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.

     

Functional response of the rat kidney to inhibition of nitric oxide synthesis: role of cytochrome P450-derived arachidonate metabolites

1. We tested the hypothesis that nitric oxide (NO) exerts a tonic inhibitory influence on cytochrome P450 (CYP450)-dependent metabolism of arachidonic acid (AA).
2. N^ω-nitro-L-Arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), increased mean blood pressure (MBP), from 91±6 to 137±5 mmHg, renal vascular resistance (RVR), from 9.9±0.6 to 27.4±2.5 mmHg ml⁻¹ min⁻¹, and reduced renal blood flow (RBF), from 9.8±0.7 to 6.5±0.6 ml min⁻¹) and GFR from 1.2±0.2 to 0.6±0.2 ml 100 g⁻¹ min⁻¹) accompanied by diuresis (UV, 1.7±0.3 to 4.3±0.8 μl 100 g⁻¹ min⁻¹), and natriuresis (U_NaV, 0.36±0.04 to 1.25±0.032 μmol 100 g⁻¹ min⁻¹).
3. 12, 12 dibromododec-enoic acid (DBDD), an inhibitor of ω hydroxylase, blunted L-NAME-induced changes in MBP, RVR, UV and U_NaV by 63±8, 70±5, 45±8 and 42±9%, respectively, and fully reversed the reduction in GFR by L-NAME. Clotrimazole, an inhibitor of the epoxygenase pathway of CYP450-dependent AA metabolism, was without effect.
4. BMS182874 (5-dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamide), an endothelin (ET)_A receptor antagonist, also blunted the increases in MBP and RVR and the diuresis/natriuresis elicited by L-NAME without affecting GFR.
5. Indomethacin blunted L-NAME-induced increases in RVR, UV and U_NaV. BMS180291 (1S-(1α,2α,3α.4α)]-2-[[3-[4-[(pentylamino)carbonyl]-2-oxazolyl] - 7 - oxabicyclo[2.2.1]hept - 2 -yl]methyl]benzenepropanoic acid), an endoperoxide receptor antagonist, attenuated the pressor and renal haemodynamic but not the renal tubular effects of L-NAME.
6. In conclusion, the renal functional effects of the CYP450-derived mediator(s) expressed after inhibition of NOS with L-NAME were prevented by inhibiting either CYP450 ω hydroxylase or cyclo-oxygenase or by antagonizing either ET_A or endoperoxide receptors. 20-hydroxyeicosatetraenoic acid (20-HETE) fulfils the salient properties of this mediator.

     

The effect of chronic treatment with trandolapril on cyclic AMP- and cyclic GMP-dependent relaxations in aortic segments of rats with chronic heart failure

1. Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation.
2. Cardiac output indices of coronary artery-ligated and sham-operated rats were 125±8 and 189±10 ml min⁻¹ kg⁻¹, respectively (P<0.05), indicating the development of CHF at this period.
3. The maximal relaxant response of aortic segments to 10 μM acetylcholine in rats with CHF and sham-operated rats was 64.0±5.7 and 86.9±1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats.
4. In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 μM isoprenaline (44.5±6.7%) was lower that sham-operated rats (81.3±2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by N^G-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 μM dibutyryl cyclic AMP in rats with CHF (26.8±2.7%) was lower than that in sham-operated rats (63.4±11.8%, P<0.05).
5. Trandolapril (3 mg kg⁻¹ day⁻¹) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5±3.6 ml min⁻¹) was lower than that of sham-operated rats (55.0±3.0 ml min⁻¹), and this reduction was reversed (54.1±3.4 ml min⁻¹) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7±1.0%; that to isoprenaline, 80.5±2.8%; that to dibutyryl cyclic AMP, 54.7±6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged.
6. The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.

