Functional innervation of the biliary sphincter of the guinea-pig revealed by anti-autonomic drugs

1. The roles of excitatory and inhibitory intrinsic motor nerves on contractions reflexly evoked by wall distension were investigated in the isolated sphincter of Oddi of the guinea-pig (SO-GP). 2. Distension of the terminal bile duct for 30-60 s time periods increased the frequency of contractions from about 2 to 12 min(-1) (n = 16). 3. Hexamethonium (HEX; 300 microM) largely prevented the distension-evoked increase in contraction frequency (4.5 min(-1), n = 8) as did atropine (ATR; 1 microM) (0.8 min(-1), n = 6), while tetrodotoxin (TTX; 1 microM) blocked the contractions triggered during distension. 4. L-nitroarginine (L-NA; 100 microM) significantly increased the frequency of contractions during and in the absence distension while apamin (APAM; 0.5 microM) significantly increased their frequency and doubled their mean amplitude during distension. 5. These results suggest that distension activates excitatory cholinergic motor nerves to increase the frequency of contractions in the SO-GP. These actions are modulated by the concomitant activation of intrinsic nitrergic and non-nitrergic inhibitory motor nerves.     

Functional antagonism between nitric oxide and ATP in the motor responses of guinea‐pig ileum

1 The interaction of nitric oxide and ATP in the non‐adrenergic, non‐cholinergic (NANC) motor responses and the presence of NADPH‐diaphorase and quinacrine‐positive myenteric neurones were studied on guinea‐pig ileum using mechanographic, histochemical and quinacrine‐fluorescence techniques. In the presence of phentolamine, propranolol and atropine, the non‐precontracted longitudinally oriented organ bath preparations responded to sodium nitroprusside (1–100 μm ) or ATP (5–50 μm ) with tetrodotoxin (0.1 μm )‐resistant relaxation or contraction, respectively. The effects of ATP were suramin (50 μm )‐ and apamin (5 μm )‐sensitive.
2 The NANC motor responses elicited by electrical stimulation (0.8 ms, 1–20 Hz, 20 s) consisted of tetrodotoxin‐sensitive relaxation phase followed by a phase of twitch‐like and tonic contractions.
3 NG‐nitro‐L‐arginine (L‐NNA, 0.1–0.5 mm ) inhibited or abolished the relaxation phase. L‐arginine (0.5 mm ), but not D‐arginine (0.5 mm ), restored the relaxation phase in L‐NNA‐pretreated preparations. The relaxation phase increased after ATP‐induced desensitization of purinoceptors and in the presence of suramin (50 μm ) but was abolished by apamin (5 μm ).
4 The phase of contractions was enhanced by L‐NNA (0.1–0.5 mm ) and restored by L‐arginine (0.5 mm ). The twitch‐like and tonic contractions were decreased during ATP‐induced purinoceptor desensitization and in the presence of suramin (50 μm ). Apamin (5 μm ) inhibited the tonic contractions.
5 The desensitization of purinoceptors by ATP did not change the L‐NNA‐induced inhibition of the relaxation phase but decreased the L‐NNA‐increased phase of contractions. L‐NNA reduced the relaxation phase and increased the phase of contractions during purinoceptor desensitization.
6 We conclude that in the longitudinal muscle layer of the guinea‐pig ileum, nitric oxide mediates the relaxation phase while ATP contributes via smooth muscle P2 purinoceptors to the phase of contractions suggesting a postjunctional functional antagonism between nitric oxide and ATP. The presence of NADPH‐diaphorase‐ and quinacrine‐positive neuronal cells and processes running to the muscle cells confirms a physiological role of nitric oxide and ATP in the ileal neurotransmission.     

Effects of eicosanoids and nitric oxide on the noradrenaline‐induced contractions in the rat mesenteric bed

