Differential actions of charybdotoxin on central and daughter branch arteries of the rabbit isolated ear

1. By use of rabbit isolated perfused intact ears and isolated perfused segments of central and first generation daughter branch ear arteries, we investigated the actions of charybdotoxin (ChTX), a blocker of calcium-activated K⁺ channels (K_Ca channels), and N^ω-nitro-L-arginine methyl ester (L-NAME) on pressure-flow and diameter-flow relationships.
2. ChTX (1 nM) induced an upwards shift in the pressure-flow curve in the rabbit intact isolated ear preconstricted with 5-hydroxytryptamine (5-HT; 100 nM) with subsequent administration of L-NAME (100 μM) inducing a further upwards shift. L-NAME itself induced an upwards shift in the pressure-flow curve, but subsequent administration of ChTX was without significant effect.
3. Microangiographic analysis revealed a tendency of ChTX (1 nM) to decrease vessel diameter in the central ear artery (G₀) with little effect on the first two generations of daughter branch arteries (G₁ and G₂) in the intact ear. Subsequent addition of L-NAME (100 μM) did not significantly further decrease vessel diameter in G₀, but did decrease vessel diameter in G₁ and G₂. L-NAME itself showed a tendency to decrease vessel diameter in G₀, G₁ and G₂ vessels with subsequent addition of ChTX being without significant effect.
4. In an isolated G₀ preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) caused an upwards shift in the pressure-flow curve which was augmented by subsequent addition of L-NAME (100 μM). L-NAME (100 μM) itself caused an upwards shift in the pressure-flow curve but subsequent addition of ChTX (1 nM) had no significant effect.
5. In comparison, in an isolated G₁ preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) had no significant effect on the pressure-flow curve relative to control, but subsequent addition of L-NAME (100 μM) caused an upwards shift. L-NAME (100 μM) itself induced an upwards shift in the pressure-flow curve with subsequent addition of ChTX (1 nM) being without significant effect.
6. ChTX (10 pM–10 nM) caused a concentration-dependent increase in perfusion pressure in isolated G₀ and G₁ preparations at fixed flow rates of 2 ml min⁻¹ and 0.5 ml min⁻¹, respectively. These responses were enhanced in the presence of L-NAME (100 μM) in G₁ but not G₀ preparations.
7. We conclude that at 1 nM, ChTX exhibits differential actions on central and daughter branch arteries of the intact ear of the rabbit, which are also apparent in the corresponding arteries when studied in isolation. The action of 1 nM ChTX in G₀ vessels may reflect inhibition of either the release or action of nitric oxide as it was blocked in the presence of L-NAME. At higher concentrations of ChTX, there would appear to be a direct constrictor effect on vascular smooth muscle which is apparent in both G₀ and G₁ vessels. This observed heterogeneity could reflect different distributions of K_Ca channels between central and daughter branch arteries at either the endothelial or smooth muscle levels, or both.

     

Involvement of K+ channel permeability changes in the L-NAME and indomethacin resistant part of adenosine-5′-O-(2-thiodiphosphate)-induced relaxation of pancreatic vascular bed