     

Dose- and time-dependence of L-NAME neuroprotection in transient focal cerebral ischaemia in rats

1. In this study the effect of the dose and administration time of N^G-nitro-L-arginine methyl ester (L-NAME), an NO-synthase inhibitor, in a model of transient focal cerebral ischaemia in rats was investigated.
2. Two injections of L-NAME were given, of 1, 3 and 10 mg kg⁻¹, 5 min and 3 h after the onset of ischaemia. None of the doses gave any striatal neuroprotection, but 1 and 3 mg kg⁻¹ L-NAME reduced the infarcted volume in the cortex (by 26%, P<0.01 for 1 mg kg⁻¹ and 21%, P<0.05 for 3 mg kg⁻¹), whereas 10 mg kg⁻¹ had no neuroprotective effect.
3. Single injections of L-NAME 1 mg kg⁻¹, given 5 min or 3 h after ischaemia onset, had similar neuroprotective effects on the cortical infarction as did the repeated injections.
4. L-NAME 1 mg kg⁻¹ given 3, 6 or 9 h after ischaemia induction reduced the cortical infarct volume by 19% (P<0.01) when given 3 h after ischaemia, by 21% (P<0.01) when given at 6 h, and by 16% (P<0.5) when given at 9 h, but had no neuroprotective activity when given 12 h after ischaemia.
5. Thus a low dose of L-NAME is neuroprotective in a model of transient focal ischaemia, with a wide therapeutic window, much larger than that found for MK-801.

     

Superoxide and endothelium-dependent constriction to flow in porcine small pulmonary arteries

1. The aim of this study was to determine the response of porcine small pulmonary arteries to intralumenal flow and to identify the cellular mechanisms and potential mediators involved in the response.
2. Porcine small pulmonary arteries were isolated from a branch of the main intrapulmonary artery of the lower lung lobe and studied in a perfusion myograph system that allowed independent control of transmural pressure and intralumenal flow. At a transmural pressure of 20 mmHg, the baseline internal diameter (BID) of the arteries was 251.2±16.1 μm (n=16).
3. Under quiescent conditions or during constriction with U46619 to ∼60% of BID, intralumenal flow caused reversible constriction in arteries with endothelium (in the presence of U46619, flow decreased diameter from 60.0±2.5% to 49.5±3.0% BID at 10 μl min⁻¹, n=16, P<0.05) but no change in diameter of arteries without endothelium.
4. In the presence of superoxide dismutase (SOD, 150 u ml⁻¹), the response to flow was converted from constriction to vasodilatation (in presence of U46619 and SOD, flow increased diameter from 54.2±3.4% to 76.7±4.5% BID at 10 μl min⁻¹, n=10, P<0.05). Inhibition of NO synthase with L-NAME (3×10⁻⁵ M) abolished the flow-induced vasodilatation occurring in the presence of SOD and the flow-induced constriction occurring in the absence of SOD. In arteries with endothelium, L-NAME (3×10⁻⁵ M) caused significant vasoconstriction, whereas SOD did not alter vasomotor tone.
5. Acetylcholine (10⁻⁸ to 10⁻⁶  M) caused endothelium-dependent relaxation of small pulmonary arteries that was not significantly affected by SOD (150 u ml⁻¹) but was inhibited by L-NAME (3×10⁻⁵ M).
6. These results suggest that in small, porcine, isolated pulmonary arteries, intralumenal flow increases the production of NO but this is obscured by the generation of superoxide which causes vasoconstriction.

     

Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle.
2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO₂Me) and a recently developed human chymase substrate Pro¹¹-,D-Ala¹²)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used.
3. Angiotensin I (AI) (0.3 μM) induced a contractile response amounting to 58±5% (n=12) of the initial K⁺ (124 mM)-induced contractions. This response was reduced to 36±3% (n=8) by the ACE-inhibitor enalaprilat (10 μM), while pretreatment with soybean trypsin inhibitor (STI 200 μg ml⁻¹) or CH 5450 (10 μM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19±5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1±1% (n=6).
4. The substrate (Pro¹¹-,D-Ala¹²)-AI (3 μM) produced contractions which amounted to 57±4% (n=13) of the initial K⁺ (124 mM) contractions. These contractions were not affected by enalaprilat (10 μM). On the other hand, STI (200 μg ml⁻¹) and CH 5450 (10 μM) added separately, depressed the (Pro¹¹-,D-Ala¹²)-AI-induced contractions to 34±5% (n=6) and 24±4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition.
5. Experiments with detrusor membrane fractions incubated with AI (50 μM) were performed. In the presence of enalaprilat (100 μM), carboxypeptidase inhibitor CPI (10 μg ml⁻¹) and aprotinin (15 μM), CH 5450 (10 nM–1 μM) caused a concentration-dependent inhibition of AII formation.
6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

     

Endothelium-independent relaxation of aortic rings by the nitric oxide synthase inhibitor diphenyleneiodonium