1 The effects of the inhibition of the metabolism of arachidonic acid (AA) on the constrictor responses to noradrenaline (NA) were studied in the rat perfused mesenteric bed. The inhibitor of all the pathways of AA metabolism, 10 μm eicosatetraynoic acid (ETYA), reduced the constrictor responses to all the concentrations of NA assayed.
2 The constrictor responses to NA were also reduced by the cyclooxygenase (COX) inhibitor, indomethacin (10 μm) , as well as by the lipoxygenase inhibitor, nordihidroguaiaretic acid (1 μm; NDGA), whereas they were unmodified by the cytochrome P450 monooxigenase inhibitors, clotrimazole (10 μm ), metyrapone (10 μm ) and proadifen (10 μm ).
3 The reduction in NA contractility induced by indomethacin was reverted with a decreasing order of potency by the thromboxane A₂ analogue, U‐46619>prostaglandin (PG) E₂>PGF₂α. The exposure of the mesenteric bed to NA increased the production of PGF₂α, whereas it did not modify the production of the remaining AA metabolites.
4 The increase in the NA‐induced contractions caused by endothelium removal, as well as by the inhibition of nitric oxide synthase (NOs) with N^G‐nitro‐l ‐arginine methyl ester (400 μm; L‐NAME), was suppressed by indomethacin but not by NDGA. These observations suggest that the lipoxygenase‐derived metabolites are formed in the endothelium, whereas the COX‐derived metabolites are formed in the vascular smooth muscle.
5 The TP receptor antagonist, SQ29548, did not modify the NA‐induced contractions, either in the presence or in the absence of the endothelium.
6 Contractions elicited by KCl (60–100 mm ) were unmodified by the AA metabolism inhibitors, ETYA, NDGA and indomethacin.
7 In summary, these results show that metabolites of AA, through both the COX and the lipoxygenase pathways, are involved in the NA‐induced contractions in the rat mesenteric bed. The lipoxygenase metabolites are likely to be formed in the vascular endothelium, whereas the COX metabolite, which could be PGF₂α, is apparently formed within the vascular smooth muscle.     

Guanylate cyclase regulates ileal longitudinal muscle contractions induced by neurogenic nitrergic activity in the rat

1. Nitrergic neurons regulate gastrointestinal (GI) activity and their dysfunction has been associated with various GI diseases. Nitric oxide (NO) typically relaxes GI smooth muscle, but nitrergic contractions also occur. Although guanylate cyclase is well established as mediating nitrergic GI relaxation, its role in contraction remains uncertain. 2. We used electrical field stimulation (EFS; 0.3 msec pulses, three trains of 1.2 s width, 2 Hz, at 30 s intervals) to evoke biphasic contraction–relaxation responses in rat ileum strips (longitudinal muscle–myenteric plexus preparations), mediated by the endogenous nitrergic transmitter, under non‐adrenergic, non‐cholinergic (NANC) conditions (1 μmol/L atropine and 4 μmol/L guanethidine). 3. All EFS responses were abolished by tetrodotoxin (1 μmol/L). Inhibition of NO synthase with N^ω‐nitro‐l ‐arginine‐methyl‐ester (l ‐NAME; 100 and 300 μmol/L) prevented both EFS‐evoked contractions and relaxations. l ‐Arginine (3 mmol/L) reversed l ‐NAME inhibition, primarily restoring contractions and suggesting that these require lower nitrergic transmitter levels than relaxations. 4. Pretreatment of preparations with subrelaxant concentrations of sodium nitroprusside (1 μmol/L) selectively desensitized EFS‐evoked contractions without affecting relaxations, suggesting different downstream mechanisms. Nevertheless, the selective guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (3 and 10 μmol/L) inhibited both nitrergic contractions and relaxations, indicating that guanylate cyclase activation is required for both responses. 5. The results of the present study support the hypothesis that the endogenous nitrergic transmitter differentially regulates guanylate cyclase, leading to either contractions or relaxations depending on its concentrations, thus providing additional insight into the regulation of ileum contractility by nitrergic activity.     

Non‐adrenergic,non‐cholinergic,non‐purinergic contractions of the urothelium/lamina propria of the pig bladder

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Metabolic dependency of ionophore A23187‐induced contraction of ileal longitudinal smooth muscle

1 Tonic contraction in response to K+ is well known to be highly dependent on aerobic metabolism in ileal muscle. The ionophore A23187 (10?5 m ) induced an initial contraction and successive rhythmic contraction in ileal muscle, but did not induce tonic contraction. This study, therefore, was performed to investigate the metabolic dependency during contraction induced by A23187.
2 Under hypoxic conditions, A23187 (10?5 m ) induced an initial contraction accompanied by an increase in lactate release. However, it induced only small rhythmic contractions and decreases in ATP, phosphocreatinine (PCr) and glycogen contents.
3 In glucose‐free medium, A23187 (10?5 m ) induced an initial contraction and concomitant significant decrease in the ATP and glycogen contents. However, it did not induce successive rhythmic contractions. In ‘glycogen‐depleted muscle’ stimulated repeatedly with 60 m m K+ in glucose‐free medium, 60 m m K+ induced a phasic contraction without tonic contraction. A23187 induced no contraction under these conditions.
4 These results suggested that the initial contraction induced by A23187 was dependent on endogenous glycogen, while successive rhythmic contractions were dependent on aerobic metabolism after supply of external glucose to the ileal muscle.     