1. We have previously demonstrated that adenosine-5′-O-(2-thiodiphosphate) (ADPβS), a potent P2Y-purinoceptor agonist, relaxed pancreatic vasculature not only through prostacyclin (PGI₂) and nitric oxide (NO) release from the endothelium but also through other mechanism(s). In this study, we investigated the effects of an inhibitor of the Na⁺/K⁺ pump, of ATP-sensitive K⁺ (K_ATP) channels and of small (SK_Ca) or large (BK_Ca) conductance Ca²⁺-activated K⁺ channels. Experiments were performed at basal tone and during the inhibition of NO synthase and cyclo-oxygenase.
2. In control conditions, ADPβS (15 μM) induced an initial transient vasoconstriction followed by a progressive and sustained vasodilatation. In the presence of N^ω-nitro-L-arginine methyl ester (L-NAME, 200 μM) the transient vasoconstriction was reversed into a one minute vasodilator effect, which was then followed by a progressive and sustained vasodilatation similar to that observed with ADPβS alone. The addition of indomethacin (10 μM) did not significantly modify the profile of ADPβS-induced vasodilatation.
3. Ouabain (100 μM) decreased basal pancreatic flow rate and did not modify ADPβS-induced relaxation. This inhibitor of the Na⁺/K⁺ pump increased the pancreatic vasoconstriction induced by L-NAME or by the co-administration of L-NAME and indomethacin. Ouabain did not modify either the L-NAME or the L-NAME/indomethacin resistant part of the ADPβS vasodilatation.
4. The K_ATP inhibitor tolbutamide (185 μM) did not significantly modify basal pancreatic flow rate and ADPβS-induced relaxation. This inhibitor which did not change L-NAME-induced vasoconstriction, significantly diminished the L-NAME resistant part of ADPβS-induced vasodilatation. Tolbutamide intensified the vasoconstriction induced by the co-administration of L-NAME and indomethacin. In contrast, the L-NAME/indomethacin resistant part of ADPβS vasodilatation was not changed by the closure of K_ATP.
5. The SK_Ca inhibitor apamin (0.1 μM) did not significantly change pancreatic vascular resistance whatever the experimental conditions (in the absence or in presence of L-NAME or L-NAME/indomethacin). In the presence of L-NAME, the closure of SK_Ca channels changed the one minute vasodilator effect of ADPβS into a potent vasoconstriction and thereafter modified only the beginning of the second part of the L-NAME-resistant part of the ADPβS-induced vasodilatation. In contrast, the L-NAME/indomethacin resistant part of ADPβS-induced relaxation remained unchanged in the presence of apamin.
6. Charybdotoxin (0.2 μM), an inhibitor of BK_Ca, increased pancreatic vascular resistance in the presence of L-NAME/indomethacin. In the presence of L-NAME, the closure of BK_Ca channels reversed the one minute vasodilator effect of ADβPS into a potent vasoconstriction and drastically diminished the sustained vasodilatation. In contrast the L-NAME/indomethacin resistant part of ADPβS-induced relaxation was not modified by the presence of charybdotoxin. Under L-NAME/indomethacin/charybdotoxin/apamin infusions, ADPβS evoked a drastic and transient vasoconstriction reaching a maximum at the second minute, which was followed by a sustained increase in the flow rate throughout the ADPβS infusion. The maximal vasodilator effect of ADPβS observed was not modified by the addition of apamin.
7. The results suggest that the L-NAME-resistant relaxation induced by ADPβS in the pancreatic vascular bed involves activation of BK_Ca, K_ATP and to a lesser extent of SK_Ca channels, but the L-NAME/indomethacin resistant part of ADPβS-induced relaxation is insensitive to the closure of K_ATP, SK_Ca and BK_Ca channels.

     

Characterization of endothelium-derived relaxing factors released by bradykinin in human resistance arteries

1. Relaxing factors released by the endothelium and their relative contribution to the endothelium-dependent relaxation produced by bradykinin (BK) in comparison with different vasodilator agents were investigated in human omental resistance arteries.
2. BK produced an endothelium-dependent relaxation of arteries pre-contracted with the thromboxane A₂ agonist, U46619. The B₂ receptor antagonist, Hoe 140 (0.1, 1 and 10 μM), produced a parallel shift to the right of the concentration-response curve to BK with a pA₂ of 7.75.
3. Neither the cyclo-oxygenase inhibitor, indomethacin (10 μM) alone, the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 300 μM) alone, the nitric oxide scavenger, oxyhaemoglobin (Hb, 10 μM) alone, nor the combination of L-NAME plus Hb affected the concentration-response curve to BK. Conversely, the combination of indomethacin with either L-NAME or Hb attenuated but did not abolish the BK-induced relaxation. By contrast, the relaxations produced by the Ca²⁺ ionophore, calcimycin ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187), and by the inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase, thapsigargin (THAPS), were abolished in the presence of indomethacin plus L-NAME. Also, the presence of indomethacin plus L-NAME produced contraction of arteries with functional endothelium.
4. The indomethacin plus L-NAME resistant component of BK relaxation was abolished in physiological solution (PSS) containing 40 mM KCl and vice versa. However, in the presence of KCl 40 mM, indomethacin plus L-NAME did not affect the nitric oxide donor, S-N-acetylpenicillamine-induced relaxation.
5. The indomethacin plus L-NAME resistant component of the relaxation to BK was significantly attenuated by the K⁺ channel blocker tetrabutylammonium (TBA, 1 mM). However, it was not affected by other K⁺ channel blockers such as apamin (10 μM), 4-aminopyridine (100 μM), glibenclamide (10 μM), tetraethylammonium (10 mM) and charybdotoxin (50 nM).
6. In the presence of indomethacin plus L-NAME, the relaxation produced by BK was not affected by the phospholipase A₂ inhibitor, quinacrine (10 μM) or by the inhibitor of cytochrome P450, SKF 525a (10 μM). Another cytochrome P450 inhibitor, clotrimazole (10 μM) which also inhibits K⁺ channels, inhibited the relaxation to BK.
7. These results show that BK induces endothelium-dependent relaxation in human small omental arteries via multiple mechanisms involving nitric oxide, cyclo-oxygenase derived prostanoid(s) and another factor (probably an endothelium-derived hyperpolarizing factor). They indicate that nitric oxide and cyclo-oxygenase derivative(s) can substitute for each other in producing relaxation and that the third component is not a metabolite of arachidonic acid, formed through the cytochrome P-450 pathway, in these arteries.