1. The flavoprotein binder diphenyleneiodonium (DPI) is a potent, irreversible inhibitor of nitric oxide synthase (NOS), but produces only a transient pressor response following systemic administration to animals, despite evidence of persistent NOS inhibition. To characterize further the effects of DPI on vascular tone, isometric tension was recorded from rat isolated aortic rings mounted between steel wires in an organ bath.
2. The NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME, 1 mM) initiated an additional contraction of prostaglandin F_2α-preconstricted rings with endothelium which was sustained throughout the period of L-NAME exposure (+234±39% at 15 min). In contrast, addition of DPI (5 μM) to rings with endothelium produced a transient initial contraction (+111±27% at 2 min) followed by a more sustained relaxation (−27±19% at 15 min, P<0.001 vs L-NAME).
3. The contraction to DPI was also observed in rings without endothelium, was abolished by L-NAME pretreatment, and was unaffected by the α-adrenoreceptor inhibitor prazosin. Relaxation in response to DPI was not inhibited by endothelium removal or by pretreatment with either L-NAME or with the ATP-sensitive potassium channel blocker glibenclamide.
4. The endothelium-independent relaxation to DPI was inhibited at 23°C and its time course was delayed by pretreatment with the guanylate cyclase inhibitor methylene blue.
5. Thus, in addition to a transient initial contraction due to NOS inhibition, DPI produces an endothelium-independent, temperature-dependent relaxation which appears in part due to activation of guanylate cyclase. This relaxant effect of DPI may explain the transient nature of its pressor effect in vivo despite sustained NOS inhibition.

     

Comparison of the acute cardiotoxicity of the antimalarial drug halofantrine in vitro and in vivo in anaesthetized guinea-pigs

1. Several unrelated drugs have pro-arrhythmic activity associated with an ability to prolong the QT interval of the ECG. The aim of this work was to examine the effects of the antimalarial drug halofantrine in vivo and in vitro.
2. In anaesthetized guinea-pigs consecutive bolus doses of halofantrine (0.3, 1, 3, 10 and 30 mg kg⁻¹, i.v.) at 25 min intervals caused dose-dependent prolongation of the rate corrected QTc interval and bradycardia. The change in heart rate became significant after administration of 10 mg kg⁻¹ halofantrine (−23±9 beats min⁻¹) whereas the increase in QTc was significant with only 1 mg kg⁻¹ halofantrine (22±10 ms). It was only with the highest dose of halofantrine that the PR interval was increased (from 52±3 to 67±4 ms) and second degree atrioventricular (AV) block (type 1 Mobitz) occurred in all animals. No changes were observed in any parameters in a separate group of guinea-pigs which received vehicle (dimethylacetamide 60% propylene glycol 40%) at equivalent time points.
3. The blood concentrations of halofantrine ranged from 0.26±0.17 μM after administration of 0.3 mg kg⁻¹ to 2.79±0.87 μM after 30 mg kg⁻¹, i.v. There was a significant correlation between the blood concentrations of halofantrine and the changes in QTc interval.
4. In guinea-pig left papillary muscles the effective refractory period was increased significantly 60 min after addition of halofantrine; from 161±4 to 173±6 ms with 10 μM, 156±8 to 174±6 ms with 30 μM and 165±6 to 179±5 ms with 100 μM halofantrine. However, the vehicle (0.1% Tween 80 in DMSO; final concentration of vehicle in Krebs, 1%) also increased the effective refractory period from 164±5 to 173±6 ms. Similar results were obtained in right ventricular strips but left atrial effective refractory periods were not altered by either the vehicle or halofantrine.
5. The results of these experiments suggest that any direct effects that halofantrine may have had on the effective refractory period of cardiac muscle cannot be separated from those of the vehicle. The prolongation of QTc and consistent observation of AV block with halofantrine in anaesthetized guinea-pigs suggest that in vivo models may be more useful for further studies investigating the mechanisms underlying the cardiotoxicity of halofantrine.