Effects of clofilium on cardiovascular tissues from normo‐ and hypertensive rats

1 The overall aim was to test whether clofilium has some potential as a positive inotrope for heart failure. We used Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) and studied the effects of clofilium on isolated blood vessels, left ventricular action potentials and left ventricular contractility.
2 Clofilium at ≤10^?6 m had no effect on WKY portal vein contractions and at ≤3×10?4 m had no effect on WKY or SHR quiescent mesenteric and intralobar pulmonary arteries.
3 Clofilium at 10^?7–10^?5 m prolonged the WKY left ventricular action potentials and with 10^?6 and 10^?5 m this included after‐depolarizations.
4 Clofilium at ≤3×10^?5 m augmented the peak force, prolonged the contractions and did not cause arrhythmias in the absence and presence of isoprenaline on left ventricle strips from 12‐month‐old WKY.
5 The 12‐month‐old SHR has hypertrophy of the left ventricle with reduced peak force and prolongation of relaxation. The effects of clofilium on 12‐month‐old SHR left ventricle contractility were similar to those in the age‐matched WKY. 6 In summary, clofilium has positive inotropic effects on the rat left ventricle that are maintained in hypertrophy. Clofilium does not have effects on blood vessels that would be detrimental in heart failure. Clofilium prolongs the rat left ventricle action potential and causes after‐depolarizations. The pro‐arrhythmic potential of clofilium, however, makes it unlikely that it could be used as a positive inotrope in the treatment of heart failure.     

Lack of Ca2+‐ and ATP‐dependent priming stage in caffeine‐induced exocytosis in bovine adrenal chromaffin cells: comparison with Ca2+

1 Caffeine (20–40 m m ) secreted catecholamines from β‐escin‐permeabilized bovine adrenal chromaffin cells in the presence or absence of 2 m m MgATP. The caffeine‐induced catecholamine secretion in the presence of MgATP was to the same extent as that in the absence of MgATP.
2 Ca2+ (0.1–10 μm ) induced a significantly greater secretion of catecholamines in the presence of MgATP than in the absence of MgATP.
3 ML‐9 (100 μm ) and ML‐7 (100 μm ), myosin light chain kinase inhibitors, and W‐7 (100 μm ) and trifluoperazine (TFP; 30 μm ), calmodulin antagonists, inhibited the Ca2+‐induced catecholamine secretion in the presence of MgATP but not in the absence of MgATP. They did not inhibit the caffeine‐induced catecholamine secretion in the presence of MgATP.
4 The ATP‐independent phase in Ca2+‐dependent exocytosis is thought to be associated with the final step that ultimately leads to fusion, while the ATP‐dependent phase is thought to be associated with a vesicle priming reaction. Therefore, these results suggest that the ATP‐requiring priming stage is lacking in the process of caffeine‐induced exocytosis in bovine adrenal chromaffin cells.     

Probing the selectivity of allosteric modulators of muscarinic receptors at other G‐protein‐coupled receptors

1 The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane‐bis‐ammonium compound W84 (hexane‐1,6‐bis[dimethyl‐3′‐phthalimidopropyl‐ammonium bromide]), may have allosteric effects on radioligand‐binding characteristics at other G‐protein‐coupled receptors, such as cerebral A1 adenosine receptors (Gi‐coupled), cardiac left ventricular α1‐adrenoceptors (Gq), and β‐adrenoceptors (Gs).
2 The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [³H]‐N‐methylscopolamine (NMS) from muscarinic M2‐receptors: 30 μmol l?1 W84, 30 μmol l?1 alcuronium, 1000 μmol l?1 gallamine. As radioligands, we used the adenosine A1‐receptor ligand [³H]‐cyclopentyl‐dipropylxanthine (CPX), the α1‐adrenoceptor ligand [³H]‐prazosin (PRAZ), and the β‐adrenoceptor ligand (?)‐[¹²⁵I]‐iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively.
3 CPX and PRAZ showed a monophasic dissociation with half‐lives of 5.88±0.15 and 12.27±0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36±2% of the control value with alcuronium and to 42±2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half‐life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve.
4 In summary, the allosteric action of the applied agents is not a common feature of G‐protein‐coupled receptors and appears to be specific for muscarinic receptors.     