     

Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade

1. The pulmonary vasculature is normally in a low resting state of tone. It has been hypothesized that this basal tone is actively maintained by the continuous release of a vasodilator in the resting state. However, evidence for basal release of nitric oxide (NO) is inconclusive.
2. We studied the release of NO in arteries from the pulmonary circulation of male Wistar-Kyoto rats by examining the effects of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME) on resting pulmonary arteries and on vessels pre-contracted with prostaglandin F_2α (PGF_2α).
3. Rats (n=21) were killed by an overdose with pentobarbitone. Pulmonary arteries were dissected (mean internal diameter 459±11 μm) and mounted in a small vessel wire myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg.
4. L-NAME (100 μM) was found to produce a contraction of 0.64±0.09 mN mm⁻¹ in resting pulmonary arteries when added alone to the myograph bath. This contraction was not produced following removal of the endothelium. Vessel contraction to PGF_2α (100 μM) was found to be significantly greater when carried out in the presence of L-NAME (100 μM)–1.37±0.15 mN mm⁻¹ compared with 1.96±0.17 mN mm⁻¹. Dilatation following acetylcholine (ACh) (1 μM) was abolished in the presence of L-NAME (100 μM).
5. Rat pulmonary artery contraction in response to the addition of L-NAME and the absence of contraction upon removal of the endothelium provides supportive evidence of the active release of nitric oxide for the maintenance of resting tone.

     

NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery

1. The nature and cellular mechanisms that are responsible for endothelium-dependent relaxations resistant to indomethacin and N^G-nitro-L-arginine methyl ester (L-NAME) were investigated in phenylephrine (PE) precontracted isolated carotid arteries from the rabbit.
2. In the presence of the cyclo-oxygenase inhibitor, indomethacin (10 μM), acetylcholine (ACh) induced a concentration- and endothelium-dependent relaxation of PE-induced tone which was more potent than the calcium ionophore A23187 with pD₂ values of 7.03±0.12 (n=8) and 6.37±0.12 (n=6), respectively. The ACh-induced response was abolished by removal of the endothelium, but was not altered when indomethacin was omitted (pD₂ value 7.00±0.10 and maximal relaxation 99±3%, n=6). Bradykinin and histamine (0.01–100 μM) had no effect either upon resting or PE-induced tone (n=5).
3. In the presence of indomethacin plus the NO synthase inhibitor, L-NAME (30 μM), the response to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was abolished. However, the response to ACh was not abolished, although it was significantly inhibited with the pD₂ value and the maximal relaxation decreasing to 6.48±0.10 and 67±3%, respectively (for both P<0.01, n=8). The L-NAME/indomethacin insensitive vasorelaxation to ACh was completely abolished by preconstriction of the tissues with potassium chloride (40 mM, n=8).
4. The Ca²⁺-activated K⁺ (K_Ca) channel blockers, tetrabutylammonium (TBA, 1 mM, n=5) and charybdotoxin (CTX, 0.1 μM, n=5), completely inhibited the nitric oxide (NO) and prostacyclin (PGI₂)-independent relaxation response to ACh. However, iberiotoxin (ITX, 0.1 M, n=8) or apamin (1–3 μM, n=6) only partially inhibited the relaxation.
5. Inhibitors of the cytochrome P450 mono-oxygenase, SKF-525A (1–10 μM, n=6), clotrimazole (1 μM, n=5) and 17-octadecynoic acid (17-ODYA, 3 μM, n=7) also reduced the NO/PGI₂-independent relaxation response to ACh.
6. In endothelium-denuded rings of rabbit carotid arteries, the relaxation response to exogenous NO was not altered by either K_Ca channel blockade with apamin (1 μM, n=5) or CTX (0.1 μM, n=5), or by the cytochrome P450 mono-oxygenase blockers SKF-525A (10 μM, n=4) and clotrimazole (10 μM, n=5). However, the NO-induced response was shifted to the right by LY83583 (10 μM, n=4), a guanylyl cyclase inhibitor, with the pD₂ value decreasing from 6.95±0.14 to 6.04±0.09 (P<0.01).
7. ACh (0.01–100 μM) induced a concentration-dependent relaxation of PE-induced tone in endothelium-denuded arterial segments sandwiched with endothelium-intact donor segments. This relaxation to ACh was largely unaffected by indomathacin (10 μM) plus L-NAME (30 μM), but abolished by the combination of indomethacin, L-NAME and TBA (1 mM, n=5).
8. These data suggest that in the rabbit carotid artery: (a) ACh can induce the release of both NO and EDHF, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 only evokes the release of NO from the endothelium, (b) the diffusible EDHF released by ACh may be a cytochrome P450-derived arachidonic acid metabolite, and (c) EDHF-induced relaxation involves the opening of at least two types of K_Ca channels, whereas NO mediates vasorelaxation via a guanosine 3′: 5′-cyclic monophosphate (cyclic GMP)-mediated pathway, in which a cytochrome P450 pathway and K_Ca channels do not seem to be involved.