     

The influence of antibodies to TNF-α and IL-1β on haemodynamic responses to the cytokines,and to lipopolysaccharide,in conscious rats

1. Male, Long Evans rats (350–450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-α and human recombinant interleukin-1 (IL-1)β, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-α and IL-1β on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-α and IL-1β had any influence on the responses to lipopolysaccharide (LPS).
2. TNF-α (10, 100 and 250 μg kg⁻¹, in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum Δ, +101±9 beats min⁻¹) and modest hypotension (maximum Δ, −10±2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum Δ, +6±6, +27±9, and +61±12% at 10, 100 and 250 μg kg⁻¹, respectively).
3. IL-1β (1, 10, and 100 μg kg⁻¹ in separate groups, n=8, 8 and 9, respectively) evoked changes similar to those of TNF-α (maximum Δ heart rate, +69±15 beats min⁻¹; maximum Δ mean blood pressure, −14±2 mmHg; maximum Δ hindquarters vascular conductance, +49±17%), but with no clear dose-dependency.
4. TNF-α (250 μg kg⁻¹) and IL-1β (10 μg kg⁻¹) together caused tachycardia (maximum Δ, +76±15 beats min⁻¹) and hypotension (maximum Δ, −24±2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52±6%, +23±8%, and +52±11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum Δ, −50±3% and −58±3%, respectively). Although bolus injection of LPS (3.5 mg kg⁻¹) caused an initial hypotension (maximum Δ, −27±11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-α and IL-1β or after bolus injection of LPS, the secondary reduction in blood pressure was similar (−16±2 and −13±3 mmHg, respectively), but in the former group the tachycardia (+117±14 beats min⁻¹) and increase in hindquarters vascular conductance (+99±21%) were greater than after bolus injection of LPS (+54±16 beats min⁻¹ and +43±9%, respectively).
5. Pretreatment with antibodies to TNF-α and IL-1β (300 mg kg⁻¹) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h.
6. Pretreatment with antibodies to TNF-α and IL-1β before infusion of LPS (150 μg kg⁻¹ h⁻¹ for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion.
7. The results indicate that although co-administration of TNF-α and IL-1β can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.

     

Cigarette smoke-inhibition of neurogenic bronchoconstriction in guinea-pigs in vivo: involvement of exogenous and endogenous nitric oxide

1. We investigated the effect of acute inhalation of cigarette smoke on subsequent non-adrenergic, non-cholinergic (NANC) neural bronchoconstriction in anaesthetized guinea-pigs in vivo by use of pulmonary insufflation pressure (PIP) as an index of airway tone. The contribution of endogenous nitric oxide (NO) was investigated with the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME). The contribution of plasma exudation to the response was investigated with Evans blue dye as a plasma marker.
2. Inhalation of 50 tidal volumes of cigarette smoke or air had no significant effect on baseline PIP. In the presence of propranolol and atropine (1 mg kg⁻¹ each), electrical stimulation of the vagus nerves in animals given air 30 min previously induced a frequency-dependent increase in PIP above sham stimulated controls (16 fold increase at 2.5 Hz, 24 fold increase at 10 Hz). In contrast, in smoke-exposed animals, the increase in subsequent vagally-induced PIP was markedly less than in the air controls (90% less at 2.5 Hz, 76% less at 10 Hz).
3. L-NAME (10 mg kg⁻¹), given 10 min before air or smoke, potentiated subsequent vagally-induced (2.5 Hz) NANC bronchoconstriction by 338% in smoke-exposed animals, but had no significant effect in air-exposed animals. The inactive enantiomer D-NAME (10 mg kg⁻¹) had no effect, and the potentiation by L-NAME was partially reversed by the NO-precursor L-arginine (100 mg kg⁻¹). Vagal stimulation did not affect the magnitude of vagally-induced bronchoconstriction 30 min later.
4. Cigarette smoke exposure reduced the magnitude of subsequent bronchoconstriction induced by neurokinin A (NKA) by 37% compared with the effect of NKA in air-exposed animals. L-NAME had no significant effect on the smoke-induced inhibition of NKA-induced bronchoconstriction.
5. Vagally-induced plasma exudation in the main bronchi was greater in smoke-exposed animals compared with air-exposed animals (120% greater at 2.5 Hz, 82% greater at 10 Hz).
6. We conclude that cigarette smoke-induced inhibition of subsequent NANC neurogenic bronchoconstriction is not associated with inhibition of airway plasma exudation and is mediated in part via exogenous smoke-derived NO, or another bronchoprotective molecule, and by endogenous NO.