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

The inhibitory effect of the selective M₃ musarinic acetylcholine receptor antagonist, 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP, 0.1–10 μM) on nicotine (100 μM)‐induced nitrergic relaxation was investigated in comparison to d‐tubocurarine (0.1–10 μM) and hexamethonium (0.1–10 μM) by using phenylephrine (1 μM)‐precontracted rat anococcygeus muscles in vitro. Nicotine produced a 60.1± 2.4% (n = 40) inhibition of phenylephrine precontractions. But this relaxant response was at a significantly lower magnitude of 20.2± 4.6% (n = 18, P < 0.01 vs. control) in the presence of the nitric oxide synthase (NOS) blocker N^G‐nitro‐L ‐arginine methyl ester (L ‐NAME, 30 μM), and it was 26.5± 5.5% (n = 8, P < 0.01 vs. control) in the presence of the soluble guanylate cyclase inhibitor methylene blue (30 μM). However, aminoguanidine (100 μM), a relatively selective blocker of the inducible nitric oxide synthase (iNOS), had no significant effect. Similarly, other iNOS inhibitors such as dexamethasone (5 mg/kg) or L ‐canavanine (100 mg/kg) did not modify contractile nor relaxant responses when they were given in vivo, concomitantly with Escherichia coli endotoxin (1 mg/kg, ip) 4 h before the isolation of the tissues. 4‐DAMP, hexamethonium, and d‐tubocurarine inhibited nicotine‐induced relaxation in a concentration‐dependent manner with the following order of potency: 4‐DAMP > hexamethonium > d‐tubocurarine with IC₅₀ values being 0.47± 0.04 μM, 0.75± 0.06 μM, and 1.02± 0.05 μM, respectively. Therefore, it was concluded that the selective M₃ muscarinic acetylcholine receptor antagonist 4‐DAMP also possesses potent antagonistic action on nicotinic receptors of peripheral nitrergic neurons that innervate the rat anococcygeus muscle. Drug Dev. Res. 46:148–154, 1999. © 1999 Wiley‐Liss, Inc.     

Unravelling the cardiovascular effects induced by α‐terpineol: A role for the nitric oxide–cGMP pathway

1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats. 2. In normotensive rats, administration of α‐terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose‐dependent hypotension (?10 ± 3, ?20 ± 8, ?39 ± 16, ?52 ± 21 and ?57 ± 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., α‐terpineol were significantly attenuated following the administration of N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 20 mg/kg, i.v.; ?2 ± 1, ?5 ± 2, ?7 ± 3, ?22 ± 9 and ?22 ± 10 mmHg, respectively; P < 0.05; n = 5). 3. In 10 μmol/L phenylephrine (PE)‐precontracted mesenteric artery rings, α‐terpineol (10^?12 to 10^?5 mol/L) caused a concentration‐dependent relaxation (maximum relaxation 61 ± 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by α‐terpineol was attenuated (maximum relaxation 20 ± 1%; P < 0.05; n = 7). In addition, vasorelaxation induced by α‐terpineol in rings pretreated with 100 or 300 μmol/L l ‐NAME, 30 μmol/L hydroxocobalamin or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one was attenuated (maximum relaxation 18 ± 3, 23 ± 3, 24 ± 7 and 21 ± 1%, respectively; n = 6; P < 0.05). 4. Furthermore, in a rabbit aortic endothelial cell line, 10^?6, 10^?5 and 10^?4 mol/L α‐terpineol induced concentration‐dependent increases in nitric oxide (NO) levels (12 ± 6, 18 ± 9 and 34 ± 12%Δ fluorescence, respectively; n = 3). 5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that α‐terpineol‐induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO–cGMP pathway.     