     

Hypoxic dilatation of porcine small coronary arteries: role of endothelium and KATP-channels

1. The aim of the present study was to determine the cellular mechanims and potential mediators involved in hypoxic dilatation of porcine small coronary arteries.
2. Small coronary arteries were isolated from a branch of the left anterior descending artery of porcine hearts, cannulated with glass micropipettes and studied in a perfusion myograph system. At a transmural pressure of 40 mmHg, the arteries had an internal diameter of 167.8±6.6 μm (n=37).
3. In arteries contracted with acetylcholine (ACh), hypoxia (0% O₂, 30 min) caused dilatation (86.9±6.7% relaxation, n=6) in vessels with endothelium but constriction in endothelium-denuded vessels.
4. Hypoxic vasodilatation occurring in arteries with endothelium was abolished by the K_ATP channel inhibitor, glibenclamide (0.44 μM), but was not affected by inhibition of nitric oxide synthase (L-NAME, 44 μM) or cyclo-oxygenase (indomethacin, 4.4 μM).
5. Bradykinin evoked endothelium-dependent relaxation that was inhibited by L-NAME (44 μM) but not glibenclamide 0.44 μM). Cromakalim (0.1–0.3 μM), a K_ATP channel opener, caused relaxation that was inhibited by glibenclamide, but was not affected by L-NAME (44 μM) and/or indomethacin (4.4 μM).
6. Endothelium-removal inhibited vasodilatation evoked by cromakalim, but increased vasodilator responses to the NO donor, SIN-1 (10⁻⁸ to 10⁻⁵ M).
7. These results indicate that hypoxia acted directly on vascular smooth muscle of small coronary arteries to cause contraction. However, this effect was overwhelmed by endothelium-dependent relaxation in response to hypoxia. This relaxation was most likely mediated by release of an endothelium-derived factor, distinct from nitric oxide or prostacyclin, that activated smooth muscle K_ATP-channels.

     

Alteration of flow-induced dilatation in mesenteric resistance arteries of L-NAME treated rats and its partial association with induction of cyclo-oxygenase-2