     

A comparison of the effects of L-NAME, 7-NI and L-NIL on carrageenan-induced hindpaw oedema and NOS activity

1. Intraplantar injection of carrageenan (150 μl, 1–3% w/v) in the rat resulted in a dose-related increase in hindpaw weight (oedema) characterized by a rapid ‘early'' phase (up to 2.5 h) response followed by a more sustained ‘late'' phase (2–6 h) response. No change in weight of either the contralateral (i.e. noninjected) hindpaw or hindpaws injected with saline was observed.
2. Six hours after intraplantar injection of carrageenan (1–3% w/v) hindpaw constitutive (i.e. calcium-dependent) nitric oxide synthase (cNOS) activity (determined ex vivo as the conversion of radiolabelled L-arginine to radiolabelled citrulline) was increased (e.g. 2% w/v; 0.64±0.08 pmol citrulline mg⁻¹ protein 15 min⁻¹ c.f. 0.08±0.04 pmol citrulline mg⁻¹ protein 15 min⁻¹ in saline-injected, control animals, n=4, P<0.05). Carrageenan injection also resulted in the appearance in hindpaw homogenates of inducible (i.e. calcium-independent) nitric oxide synthase (iNOS, e.g. 2% w/v; 0.67±0.14 pmol citrulline mg⁻¹ protein 15 min⁻¹, n=4). Hindpaw cyclic GMP concentration was also significantly increased 6 h after intraplantar injection of carrageenan (e.g. 2% w/v; 379.6±26.8 fmol mg⁻¹ protein c.f. 261.8±42.2 fmol mg⁻¹ protein, in saline-injected, control animals, n=4, P<0.05).
3. Pretreatment (5–25 mg kg⁻¹, i.p., 30 min before carrageenan, 2% w/v) of animals with L-N^G nitro arginine methyl ester (L-NAME; isoform nonselective inhibitor of NOS) or 7-nitro indazole (7-NI; inhibitor of neuronal NOS, nNOS) caused dose-related inhibition of both the early (2 h) and late (6 h) phase hindpaw oedema, associated with reduced hindpaw iNOS and cNOS activity and cyclic GMP concentration in animals killed at 6 h. Administration of 7-NI (5–25 mg kg⁻¹, i.p.) to animals 2.5 h after intraplantar carrageenan (2% w/v) injection (i.e. at the end of the early phase oedema response) produced dose-related inhibition of the late phase response.
4. Pretreatment (5–25 mg kg⁻¹, i.p., 30 min before carrageenan, 2% w/v) of animals with L-N6-iminoethyllysine (L-NIL, selective inhibitor of iNOS) (5–25 mg kg⁻¹) failed to affect the early phase hindpaw oedema response but did produce a dose-related inhibition of the late phase oedema. L-NIL pretreatment also inhibited the carrageenan-induced increase in both hindpaw iNOS and cNOS activity as well as the rise in hindpaw cyclic GMP concentration.
5. The present experiments demonstrate an anti-inflammatory effect of 7-NI as evidenced by inhibition of carrageenan-induced hindpaw oedema in the rat. Inhibition of nNOS (early phase) and iNOS (late phase) at the site of inflammation most probably accounts for the anti-inflammatory activity observed. These data suggest a role for nitric oxide synthesized by the nNOS isoform (most probably within sensory nerves) in this model of inflammation.

     

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

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus.
2. ODQ (10⁻⁶ and 10⁻⁵ M) concentration-dependently inhibited the short-lasting relaxations by NO (2×10⁻⁶ M–10⁻⁴ M) administered as a bolus without influencing the relaxation by 3×10⁻⁸ M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10⁻⁶ and 10⁻⁵ M ODQ.
3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5–16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10⁻⁶ and 10⁻⁵ M ODQ, and by the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 3×10⁻⁴ M).
4. ODQ (10⁻⁶ M) and L-NAME (3×10⁻⁴ M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz).
5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F_2α (PGF_2α), the inhibitory effect of 3×10⁻⁴ M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3×10⁻⁷ M PGF_2α.
6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.