The α‐adrenoceptor antagonist,zolertine, inhibits α1D‐ and α1A‐adrenoceptor‐mediated vasoconstriction in vitro

1 The antagonist effect of zolertine (4‐phenyl‐1‐[2‐(5‐tetrazolyl)ethyl]piperazine trihydrochloride), on vascular contraction elicited by noradrenaline in aorta, carotid (α1D‐adrenoceptors), mesenteric (α1A/D‐adrenoceptors) and caudal arteries (α1A‐adrenoceptors) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats and rabbit aorta (α1B‐adrenoceptors), was investigated in endothelium‐denuded arterial rings.
2 The selective α1D‐adrenoceptor agonist, noradrenaline, elicited concentration‐dependent contractions in all arterial rings from both species. Noradrenaline selectivity was: carotid=aorta>>.Gt;mesenteric=rabbit aorta>caudal arteries.
3 The contractile responses induced by noradrenaline were competitively antagonized by zolertine in rat carotid and aorta arteries, yielding pA2 values of WKY, 7.48±0.18; SHR, 7.43±0.13 and WKY, 7.57±0.24; SHR, 7.40±0.08, respectively. Zolertine was a non‐competitive antagonist in some blood vessels as Schild plot slopes were lower than unity. The pKb estimates for zolertine were WKY, 6.98±0.16; SHR, 6.81±0.18 in the mesenteric artery, WKY, 5.73±0.11; SHR, 5.87±0.25 in the caudal artery and 6.65±0.09 in rabbit aorta.
4 Competition binding experiments using the α1‐adrenoceptor antagonist [³H]prazosin showed a zolertine pKi of 6.81±0.02 in rat liver (α1B‐adrenoceptors) and 6.35±0.04 in rabbit liver (α1A‐adrenoceptors) membranes.
5 Zolertine showed higher affinity for α1D‐adrenoceptors compared to α1A‐adrenoceptors, while it had an intermediate affinity for α1B‐adrenoceptors. The ability of the α1‐adrenoceptor antagonist zolertine to block α1D‐adrenoceptor‐mediated constriction in different vessels of WKY and SHR rats may explain its antihypertensive efficacy despite its low order of potency.     

Syntheses,calcium channel modulation effects,and nitric oxide release studies of O2‐alkyl‐1‐(pyrrolidin‐1‐yl) diazen‐1‐ium‐1,2‐diolate 4‐aryl(heteroaryl)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates

A group of racemic 4‐aryl(heteroary)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxy‐lates possessing a potential nitric oxide donor C‐5 O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester [alkyl=(CH₂)_n, n=1–4] substituent were synthesized using a modified Hantzsch reaction. Compounds having a C‐4 2‐trifluoromethylphenyl ( 16 ), 2‐pyridyl ( 17 ), or benzofurazan‐4‐yl ( 20 ) substituent generally exhibited more potent smooth‐muscle calcium channel antagonist activity (IC₅₀ values in the 0.55 to 38.6 μM range) than related analogs having a C‐4 3‐pyridyl ( 18 ), or 4‐pyridyl ( 19 ) substituent with IC₅₀ values > 29.91 μM, relative to the reference drug nifedipine (IC₅₀=0.0143 μM). The point of attachment of C‐4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2‐pyridyl > 3‐pyridyl and 4‐pyridyl. Subgroups of compounds 16a–d , 17a–d , and 20a–d having alkyl spacer groups of variable chain length [–CO₂(CH₂)_nO–, n=1–4] exhibited small differences in calcium channel antagonist potency. Replacement of the ester “methyl” moiety of Bay K 8644 by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate group provided the Bay K 8644 group of analogs 16a‐d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O²‐ethyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)pyridine‐5‐carboxylate ( 16b , EC₅₀=0.096 μM) is about eightfold more potent positive inotrope (cardiac calcium channel agonist) than the reference compound Bay K 8644 (EC₅₀=0.77 μM). A similar replacement of the ester “isopropyl” group in the C‐4 benzofurazan‐4‐yl group of compounds by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester substituent provided compounds 20 (n=1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202‐791 ( 3 , EC₅₀=9.40 μM). The O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester moiety present in 1,4‐dihydropyridine calcium channel modulating compounds 16–20 is not a suitable ?NO donor moiety because the percent nitric oxide released upon in vitro incubation with either l ‐cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204–216, 2003. © 2003 Wiley‐Liss, Inc.     