1. We investigated the response to pressure (myogenic tone) and flow of rat mesenteric resistance arteries cannulated in an arteriograph which allowed the measurement of intraluminal diameter for controlled pressures and flows. Rats were treated for 3 weeks with N^G-nitro-L-arginine methyl ester (L-NAME, 50 mg kg⁻¹ day⁻¹) or L-NAME plus the angiotensin I converting enzyme inhibitor (ACEI) quinapril (10 mg kg⁻¹ day⁻¹).
2. Mean blood pressure increased significantly in chronic L-NAME-treated rats (155±4 mmHg, n=8, vs control 121±6 mmHg, n=10; P<0.05). L-NAME-treated rats excreted significantly more dinor-6-keto prostaglandin F_1α (dinor-6-keto PGF_1α), the stable urinary metabolite of prostacyclin, than control rats. The ACEI prevented the rise in blood pressure and the rise in urinary dinor-6-keto PGF_1α due to L-NAME.
3. Isolated mesenteric resistance arteries, developed myogenic tone in response to stepwise increases in pressure (42±6 to 847±10 mN mm⁻¹, from 25 to 150 mmHg, n=9). Myogenic tone was not significantly affected by the chronic treatment with L-NAME or L-NAME+ACEI.
4. Flow (100 μl min⁻¹) significantly attenuated myogenic tone by 50±6% at 150 mmHg (n=10). Flow-induced dilatation was significantly attenuated by chronic L-NAME to 22±6% at 150 mmHg (n=10, P=0.0001) and was not affected in the L-NAME+ACEI group.
5. Acute in vitro N^G-nitro-L-arginine (L-NOARG, 10 μM) significantly decreased flow-induced dilatation in control but not in L-NAME or L-NAME+ACEI rats. Both acute indomethacin (10 μM) and acute NS 398 (cyclo-oxygenase-2 (COX-2) inhibitor, 1 μM) did not change significantly flow-induced dilatation in controls but they both decreased flow-induced dilatation in the L-NAME and L-NAME+ACEI groups. Acute Hoe 140 (bradykinin receptor inhibitor, 1 μM) induced a significant contraction of the isolated mesenteric arteries which was the same in the 3 groups.
6. Immunofluorescence analysis of COX-2 showed that the enzyme was expressed in resistance mesenteric arteries in L-NAME and L-NAME+ACEI groups but not in control. COX-1 expression was identical in all 3 groups.
7. We conclude that chronic inhibition of nitric oxide synthesis is associated with a decreased flow-induced dilatation in resistance mesenteric arteries which was compensated by an overproduction of vasodilator prostaglandins resulting in part from COX-2 expression. The decrease in flow-induced dilatation was prevented by the ACEI, quinapril.

     

Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon

1. We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.
2. Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μM). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).
3. L-Nitro-arginine-methyl ester (L-NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2–2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.
4. L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to L-arginine.
5. Higher concentrations of L-citrulline (e.g. 2 mM) had time-dependent effects. Brief exposure (15 min) reversed the effects of L-NAME, but during longer exposures (30 min) the effects of L-NAME gradually returned. In the continued presence of L-citrulline, L-arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L-citrulline, the ability to generate arginine from citrulline was reduced.
6. Aspartate (2 mM) had no effect on i.j.ps, the effects of L-NAME, or the actions of L-citrulline in the presence of L-NAME. L-Citrulline (0.2–2 mM) alone had no effect on i.j.ps under control conditions.
7. S-methyl-L-thiocitrulline (10 μM), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L-arginine (2 mM), but not by L-citrulline (2 mM).
8. These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L-citrulline to L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L-citrulline are elevated.

     

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03–30 μM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded.
2. Sodium rhein (0.3, 3 and 30 μM) significantly potentiated (52±11% at 30 μM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 μM) or ω-conotoxin GVIA (10 nM) sodium rhein (3 and 30 μM) did not enhance, but significantly reduced (49±10% and 44±8%, respectively, at 30 μM) acetylcholine-induced contractions.
3. Sodium rhein (0.3, 3 and 30 μM) significantly increased (65±11% at 30 μM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 μM), ω-conotoxin GVIA (10 nM) or atropine (0.1 μM), sodium rhein (3 and 30 μM) significantly reduced (50±10%, 55±8% and 46±10%, respectively, at 30 μM) substance P-induced contractions.
4. N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 μM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30±6%)- or substance P (36±6%)-induced contractions.
5. Sodium rhein (30 μM) significantly potentiated (29±9%) the electrically-evoked contractions. L-NAME (100 μM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 μM) significantly increased (32±9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment.
6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca²⁺ entry through the N-type Ca²⁺ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Endothelium-dependent sensory NANC vasodilatation: involvement of ATP,CGRP and a possible NO store