     

Study of the in vivo and in vitro cardiovascular effects of a hydralazine-like vasodilator agent (HPS-10) in normotensive rats

1. In this work, the cardiovascular effects of HPS-10, a new vasodilator agent, were studied in rats.
2. In conscious normotensive rats, oral administration of HPS-10 (4–9 mg kg⁻¹) produced a dose-related and long-lasting fall in systolic arterial blood pressure (ED₃₀ of 5.32 mg kg⁻¹), accompanied by an increase in heart rate (ED₃₀ of 8.43 mg kg⁻¹). This tachycardia was totally inhibited by pretreatment with (±)-propranolol (10 mg kg⁻¹, p.o.).
3. In anaesthetized normotensive rats, HPS-10 (0.3–0.6 mg kg⁻¹, i.v.) produced a gradual, dose-dependent and sustained decrease in systolic, diastolic and mean arterial pressure (MAP) (ED₃₀ for MAP of 0.41 mg kg⁻¹, i.v.), accompanied by a significant bradycardia at high doses (>0.4 mg kg⁻¹; ED₂₀ of 0.61 mg kg⁻¹, i.v.). HPS-10 (0.5 mg kg⁻¹, i.v.) did not modify the positive chronotropic effects induced by intravenous administration of noradrenaline (NA; 5 μg kg⁻¹), angiotensin II (AII; 0.2 μg kg⁻¹) and nicotine (200 μg kg⁻¹) but markedly inhibited the hypertensive response produced by these agents.
4. In rat isolated rubbed aorta, HPS-10 (0.1–1 mM) non-competitively and with almost equal effectiveness antagonized the contractions induced by NA, AII (in normal Krebs solution) and Ca²⁺ (in depolarizing Ca²⁺-free high-K⁺ 50 mM solution). In the experiments in Ca²⁺-free medium, HPS-10 (1 mM) considerably inhibited the contractions induced by NA, AII and caffeine in rat aorta.
5. Furthermore, in the studies with radioactive Ca²⁺, HPS-10 (1 mM) did not modify the basal uptake of ⁴⁵Ca²⁺ but strongly decreased the influx of ⁴⁵Ca²⁺ induced by NA, AII and K⁺ in rat aortic rings.
6. In rat isolated atria, HPS-10 (1 mM) produced a positive inotropic/negative chronotropic effect.
7. HPS-10 (0.3 mM) significantly inhibited the sustained and transient Ba²⁺ inward current (I_Ba) recorded in whole-cell clamped rat aortic myocytes.
8. These results indicate that the non-selective vasorelaxant effects of HPS-10 in rat aortic rings can be attributed to transmembrane Ca²⁺-antagonist activity and an intracellular action on smooth muscle cells. The direct vasodilator action of HPS-10 observed in rat isolated aorta may be responsible for the HPS-10 hypotensive activity in anaesthetized normotensive rats.

     

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.

     

Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats

1. We investigated the effect of the non-peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound.
2. In fed animals, SR 48692 dose-dependently (0.5 to 12.5 mg kg⁻¹, p.o., 0.03 to 1 mg kg⁻¹, i.p. and 0.1 to 1 μg/rat, i.c.v.) increased urine output and urinary excretion of Na⁺, K⁺ and Cl⁻ and reduced urine osmolality. The diuretic activity was also evident in water-deprived, fasted animals and in fasted, water-loaded rats.
3. NT (0.1 μg/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 μg/rat, i.c.v.). The opposite result was obtained in fasted, water-loaded animals: NT dose-dependently (0.01 and 0.1 μg/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on-going diuresis.
4. The NO synthesis inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME; 30 mg kg⁻¹, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; L-arginine (1 g kg⁻¹, i.p.) but not D-arginine (1 g kg⁻¹, i.p.) restored the SR 48692-dependent increase in diuresis. L-NAME had no effect on furosemide-stimulated diuresis.
5. Systemically administered L-NAME or i.c.v. NT in fasted, water-loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. L-arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine.
6. Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT-receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692.
7. In fed rats, the NO donor isosorbide-dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose-dependently (1 and 5 mg kg⁻¹, i.p.) stimulated urine output.
8. The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na⁺, K⁺ and Cl⁻.

     

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.