Adrenoceptor‐mediated modulation of endothelial‐dependent vascular smooth muscle growth

1 Signals derived from endothelial cells (EC) and the sympathetic nervous system are known to independently modulate the growth of vascular smooth muscle (VSM). It is not known if and how these signals are integrated. The purpose of the present study was to test the hypothesis that activation of adrenoceptors by sympathetic‐derived catecholamines modulates EC regulation of VSM growth.
2 The effects of adrenergic agonists on VSM growth were studied in vitro in EC/VSM cocultures. EC stimulated VSM growth in EC/VSM cocultures. Activation of β‐adrenoceptors inhibited this stimulation. EC stimulation of VSM growth was 225±31% in the absence and 127±27% in the presence of 10 μm isoprenaline. Activation of α‐adrenoceptors had no effect on EC stimulation of VSM growth in coculture.
3 Isoprenaline did not affect the growth of VSM grown in the absence of EC, suggesting that it did not inhibit EC stimulation of VSM growth by directly inhibiting VSM growth.
4 Isoprenaline did not affect EC production of growth factors, as media conditioned by EC grown in the absence or presence of isoprenaline, stimulated VSM growth to the same extent. Isoprenaline did not alter EC, VSM or EC/VSM production of the VSM growth inhibitor transforming growth factor β‐1 (TGF‐β1).
5 These data provide evidence that catecholamines, via activation of β‐adrenoceptors, can modulate EC‐dependent VSM growth, and suggest that the sympathetic nervous system and EC coordinately regulate VSM growth.     

In vitro effect of nickel on bovine spermatozoa motility and annexin V‐labeled membrane changes

In this study the effect of in vitro culture of bovine spermatozoa with nickel (NiCl₂) on spermatozoa motility and membrane changes was analyzed. The spermatozoa motility significantly decreased after 120 min of culture at the concentration of 1000 μm Ni ml^?1 (P < 0.05) and after 240 min of culture at the concentration of 500 and 1000 μm Ni ml^?1 (P < 0.001) as compared with control. The progressive motility was the highest in the control group and in the groups with the lowest nickel concentrations (7.8 and 125 μm Ni ml^?1). The progressive spermatozoa motility was significantly altered even after 30 min of culture in the group with the highest nickel concentration (1000 μm Ni ml^?1). A significant decrease in progressive motility from the concentration of 250 μm Ni ml^?1 was detected after 240 min of culture. Concentrations from 125 μm Ni ml^?1 in various time periods of culture stimulated spermatozoa motility after 30 min (P < 0.001), but later an inhibitory effect was noted. After 240 min of in vitro spermatozoa culture with 125 μm Ni ml^?1 a typical Annexin V fluorescence reaction was detected. Fluorescence was detected in mitochondrial segment of bovine spermatozoa. In spermatozoa exposed to higher nickel concentrations the Annexin V‐positive reaction was detected also on the spermatozoa head membrane. In the group with the highest concentration and the longest time of exposure (1000 μm Ni ml^?1; 240 min) the apoptotic Annexin‐positive regions were detected not only in the mitochondrial part, but also in the spermatozoa head (acrosomal and postacrosomal part), showing significant alteration of spermatozoa membrane integrity. Copyright © 2010 John Wiley & Sons, Ltd.     

Different sensitivity of isoprenaline‐induced responses in ventricular muscle to sodium nitroprusside in normotensive and spontaneously hypertensive rats 1

1 The aim of the present work was to study the possible modulatory role of nitric oxide (NO) on the positive inotropic effect induced by the β‐adrenoceptor agonist isoprenaline in myocardial contractility, and whether this modulation is altered by hypertension.
2 The study was performed using right ventricular strips from the hearts of 6‐month‐old male Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The contractile force of electrically‐stimulated ventricular strips was measured by a force‐displacement transducer.
3 Isoprenaline (from 10 nmol l?1 to 10 μmol l?1) induced a concentration‐dependent increase in cardiac contractility in strips from both rat strains. This positive inotropic effect to isoprenaline was reduced by the NO donor sodium nitroprusside (SNP, 0.1 mmol l?1) in muscles from WKY rats and slightly increased in those from SHR. The SNP‐induced increase in strips from SHR was abolished by superoxide dismutase (100 U ml?1).
4 NG‐nitro‐arginine‐methyl ester (L‐NAME, 0.1 mmol l?1) and 1H‐[1,2,4]oxadiazolo[4,3]quinoxalin‐1‐one (ODQ, 10 μmol l?1), respective inhibitors of NO synthase and guanylate cyclase, increased the response to isoprenaline in muscles from WKY rats, whereas it was unaltered in strips from SHR.
5 In strips from WKY rats, the combination of ODQ and SNP produced an increase in the response elicited by isoprenaline, which was similar to that observed with ODQ or L‐NAME. 8‐Br‐cyclicGMP (8‐Br‐cGMP, 0.1 mmol l?1), a permeable and structural cGMP analogue, decreased the effect induced by isoprenaline only in muscles from WKY rats.
6 These results suggest that the positive inotropic response to isoprenaline in ventricular strips from WKY rats is negatively modulated by NO, and positively by superoxide anions in those from SHR. The lack of a modulatory response to NO in ventricular strips from SHR is probably a result of an alteration of mechanisms in NO‐signalling pathway downstream of cGMP formation in SHR hearts.     