1. Non-adrenergic non-cholinergic (NANC) vasodilator nerves regulate tone in certain vascular beds. We have investigated the mechanisms of the NANC dilator response in the isolated small mesenteric artery of the rabbit by use of the tension myograph.
2. Small second or third order (150–300 μm in diameter) arteries of the rabbit mesenteric bed were mounted in a Mulvany tension myograph. Responses to electrical field stimulation (EFS) and exogenous vasodilators were investigated.
3. EFS (0.5–16 Hz, 10 V, 0.3 ms for 5 s), in the presence of guanethidine (5 μM) and atropine (1 μM) produced frequency-dependent relaxation of small arteries. Pretreatment with tetrodotoxin (1 μM) abolished the relaxation and desensitization with capsaicin (10 μM) strongly inhibited the relaxation.
4. Pretreatment with a P2Y-purinoceptor antagonist, basilen blue (3 μM) or a human calcitonin gene-related peptide (hCGRP) receptor antagonist, hCGRP_8–37 (1 μM) suppressed the NANC relaxation by approximately 40–60 % in each case and combined pretreatment almost abolished the relaxation.
5. The EFS-induced relaxation was suppressed by endothelium-removal, pretreatment with the soluble guanylyl cyclase inhibitor ODQ (1 μM) and the NO scavenger oxyhaemoglobin (OxyHb; 20 μM) but not by NO synthase inhibitors N^G-nitro-L-arginine methyl ester (L-NAME; 300 μM) or N^G-nitro-L-arginine (L-NOARG; 300 μM). Combined pretreatment with ODQ and CGRP_8–37 almost abolished the relaxation.
6. A P2Y-purinoceptor agonist, 2-methylthio ATP, produced endothelium-dependent relaxation which was inhibited by L-NAME and ODQ (1 μM), whilst hCGRP produced endothelium-independent and ODQ-insensitive relaxation.
7. Ultraviolet light (320 nm, 5 shots over 20 s) produced relaxation that was blocked by both OxyHb and ODQ but not by N^G-monomethyl-L-arginine (L-NMMA, 300 μM).
8. The present study suggests that EFS-induced NANC relaxation of the mesenteric small artery of the rabbit is mediated mainly by capsaicin-sensitive sensory C-fibres and that both ATP and CGRP are involved. The action of ATP released by EFS appears to be endothelium-dependent and involve activation of soluble guanylyl cyclase, but is resistant to inhibitors of NO synthase. The response to CGRP is endothelium-independent. These results show that ATP and CGRP account fully for the NANC relaxation of this vessel type and that the endothelium is involved in NANC-induced relaxation. The endothelium-dependent part of the response is consistent with the release of NO, either from NO synthase, incompletely inhibited by the NO synthase inhibitors, or by some preformed stores.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres

1. The effect of Tityus serrulatus scorpion venom and its toxin components on the rabbit isolated corpus cavernosum was investigated by use of a bioassay cascade.
2. Tityus serrulatus venom (3–100 μg), acetylcholine (ACh; 0.3–30 nmol) and glyceryl trinitrate (GTN; 0.5–10 nmol) dose-dependently relaxed rabbit isolated corpus cavernosum preparations precontracted with noradrenaline (3 μM). The selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ; 30 μM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 μM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible.
3. The non-selective NO synthase (NOS) inhibitors N^Ω-nitro-L-arginine methyl ester (L-NAME; 10 μM) and N^G-iminoethyl-L-ornithine (L-NIO; 30 μM) also increased the tone of the rabbit isolated corpus cavernosum and markedly reduced both ACh- and Tityus serrulatus venom-induced relaxations without affecting those evoked by GTN. The inhibitory effect was reversed by infusion of L-arginine (300 μM), but not D-arginine (300 μM). The neuronal NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM, 100 μM) did not affect either the tone of the rabbit isolated corpus cavernosum or the relaxations induced by ACh, bradykinin (Bk), Tityus serrulatus venom and GTN. TRIM was approximately 1,000 times less potent than L-NAME in inhibiting rabbit cerebellar NOS in vitro, as measured by the conversion of [³H]-L-arginine to [³H]-L-citrulline.
4. The protease inhibitor aprotinin (Trasylol; 10 μg ml⁻¹) and the bradykinin B₂ receptor antagonist Hoe 140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]-BK; 50 nM) did not affect the rabbit isolated corpus cavernosum relaxations induced by Tityus serrulatus venom. The ATP-dependent K⁺ channel antagonist glibenclamide (10 μM) and the Ca²⁺-activated K⁺  channel antagonists apamin (0.1 μM) and charybdotoxin (0.1 μM) also failed to affect the venom-induced relaxations. Similarly, the K⁺ channel blocker tetraethylammonium (TEA; 10 μM) had no effect on the venom-induced relaxations.
5. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose-dependent and non-tachyphylactic manner. Ruthenium red (30 μM), an inhibitor of capsaicin-induced responses, markedly reduced the relaxations caused by capsaicin, but failed to affect those induced by Tityus serrulatus venom. L-NAME (10 μM) had no effect on the capsaicin-induced relaxations of the rabbit isolated corpus cavernosum.
6. The sodium channel blocker tetrodotoxin (TTX; 1 μM) abolished the relaxations of the rabbit isolated corpus cavernosum induced by Tityus serrulatus venom without affecting those evoked by capsaicin, ACh and GTN. Tetrodotoxin (1 μM) also promptly reversed the response to the venom when infused during the relaxation phase.
7. The bioassay cascade of the toxin components purified from Tityus serrulatus venom revealed that only fractions X, XI and XII caused dose-dependent relaxations of the rabbit isolated corpus cavernosum and these were markedly reduced by either TTX (1 μM) or L-NAME (10 μM).
8. Our results indicate that Tityus serrulatus scorpion venom (and the active fractions X, XI and XII) relaxes rabbit corpus cavernosum via the release of NO. This release is specifically triggered by the activation of capsaicin-insensitive cavernosal non-adrenergic non-cholinergic (NANC) fibres, that may possibly be nitrergic neurones. Tityus serrulatus venom may therefore provide an important tool for understanding further the mechanism of NANC nitrergic nerve activation.

