but not that of ODQ, was partially reversed by the addition of L-arginine (1 mM). Sodium nitroprusside (10 nM - 10 microM) caused concentration-dependent inhibition of myogenic tone and this effect was significantly reduced by ODQ. Calcium-free Krebs-Henseleit solution also reduced myogenic tone by 85%. Transmural electrical stimulation of the sheep isolated internal anal sphincter causes a transient relaxation of myogenic tone that appears to involve nitric oxide from non-adrenergic, non-cholinergic nerves and, to a lesser degree, noradrenaline from sympathetic nerves. The characteristics of the preparation compares well with that of human tissue and may prove to be a suitable animal based model for further studies.

Pharmacological comparison of the effect of ibogaine and 18-methoxycoronaridine on isolated smooth muscle from the rat and guinea-pig.

Ibogaine and 18-methoxycoronaridine are naturally occurring alkaloids reported to possess antiaddictive properties in several models of drug dependence. We have examined their effect at mu-opioid receptors regulating neurogenic contractions of several smooth muscle preparations and also against spontaneous contractions of the rat isolated portal vein. Ibogaine (pIC(50) 5.28) and 18-methoxycoronaridine (pIC(50) 5.05) caused a concentration-dependent inhibition of cholinergic contractions of the guinea-pig ileum which was not affected by the opioid receptor antagonist naloxone (1 microM). In the rat isolated vas deferens ibogaine and 18-methoxycoronaridine caused a concentration-dependent enhancement of purinergic contractions. Both agents (30 microM) caused a 3 - 5 fold rightward displacement of DAMGO-induced inhibition of purinergic contractions, but similar effects were observed for ibogaine against alpha(2)-adrenoceptor-mediated inhibition of neurogenic responses. In the guinea-pig isolated bladder both ibogaine (10 microM) and 18-methoxycoronaridine (10 microM) caused a 2 fold increase in the purinergic component of neurogenic contractions without significantly altering cholinergic contractions or responses to exogenous ATP. In contrast, ibogaine (1 - 30 microM), but not 18-methoxycoronaridine, caused a concentration-dependent enhancement of spontaneous contractions of the rat isolated portal vein. In summary, while ibogaine and 18-methoxycoronaridine modulated electrically-evoked contractions in the three preparations examined, we have no evidence for a selective interaction with pre-junctional mu-opioid receptors. The pronounced enhancement of purinergic contractions produced by both agents is a novel finding and worthy of further investigation.

Electrophysiological effects of opioid receptor activation on Syrian hamster suprachiasmatic nucleus neurones in vitro.

Entrainment of the dominant circadian pacemaker localised to the hypothalamic suprachiasmatic nuclei (SCN) is mediated partially via the indirect retino-geniculo-hypothalamic projection to the SCN, which is presumed to utilise enkephalin and other neurotransmitters, to modulate circadian rhythmicity. In the present study, we have investigated electrophysiologically the currently unknown functional effects of enkephalin, and another opioid receptor agonist morphine, on hamster SCN neuronal activity in vitro. Basal or N-methyl-D-aspartate-evoked firing rates of SCN neurones were generally unresponsive (86%) to the opioid receptor agonists leucine-enkephalin, methionine-enkephalin, or morphine. Washout of the enkephalins or morphine resulted in a rebound excitatory response ("withdrawal activation") in 39% of neurones tested. Withdrawal activation was also elicited by administration of the opioid receptor antagonist naloxone, following pre-exposure to morphine, in 59% of neurones tested. These withdrawal responses were blocked or attenuated by the alpha2-adrenoceptor agonist clonidine, results which suggest a functional interaction exists between opioid receptors and alpha2-adrenoceptors in the SCN. Our observations show that opioid receptor agonists are largely devoid of actions on normal hamster SCN circadian pacemaker activity, while the occurrence of withdrawal responses may have implications on circadian function during withdrawal from opiate abuse.

Selective potentiation by ouabain of naloxone-induced withdrawal contractions of isolated guinea-pig ileum following acute exposure to morphine.

