Alicia Labadia

Endothelium-dependent relaxation to acetylcholine in bovine oviductal arteries: mediation by nitric oxide and changes in apamin-sensitive K+ conductance

1 Mechamisms underlying the relaxant response to acetylcholine (ACh) were examined in bovine oviductal arteries (o.d. 300–500 μm and i.d. 150–300μm) in vitro. Vascular rings were treated with indomethacin (10 μm) to prevent the effects of prostaglandins. 2 ACh elicited a concentration‐related relaxation in ring segments precontracted with noradrenaline (NA), which was abolished by endothelium denudation. 3 The ACh‐induced relaxation was attenuated but not abolished by NG‐nitro‐L‐arginine (l‐NOARG, 1 μm‐1 mM), an inhibitor of nitric oxide (NO) formation. The inhibition caused by L‐NOARG (10 μm) was reversed by addition of excess of L‐arginine but not D‐arginine (1 mM). 4 In high K+ (40–60 mM)‐contracted rings, ACh was a much less effective vasodilator and its relaxant response was completely abolished by L‐NOARG (100 μm). 5 In NA (10 μm)‐contracted rings, ACh induced sustained and concentration‐dependent increases in cyclic GMP, which were reduced below basal values by L‐NOARG (100 μm), while potent relaxation persisted. Similar increases in cyclic GMP were evoked by ACh in high K+ (50 mM)‐treated arteries and under these conditions, both cyclic GMP accumulation and relaxation were L‐NOARG‐sensitive. 6 S‐nitroso‐L‐cysteine (NC), a proposed endogenous precursor of endothelial NO, also induced cyclic GMP accumulation in NA‐contracted oviductal arteries. 7 Methylene blue (MB, 10 μm), a proposed inhibitor of soluble guanylate cyclase, inhibited both endothelium‐dependent relaxation to ACh and endothelium‐independent response to exogenous NO, whereas relaxation to NC remained unaffected. 8 The L‐NOARG‐resistant response to ACh was not affected by either ouabain (0.5 mM), glibenclamide (3 μm), tetraethylammonium (TEA, 1 mM) or charybdotoxin (50 nM), but was selectively blocked by apamin (0.1‐1 μm). However, apamin did not inhibit either relaxation to ACh in high K+‐contracted rings or endothelium‐independent relaxation to either NO or NC. 9 Apamin and MB inhibited ACh‐induced relaxation in an additive fashion, suggesting the involvement of two separate modulating mechanisms. 10 These results suggest that ACh relaxes bovine oviductal arteries by the release of two distinct endothelial factors: a NO‐like substance derived from L‐arginine, which induces cyclic GMP accumulation in smooth muscle, and another non‐prostanoid factor acting by hyperpolarization mechanisms through alterations in apamin‐sensitive K+ conductance.

Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications.

1 To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO‐mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L‐[14C]‐arginine to L‐[14C]‐citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2 NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg−1 protein min−1): urethra (33 ± 3.3), detrusor (13.1 ± 1.1) and ureter (1.5 ± 0.2). No activity was detected in the particulate fraction of any region. 3 NOS activity was dependent on Ca2+‐calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4 NOS activity was inhibited to a greater extent by NG‐nitro‐l‐arginine (l‐NOARG) and its methyl ester (l‐NAME) than by NG‐monomethyl‐l‐arginine (l‐NMMA), while 7‐nitroindazole (7‐NI) was a weak inhibitor and L‐cannavine had no effect. 5 Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin‐sensitive manner, suggesting feedback inhibition of NOS by NO. 6 EFS induced prominent NO‐mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L‐NOARG = L‐NAME > 7‐NI > L‐NMMA. 7 In conclusion, we have demonstrated the presence of NO‐synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO‐mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.

Local regulation of oviductal blood flow.