     

Involvement of 5-hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine

1. 5-Hydroxytryptamine (5-HT; 1 nM–100 μM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC₅₀; 68–84 nM) was more sensitive than the longitudinal muscle (EC₅₀; 1.3–1.44 μM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations.
2. Pretreatment with tetrodotoxin (1 μM), propranolol (1 μM), atropine (1 μM), guanethidine (10 μM) or L-NAME (100 μM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells.
3. 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, α-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10–100 μM.
4. Inhibition by 5-HT was not decreased by either pindolol (1 μM), ketanserin (1 μM), tropisetron (10 μM), MDL72222 (1 μM) or {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (10 μM), but was antagonized by the following compounds in a competitive manner (with pA₂ values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86).
5. Ro 20-1724 (20 μM) and rolipram (10 μM) significantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 μM) nor dipyridamole (10 μM) altered the response of 5-HT.
6. 5-HT (1 nM–1 μM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle.
7. From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT₇ receptor, mediates the inhibitory effect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT₇ receptors.

     

Characterization of the potassium channels involved in EDHF-mediated relaxation in cerebral arteries

1. In the presence of N^G-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 μM), the relaxations induced by acetylcholine and the calcium (Ca) ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 are considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF) in the guinea-pig basilar artery.
2. Inhibitors of adenosine 5′-triphosphate (ATP)-sensitive potassium (K)-channels (K_ATP; glibenclamide, 10 μM), voltage-sensitive K-channels (K_V; dendrotoxin-I, 0.1 μM or 4-aminopyridine, 1 mM), small (SK_Ca; apamin, 0.1 μM) and large (BK_Ca; iberiotoxin, 0.1 μM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine.
3. Synthetic charybdotoxin (0.1 μM), an inhibitor of BK_Ca and K_V, caused a rightward shift of the concentration-response curve for acetylcholine and reduced the maximal relaxation in the presence of L-NOARG and indomethacin, whereas the relaxation induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was not significantly inhibited.
4. A combination of charybdotoxin (0.1 μM) and apamin (0.1 μM) abolished the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. However, the acetylcholine-induced relaxation was not affected by a combination of iberiotoxin (0.1 μM) and apamin (0.1 μM).
5. Ciclazindol (10 μM), an inhibitor of K_V in rat portal vein smooth muscle, inhibited the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, and the relaxations were abolished when ciclazindol (10 μM) was combined with apamin (0.1 μM).
6. Human pial arteries from two out of four patients displayed an L-NOARG/indomethacin-resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 μM) and apamin (0.1 μM), whereas each toxin had little effect alone.
7. The results suggest that K_V, but not K_ATP and BK_Ca, is involved in the EDHF-mediated relaxation in the guinea-pig basilar artery. The synergistic action of apamin and charybdotoxin (or ciclazindol) could indicate that both K_V and SK_Ca are activated by EDHF. However, a single type of K-channel, which may be structurally related to K_V and allosterically regulated by apamin, could also be the target for EDHF.