THE INHIBITION OF NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE BY DIPHENYLENE IODONIUM

1. The effects of diphenylene iodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate-dependent oxidases (which generate superoxide anions), were studied on nitric oxide (NO)-mediated responses in isolated preparations of the rat aorta and anococcygeus muscle. 2. In aortic rings, the endothelium-dependent relaxant action of acetylcholine was reduced by DPI (0.3–10 μmol/L) in a concentration-dependent manner and abolished by the NO synthase (NOS) inhibitor L-nitro-N^G-arginine methylester (l-NAME; 100 μmol/L). Relaxations induced by sodium nitroprusside (SNP) or NO were not affected by DPI or l-NAME. 3. In anococcygeus muscles, DPI (0.3–10 μmol/ L) as well as l-NAME (5–100 μmol/L) produced concentration-dependent reductions of relaxations produced by nitrergic nerve stimulation. Relaxations induced by NO and SNP were not affected by either DPI or l-NAME. l-Arginine (1 mmol/L) prevented the reduction of nitrergic relaxations by l-NAME but not by DPI. 4. Contractions of anococcygeus muscles elicited by exogenous noradrenaline (1 μmol/ L) were not affected or were inhibited by DPI (0.3–10 μmol/L), but the contractions elicited by noradrenergic nerve stimulation were significantly enhanced by DPI and l-NAME. When noradrenergic contractions had already been maximally enhanced by l-NAME (100 μmol/L), DPI produced no further enhancement. l-Arginine (1 mmol/L) prevented the enhancement of noradrenergic contractions by l-NAME but not by DPI. 5. The efflux of radioactivity induced by field stimulation from anococcygeus muscles previously incubated with [³H]-noradrenaline was not affected by either DPI or l-NAME. 6. Superoxide dismutase (SOD, 100 U/mL) had no significant effects on noradrenergic contractions, nitrergic relaxations, relaxations induced by NO or the actions of DPI in the rat anococcygeus muscle. 7. The results suggest that the effects of DPI in reducing the NO-mediated relaxations produced by acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle are due to the inhibition of NOS in these tissues. The effects of DPI were not sensitive to l-arginine, and thus the mechanism of inhibition of NOS differs from that of l-NAME.     

EFFECTS OF HYDROXOCOBALAMIN AND HAEMOGLOBIN ON NO-MEDIATED RELAXATIONS IN THE RAT ANOCOCCYGEUS MUSCLE

1. The effects of hydroxocobalamin (Vitamin B_12a) on relaxations produced by nitric oxide (NO), some NO-donating compounds and nitrergic nerve stimulation in isolated preparations of the rat anococcygeus muscle were compared with the effects of haemoglobin. 2. Hydroxocobalamin (30 μmol/L) significantly reduced relaxations induced by NO (0.1–3 μmol/L) and sodium nitroprusside (SNP; 0.01–0.3 μmol/L) but did not affect relaxations induced by glyceryl trinitrate (GTN; 0.01–1 μmol/L), S-nitrosocysteine (0.1–0.3 μmol/L) or stimulation of nitrergic nerves. A higher concentration of hydroxocobalamin (100 μmol/L) slightly reduced nitrergic nerve stimulation-induced relaxations. 3. Haemoglobin (10 μmol/L) blocked relaxation induced by NO and reduced relaxations induced by SNP, GTN, S-nitrosocysteine and nitrergic nerve stimulation. 4. When nitrergic nerve stimulation-induced relaxations had been partially reduced by the NO synthase inhibitor l-NAME (5–10 μmol/L), hydroxocobalamin had only a weak and transient inhibitory effect. 5. Noradrenergic contractions induced by field stimulation were not affected by hydroxocobalamin (30 μmol/L), but were enhanced by haemoglobin (10 μmol/L). 6. The results suggest that the transmitter released from nitrergic nerves in anococcygeus muscles resembles NO-releasing compounds such as S-nitrosocysteine and GTN but not SNP or free NO.     