1. Blood flow to the oviduct is implicated in the genesis and maintenance of oviductal fluid, in this way contributing to the creation of an adequate medium for ovum/embryo physiology. Therefore, factors controlling the tone of the vessels supplying the oviduct would be expected to affect its luminal environment. In addition, cyclic changes in oviductal blood flow have been suggested to have mechanical functions in the transport of the ovum/embryo. 2. The vascular supply to the oviduct has a prominent adrenergic vasomotor control. A dense adrenergic innervation, together with the presence of a predominant population of alpha(1)-adrenoceptors, provides a contractile regulatory mechanism of oviductal blood flow. No evidence is available on the presence of beta-adrenoceptors. The scanty cholinergic innervation of mammalian oviduct is mainly confined to the vessels, where acetylcholine (ACh) has a vasodilatory effect by releasing endothelium-derived relaxing factors. 3. The presence of nerves containing neuropeptides has been shown in the oviduct. Specifically, a high density of neuropeptide Y- and vasointestinal peptide-containing nerve fibers has been found in relation to blood vessels, but their role in the neutral control of the oviduct blood flow remains to be established. To date, it is not known whether or not oviductal blood vessels receive perivascular nitrergic nerves. 4. Relaxing and contracting factors derived from endothelium also seem to have a modulatory role on oviductal vascular tone. Neurotransmitters or autacoids, such as ACh and histamine, acting on endothelial receptors, release nitric oxide (NO), which relaxes oviductal arteries through guanylyl cyclase activation and accumulation of cyclic GMP. In addition, the release of an endothelium-derived hyperpolarizing factor (EDHF), distinct from NO, by ACh has been shown in oviductal arteries. It acts through the opening of low-conductance Ca(2+)-activated K+ channels leading to hyperpolarization and relaxation. Furthermore, potent and long-lasting contractions induced by the endothelium-derived contractile factor, endothelin (ET), points to its role in the long-term regulation of oviductal vascular tone. 5. A particularly high density of 5-hydroxytryptamine (5-HT) and histamine, present in mast cells clustered in the vicinity of blood vessels, has been described in the oviduct. It is known that histamine elicits a relaxation of oviductal arteries that is partially endothelium-dependent and mediated by the activation of H1-receptors. The implication of histamine in both the increase in blood flow and edema around ovulation, as well as the existence of a functional antagonism between histamine and 5-HT in the regulation of oviductal blood flow, await further investigation. 6. Other factors, such as relaxing and contracting cyclooxygenase-derived products, may also participate in the modulation of blood flow to the oviduct. 7. An overall endocrine regulation of the oviductal vascular supply exists, acting by both direct effects on smooth muscle and modulation of neural and autocrine factors. This control enables cyclic changes in blood flow to the oviduct that are tightly coupled to the reproductive functions of the tube.

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoine‐1‐oxyl 3‐oxide (carboxy‐PTIO), the specific guanylate cyclase inhibitor 1H‐[1,2,4]‐oxadiazole‐[4,3‐a]‐quinoxalin‐1‐one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 μM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml−1) inhibited NO‐mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy‐PTIO (0.1–0.5 mM) inhibited NO‐mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml−1). Relaxations to NO were reduced, but not abolished, by ODQ (10 μM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5′‐dithio‐bis (2‐nitrobenzoic acid) (DTNB, 0.5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N‐ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L‐cysteine (L‐cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD‐independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open.

Histamine receptors in isolated bovine oviductal arteries

The present in vitro study was designed to evaluate the effect of histamine on isolated rings of bovine oviductal artery and to characterize the histamine receptors involved in the histamine-induced response. Endothelial dependence of the response was also investigated. Cumulative addition of histamine and 2-pyridylethylamine (histamine H receptor agonist) induced a concentration-dependent relaxation in intact arterial segments precontracted with noradrenaline. The histamine H1 receptor antagonist mepyramine showed non-competitive antagonism in the histamine-induced concentration-response curve. However, when the response to histamine was evaluated in the presence of mepyramine and histamine H1 and H3 receptors were blocked, Schild analysis yielded a line with a slope of 1.10 and a pA2 value of 8.91, indicating simple competitive antagonism of mepyramine at histamine H1 receptor sites. The histamine H2 receptor agonist, dimaprit, caused marked dilatation only at high doses. Cimetidine, propranolol and mepyramine failed to inhibit this relaxant effect. In precontracted oviductal arteries, cimetidine did not modify the histamine-induced concentration-response curves. Combined treatment with histamine H1 and H2 receptor antagonists did not induce an additional displacement with respect to the isolated effect of mepyramine thus excluding activation of histamine H2 receptors. Histamine and (R)-alpha-methylhistamine, a selective histamine H3 receptor agonist, produced a moderate contractile effect on the resting tone of preparations. Pretreatment with the selective histamine H1 receptor antagonist decreased the (R)-alpha-methylhistamine response but increased the maximal relaxant effect and abolished the contractile effect of histamine, suggesting the presence of a limited population of contractile histamine H3 receptors. Removal of the endothelium or pretreatment with methylene blue produced a significant inhibition of the relaxant response to histamine. Remaining dilatation was practically abolished by mepyramine and also by indomethacin. The L-arginine analogue, N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of histamine and basal production of nitric oxide. L-Arginine, which on its own induced significant endothelium-dependent vasodilatation, reversed the effect of L-NAME on histamine relaxation. Indomethacin only caused a slight modification of the sensitivity of the vessels to histamine, suggesting that prostacyclin or other cyclo-oxygenase products did not make a significant contribution to the model. The absence of the endothelium did not modify the contractile effect of histamine. The results suggest that the relaxant response of isolated oviductal arteries to histamine is dependent on the functional integrity of the endothelium and is mainly mediated by histamine H1 receptors. These receptors may mask a minority presence of histamine H3 contractile receptors located on smooth muscle. The main relaxing factor released from the endothelium by mediation of histamine is nitric oxide, which may also exert an effect on vascular tone.