     

Theophylline dilates rat diaphragm arterioles via the prostaglandins pathway

1. We investigated by intravital microscopy in rats, the in vivo direct effects of theophylline on the diameters of second and third order diaphragm arterioles.
2. Theophylline (1–100 μM) dilated second and third order diaphragm arterioles significantly, and with an amplitude which was not statistically different from the one obtained with adenosine (1–100 μM). Enprofylline (1–100 μM), a theophylline analogue with poor adenosine-receptor antagonism but with similar or higher phosphodiesterases inhibition properties than theophylline, also dilated diaphragm arterioles, causing however, a significantly smaller dilatation than theophylline.
3. Neither the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 50 nM), nor the A₂ adenosine receptor antagonist 3,7-dimethyl-1-proparglyxanthine (DMPX, 10 μM) reduced significantly theophylline-induced arteriolar dilatation.
4. Theophylline (100 nM) abolished adenosine-induced arteriolar dilatation.
5. The dilatation induced by theophylline was unchanged by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine (NNA, 300 μM).
6. Theophylline-induced arteriolar dilatation was abolished by the prostaglandin synthesis inhibitors mefenamic acid or indomethacin (20 μM).
7. These findings show that theophylline induced a significant dilatation of diaphragm arterioles via the release of prostaglandins.

     

AT2 antagonist-sensitive potentiation of angiotensin II-induced constriction by NO blockade and its dependence on endothelium and P450 eicosanoids in rat renal vasculature

1. We showed earlier that NO inhibition caused a left-shift and augmented E_max of the concentration-response curve of AT₁-mediated (angiotensin II)-induced vasoconstrictions (AII-VC) in the rat kidney. The 0.01–0.1 nM AII-VC unmasked by the potentiating effect of NO inhibition, were sensitive not only to AT₁ (L158809), but also to AT₂ receptor (PD123319) antagonists. We now demonstrate the role of endothelium and eicosanoids in the NO-masked AT₁/AT₂-mediated component of the AII-VC in isolated indomethacin-perfused kidneys of the rat.
2. L-NAME increased 0.1 nM AII-VC 7.2 fold. Pretreatment of the kidneys with factor VIII antibody/complement or with the detergent CHAPS to damage endothelium, decreased carbachol-induced vasodilatation and blunted by 60 and 30% respectively, the enhancement of AII-VC during NO inhibition.
3. L-NAME also increased 3 μM noradrenaline (NA)-induced vasoconstriction (NA-VC) 8.1 fold. In contrast to AII-VC, endothelium damage was without effect on the enhancement of NA-VC by L-NAME, suggesting a dominant role of endothelium-derived NO in the enhancement of NA-VC.
4. During NO inhibition, ETYA (2 μM; an inhibitor of all arachidonic acid derived pathways) and α-naphtoflavone (10 μM; an inhibitor of the cytochrome P450 isozymes), decreased by 85% the 0.1 nM AII-VC.
5. In conclusion, during NO inhibition, the AT₁-mediated constriction to low concentrations of AII, which is sensitive to AT₂ antagonists, depends on intact endothelium, and can be blocked by inhibition of eicosanoid synthesis. The results suggest that the AII-mediated vasoconstriction through AT₁ receptors is potentiated in the absence of NO, by the release of eicosanoids from the endothelium through AT₂ receptors.

     

Evidence that both nitric oxide (NO) and a non-NO hyperpolarizing factor elicit NANC nerve-mediated relaxation in the rat isolated anococcygeus

1. Responses to electrical field stimulation (EFS; 0.5–10 Hz, 0.2 ms duration, supramaximal voltage for 20 s) of non-adrenergic, non-cholinergic, (NANC) nerves were obtained in preparations of rat anococcygeus pre-contracted with titrated concentrations of phenylephrine (0.1–1 μM) to ∼40% of their maximum contraction to phenylephrine (F_max) regardless of drug treatment.
2. With this set level of active force, NANC nerve stimulation resulted in relaxations that were maximal (peak relaxation) at 0.5–1 Hz, abolished by tetrodotoxin (1 μM) but only minimally blocked by the nitric oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine, (L-NOARG; 100 μM). Furthermore, the nitric oxide (NO) scavenger, oxyhaemoglobin (HbO; 30 μM) gave no further block alone or in combination with L-NOARG (100 μM). By comparison, in preparations contracted with phenylephrine to ∼70% F_max, relaxations to NANC nerve stimulation were markedly reduced or abolished by combined treatment with L-NOARG (100 μM) and HbO (30 μM).
3. Nifedipine (0.3 μM) significantly inhibited NANC nerve-mediated relaxations, which became frequency-dependent and abolished those resistant to L-NOARG (100 μM) and HbO (30 μM).
4. These data suggest that a non-NO, hyperpolarizing factor and NO both contribute to NANC nerve-mediated inhibitory responses in the rat anococcygeus. However, responses to the non-NO factor were observed only in preparations contracted sub-maximally by a nifedipine-sensitive mechanism.