Cross tolerance between nitroglycerin and neural relaxation of the rabbit sphincter of Oddi

We studied whether non-adrenergic, non-cholinergic (NANC) relaxation of the rabbit sphincter of Oddi was influenced by tolerance to nitroglycerin (NG)in vitro. Sphincter of Oddi (SO) muscle rings precontracted with EC₅₀concentrations of cholecystokinin octapeptide (CCK8) were exposed to cumulative increases in NG concentrations and tested for relaxation by measurement of isometric tension. A separate group of six rings was subjected to a preceding exposure to 275 μmnitroglycerin over 60 min to inducein vitrotolerance to nitroglycerin. The rings (both tolerant and non-tolerant) were subjected to electrical field stimulation (FS: 50 V, 0.1 ms, 20 Hz, 3 and 10 stimuli). The rings were then preincubated with NANC solution: phentolamine, oxprenolol and atropine (all 1 μm) for 20 min and FS was applied again. FS was repeated after additional incubation withN^G-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthase (30 μm) and after a successive incubation with 3 mml-arginine (20 min). Maximum contractions produced by CCK8 in `tolerant' and ‘non-tolerant’ sphincters were 29.9±5.8 and 28.3±5.2 mN, respectively. The sensitivity to CCK8 also was not different between the two groups with EC<sub>50</sub>(−log M) values of 8.5±0.2 and 8.3±0.1, respectively. FS evoked twitchlike contraction followed by relaxation in the ampullary SO in both `tolerant' and `non-tolerant' preparations. Incubation in NANC solution resulted in monophasic relaxations in response to FS in non-tolerant sphincters but not in tolerant ones.l-NAME (30 μm) reversed NANC relaxation in non-tolerant muscle rings whereas it failed to modify NANC contractions in the tolerant preparations.l-arginine (3 mm) reversed the inhibitory effect ofl-NAME on NANC relaxation in the `non-tolerant' rings and it was without effect on FS-induced contractions in the `tolerant' SO. As measured by radioimmunoassay, tolerance to NG was without any significant effect on tissue content of both cyclic adenosine 3′:5′ monophosphate (cAMP) and cyclic guanosine 3′:5′ monophosphate (cGMP). FS significantly increased tissue cAMP and cGMP content in ‘non-tolerant’ preparations. FS failed to increase the level of either cyclic nucleotide in `tolerant' tissue. We conclude that NANC relaxation of the ampullary part of the rabbit SO is significantly impaired in the state of tolerance to NG`in vitro'.     

Comparison of the Effects of Histamine and Nα-Methylhistamine on Neuronal Function in the Guinea-pig Oesophagus and Ileum

The effects of histamine and N^α-methylhistamine, two components of gastric juice, on vagal and transmural stimulation of the guinea-pig isolated oesophagus were compared with their effects on cholinergic and on non-adrenergic-non-cholinergic (NANC) neuronal responses in the isolated ileum, both tissues having been pretreated with mepyramine (1 μm ). Histamine (≤10 μm ) and N^α-methylhistamine (≤1 μm ) had no significant effect on either vagal or transmural stimulation in the oesophagus. Substance P, which produces a contraction by activation of cholinergic nerves in the oesophagus also was unaffected by histamine. In contrast, the agonists inhibited contractions produced by cholinergic nerve stimulation in the ileum; the inhibition produced by histamine (10 μm ) was up to 73 ± 5%, that by N^α-methylhistamine (1 μm ), 48 ± 5%. Histamine also inhibited responses to stimulation of NANC neurons by up to 37 ± 14%. The effects of histamine and N^α-methylhistamine in the ileum were inhibited by clobenpropit (01 μm ). These findings suggest that histamine and N^α-methylhistamine have no role in the modulation of neuronal function in the oesophagus, in contrast with their effect in the ileum.