Spontaneous photo-relaxation of urethral smooth muscle from sheep, pig and rat and its relationship with nitrergic neurotransmission

In the present work we have characterized the relaxant response induced by light stimulation (LS) in the lower urinary tract from sheep, pig and rat, establishing its relationship with nitrergic neurotransmission. Urethral, but not detrusor, preparations showed pronounced photo‐relaxation (PR) which declined progressively following repetitive LS. Sheep urethral PR was again restored either spontaneously or (to a greater extent) by exogenous nitric oxide (NO) addition and by electrical field stimulation (EFS) of intrinsic nitrergic nerves. Greater NO generation was detected from sheep urethral than from detrusor homogenates following illumination. Sheep urethral PR was inhibited by oxyhaemoglobin, but not by methaemoglobin, carboxy‐PTIO, extracellular superoxide anion generators or superoxide dismutase. Guanylyl cyclase but not adenylyl cyclase activation mediates urethral relaxation to LS. Urethral PR was more resistant to inhibition by L‐thiocitrulline than EFS‐induced responses, although this agent prevented PR restoration by high‐frequency EFS. Urethral PR was TTX insensitive and partially modified in high‐K+ solutions. Cold storage for 24 h greatly impaired urethral PR, although it was restored by high‐frequency EFS. Repetitive exposure to LS, EFS or exogenous NO induced changes in the shape of the EFS‐induced nitrergic relaxation, possibly by pre‐synaptic mechanisms. In conclusion, we suggest the presence of an endogenous, photo‐labile, nitro‐compound store in the urethra, which seems to be replenished by neural nitric oxide synthase activity, indicating a close functional relationship with the nitrergic neurotransmitter.

Characterization of postsynaptic α-adrenoceptors in the arteries supplying the oviduct

1 In vitro experiments were designed to characterize postjunctional α‐adrenoceptor subtypes in ring segments (1 mm length; outer diameter 300–500 μm) from arteries supplying the oviduct of the heifer. 2 Noradrenaline, adrenaline and phenylephrine evoked concentration‐dependent contractile responses. The pD2 values were 5.67, 5.89 and 5.93, respectively. Medetomidine, clonidine and B‐HT 920 (2‐amino‐6‐allyl‐5,6,7,8‐tetra‐hydro‐4H‐(thiazo)‐4,5‐d‐azepine) were ineffective. 3 The α‐adrenoceptor selective antagonists, prazosin (1 nm−0.1 μm) and rauwolscine (0.1–10 μm) competitively antagonized the response to noradrenaline. The pA2 values were 9.38 and 6.83, respectively. 4 The dissociation constant (KD) for noradrenaline calculated by use of the irreversible antagonist, dibenamine, was 3.95 (2.09–5.81) μm. The occupancy‐response relationship was non‐linear. Half‐maximal response to noradrenaline was obtained with 22% receptor occupancy while maximal response required 100% occupancy. 5 B‐HT 920 evoked a biphasic contractile concentration‐dependent response in preparations incubated in a physiological solution containing 20 mm K+, 0.1 μm prazosin and 1 μm propranolol. Rauwolscine 0.1 μm significantly (P < 0.01) blocked the first component of the B‐HT 920 concentration‐response curve with an apparent pA2 value of 8.52 (7.86–9.18). 6 These results strongly suggest that α‐adrenoceptors in oviductal arteries are mainly of the α1 subtype, although a possible role for α2‐adrenoceptors cannot be excluded.