1. Ouabain, an inhibitor of Na+/K+ ATPase induces the release of acetylcholine from central and myenteric cholinergic neurones principally due to partial depolarization of the cell membrane. The effect of ouabain has been examined on neurogenic contractions in the guinea-pig ileum arising from either electrical field stimulation or from naloxone in morphine-exposed preparations. 2. Guinea-pig isolated ileum preparations were stimulated transmurally (0.1 Hz, 0.3 ms, 200 mA) to elicit contractions of the myenteric plexus-longitudinal smooth muscle. 3. Incubation with morphine (0.3 microM, 60 min) was followed by naloxone (1 microM) which produced withdrawal contractions in 16/26 preparations (median of 10.7 [2.2-40.0]% of a maximal contracture to KCl (60 mM)). 4. In parallel experiments, ouabain (1 microM) was added to the tissue before exposure to morphine (0.3 microM, 60 min). Naloxone (1 microM) subsequently displayed a withdrawal contraction in all 26/26 tissues (57.9 [30.5-151.7]% of a maximal contracture to KCl (60 mM). 5. Ouabain neither affected the concentration-dependent contractions of guinea-pig ileum produced by carbachol nor the inhibition of electrically-evoked contraction produced by morphine (0.3 microM). 6. The muscarinic antagonist atropine (0.1 microM) antagonized control naloxone withdrawal responses. The atropine resistant component, evident in ouabain-treated tissues, was blocked by SR140333((S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenyla cetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2. 2]-octane, chloride), a substance P antagonist. 7. Clonidine (alpha2-adrenoceptor agonist) inhibited electrically-evoked contractions. Exposure to the alpha2-adrenoceptor antagonist RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline), resulted in a contracture which was not significantly enhanced by ouabain (1 microM). 8. Ouabain selectively potentiates the naloxone-induced withdrawal contraction following acute exposure to morphine the major components of which are mediated by both acetylcholine and substance P.

Effects of 8-OHDPAT and 5-HT1A antagonists WAY100135 and WAY100635, on guinea-pig behaviour and dorsal raphe 5-HT neurone firing.

1. The effects of 5-HT1A antagonists on guinea-pig behaviour and dorsal raphe neuronal activity were investigated. 2. WAY100135 (10 mg kg-1, s.c.) and WAY100635 (1 mg kg-1, s.c.) significantly reduced the behaviours induced by 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT) (1 mg kg-1, s.c.) indicative of post-synaptic 5-HT1A receptor antagonism. WAY100635 (10 mg kg-1, s.c.) alone induced ear twitches, which were antagonized by ketanserin (1 mg kg-1, s.c.), but no other overt behaviours. 3. WAY100635 (0.125 mg kg-1, i.v.) produced a right-ward shift in the dose-related inhibition of neuronal firing by 8-OHDPAT (5-100 micrograms kg-1, i.v.) but did not affect the maximum inhibition induced by 8-OHDPAT indicating competitive antagonism between 8-OHDPAT and WAY100635 at the 5-HT1A somato-dendritic autoreceptor in the dorsal raphe nucleus of the guinea-pig. WAY100635 also produced a dose-related increase in the basal firing of 5-HT neurones in the dorsal raphe nucleus and restored the firing of dorsal raphe neurones previously inhibited by 8-OHDPAT (10 micrograms kg-1, i.v.). 4. The results indicate that WAY100635 is a competitive 5-HT1A antagonist in the guinea-pig. Furthermore WAY100635 can increase 5-HT neuronal firing, suggesting that it blocks a 5-HT1A receptor-mediated inhibitory tone acting on guinea-pig 5-HT neurones resulting in increased 5-HT release and 5-HT2 receptor-mediated behaviours.

Effect of the putative 5-HT1A antagonists WAY100135 and SDZ 216-525 on 5-HT neuronal firing in the guinea-pig dorsal raphe nucleus.

The selective 5-hydroxytryptamine1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT, 0.5-35 micrograms kg-1 i.v.) produces a dose related reversible inhibition (ED50 = 6.5 micrograms kg-1 i.v.) of the firing of serotonergic neurones in the dorsal raphe nucleus of the guinea-pig. Administration of N-tert-butyl-3- (4-(2-methoxyphenyl)piperazine-1-yl)-2phenylpropanamide dihydrochloride (WAY100135, 0.5 mg kg-1 i.v.), a specific 5-HT1A antagonist, antagonized the 8-OHDPAT induced inhibition of neuronal firing whilst methyl 4-(4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1- piperazinyl) 1 H-indole-2-carboxylate (SDZ 216-525, 0.1-0.5 mg kg-1 i.v.) (also a putative 5-HT1A antagonist) reduced the basal firing of 5-HT neurones and furthermore failed to antagonize the inhibition produced by 8-OHDPAT. These results indicate that WAY 100135 is a silent and selective 5-HT1A antagonist whereas SDZ 216-525 demonstrates a partial agonist activity at the somatodendritic 5-HT1A autoreceptor in the guinea-pig DRN.