Endothelin receptor-mediated Ca2+ mobilization and contraction in bovine oviductal arteries : Comparison with noradrenaline and potassium

1. The effects of endothelin-1 (ET-1) were studied in bovine oviductal arteries and compared to those of noradrenaline (NA) and high K+ (K+). The influence of endothelium, the receptor subtypes involved, and the mechanisms of Ca2+ mobilization were assessed. 2. ET-1 (0.1-300 nM) induced concentration-dependent contractions with a potency of 10(3) and 10(2) times higher than NA (0.1 microM-0.1 mM) and K+ (9.5-119 mM), respectively. Removal of endothelium or NG-nitro-L-arginine (L-NOARG, 0.1 mM) pretreatment did not affect responses to either ET-1 or K+, whereas the NA response was significantly increased. Indomethacin (1 microM) had no effect on either of these agonists. 3. The rank order of potency for the ET isopeptides was: ET-1 = ET-2 > ET-3. The ETA receptor-selective agonist, sarafotoxin 6c (S6c), had no effect. The ETA receptor-selective antagonist, BQ-123, showed a competitive antagonism on the ET-1 response (pA2 value of 6.58 +/- 0.01), whereas contractions to ET-3 were completely abolished by BQ-123 at 0.1 microM. 4. Concentration-response curves to both ET-1 and NA were shifted to the right and their maximum response reduced to approximately 56% and 65% of controls, respectively, under 30 min of incubation in Ca(2+)-free solution, whereas responses to K+ were almost abolished by this treatment. Contractions to both NA (30 microM) and ET-1 (30 nM) were maximally inhibited after 10 min of extracellular Ca2+ deprivation. 5. Contractions to ET-1 were more potently inhibited by nickel (Ni2+, 0.3 mM), whereas nifedipine (1 microM) and cadmium (Cd2+, 0.1 mM) induced only a slight effect. In contrast, opposite effects were found for both NA and K+. 6. Treatment with ryanodine (100 microM) and caffeine (10 mM) in Ca(2+)-free solution reduced the tension measured 5 min after NA (30 microM) and ET-1 (30 nM) addition, but the sustained response (tension at 25 min) remained unaffected. 7. Calphostin C (1 microM), a specific protein kinase C (PKC) inhibitor, reduced the maximum contractile response to ET-1 by about 50% without significantly affecting its pD2 value. 8. These results suggest that ET-1 acts in bovine oviductal arteries by directly activating a homogenous population of ETA receptors in smooth muscle, without endothelial modulation. Several Ca2+ activation mechanisms seem to be involved in the contractile action of the peptide, including: (1) extracellular Ca2+ entrance through Ni(2+)-sensitive and L-type Ca2+ channels; (2) intracellular Ca2+ release from a ryanodine-sensitive Ca2+ store; and (3) sensitization of the contractile machinery to Ca2+ via PKC.

Endothelial modulation of resting and stimulated vascular tone in the pig capsular testicular artery

Abstract We have investigated the wall tension characteristics and the role of endothelium on vascular tone, at rest and under K+ depolarization, in isolated rings from the pig capsular testicular artery. The active tension reached a maximum that was significantly lower in vessels without endothelium, whereas the passive tension and the Ca2+-dependent myogenic tone were not significantly affected by endothelium removal. Both NG-nitro-l-arginine (L-NOARG, 10−4 M) and methylene blue (10−5 M), increased the basal resting tension (BRT) in vessels with endothelium, while indomethacin (10−6 M) decreased BRT in vessels both with and without endothelium. Either removal of endothelium or treatment with indomethacin (10−6 M), quinacrine (10−5 M) or ibuprofen (10−5 M), significantly depressed the K+ concentration response curve, while dazoxiben (10−5 M) and SQ 30,741 (10−5 M) had no effect. L-NOARG (10−6 M) potentiated the contractile response to K+ in vessels with endothelium, whereas L-NOARG (10−4 M) was ineffective in vessels devoid of endothelium. These results suggest that a predominating NO release from endothelium, together with a cyclooxygenase-derived vasoconstrictor, modulate vascular tone at rest. In contrast, predominant endothelial release of a cyclooxygenase-derived contractile factor, but different from TXA2, PGH2 or superoxide anions, is induced by K+ depolarization and leads to vasoconstriction.

α- and β-adrenoceptors in the sheep urinary bladder

Abstract A study was undertaken to determine the presence and distribution of α- and β-adrenoceptors in the sheep bladder body and base. In the bladder body, noradrenaline and isoproterenol induced relaxation which was significantly inhibited by propranolol, pafenolol and butoxamine. In the presence of propranolol (10 −5 M), noradrenaline induced a small contraction, as well as phenylephrine, but B-HT 920 failed to cause any effect on the bladder body. In the bladder base, noradrenaline caused a contraction that was significantly inhibited by prazosin but not by yohimbine. Phenylephrine also induced a contractile response in this structure which was inhibited by prazosin. Isoproterenol caused a relaxation that was significantly inhibited by propranolol and pafenolol but not by butoxamine. Relaxation was mediated by both β 1 and β 2 -adrenoceptors in the detrusor muscle and by β 1 -adrenoceptors in the bladder base. α 1 -adrenoceptors contributed to maintain the detrusor tone and contract the bladder base.