     

Cerebrovascular effects of nitric oxide manipulation in spontaneously hypertensive rats

1. Evidence that nitric oxide (NO) bioactivity is altered in chronic hypertension is conflicting, possibly as a result of heterogeneity in both the nature of the dysfunction and in the disease process itself. The brain is particularly vulnerable to the vascular complications of chronic hypertension, and the aim of this study was to assess whether differences in the cerebrovascular responsiveness to the NO synthase (NOS) inhibitors, N^G-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), and to the NO donor 3-morpholinosydnonimine (SIN-1) might indicate one possible source of these complications.
2. Conscious spontaneously hypertensive (SHR) and WKY rats, were treated with L-NAME (30 mg kg⁻¹, i.v.), 7-NI (25 mg kg⁻¹, i.p.), SIN-1 (0.54 or 1.8 mg kg⁻¹ h⁻¹, continuous i.v. infusion) or saline (i.v.), 20 min before the measurement of local cerebral blood flow (LCBF) by the fully quantitative [¹⁴C]-iodoantipyrine autoradiographic technique.
3. With the exception of mean arterial blood pressure (MABP), there were no significant differences in physiological parameters between SHR and WKY rats within any of the treatment groups, or between treatment groups. L-NAME treatment increased MABP by 27% in WKY and 18% in SHR groups, whilst 7-NI had no significant effect in either group. Following the lower dose of SIN-1 infusion, MABP was decreased to a similar extent in both groups (around −20%). There was no significant difference in MABP between groups following the higher dose of SIN-1, but this represented a decrease of −41% in SHR and −21% in WKY rats.
4. With the exception of one brain region (nucleus accumbens), there were no significant differences in basal LCBF between WKY and SHR. L-NAME produced similar decreases in LCBF in both groups, ranging between −10 and −40%. The effect of 7-NI upon LCBF was more pronounced in the SHR (ranging from −34 to −57%) compared with the WKY (ranging from −14 to −43%), and in seven out of the thirteen brain areas examined there were significant differences in LCBF.
5. Following the lower dose of SIN-1, in the WKY 8 out of the 13 brain areas examined showed significant increases in blood flow compared to the saline treated animals. In contrast, only 2 brain areas showed significant increases in flow in the SHR. In the rest of the brain areas examined the effects of SIN-1 upon LCBF were less marked than in the WKY.
6. Infusion of the higher dose of SIN-1 resulted in further significant increases in LCBF in the WKY group (ranging between +30% and +74% compared to saline-treated animals), but no significant effects upon LCBF were found in the SHR. As a result, there were significant differences in LCBF between SIN-1-treated WKY and SHR in six brain areas. In most brain areas examined, cerebral blood flow in SHR following the higher dose of SIN-1 was less than that measured with the lower dose of SIN-1.
7. Despite comparable reductions in MABP (∼20%) in both groups, calculated cerebrovascular resistance (CVR) confirmed that the vasodilator effects of the lower dose of SIN-1 were significantly more pronounced throughout the brain in the WKY (ranging between −3% and −50%; median=−38%) when compared to the SHR (ranging between −10% and −36%; median=−26%). In the animals treated with the higher dose of SIN-1, CVR changes were broadly similar in both groups (median=−45% in WKY and −42% in SHR), but with the reduction in MABP in SHR being twice that found in WKY, this is in keeping with an attenuated blood flow response to SIN-1 in the SHR.
8. The results of this study indicate that NO-dependent vasodilator capacity is reduced in the cerebrovasculature of SHR. In addition, the equal responsiveness to a non-specific NOS inhibitor but an enhanced effectiveness of a specific neuronal NO inhibitor upon LCBF in the SHR could be consistent with an upregulation of the neuronal NO system.

     

Endogenous angiotensin II and the reflex response to stimulation of cardiopulmonary serotonin 5HT3 receptors

1. Angiotensin (Ang) II modulates cardiovascular baroreflexes; whether or not the peptide influences chemosensitive cardiovascular reflexes is not known. We tested the hypothesis that Ang II modulates the reflex control of sympathetic nerve activity exerted by 5-hydroxytryptamine 3 (5HT₃) cardiopulmonary receptors.
2. The 5HT₃ receptor agonist phenylbiguanide (PBG), infused intravenously for 15 min, elicited a sustained reflex decrease of renal sympathetic nerve activity (RSNA) but only transient (<3 min) changes of arterial blood pressure (BP) and heart rate (HR) in methohexital-anaesthesized rats.
3. Infusion of Ang II at a dose that did not affect baseline BP, HR and RSNA enhanced the PBG-evoked reflex decrease of RSNA (−54±5% in Ang II treated versus −33±6% in control rats after 15 min PBG, P<0.05, n=6 each) in methohexital-anaesthetized rats.
4. The angiotensin converting enzyme (ACE) inhibitor lisinopril blunted the reflex responses to PBG in anaesthetized as well as conscious animals. The effect of the ACE inhibitor was abolished by concomitant infusion of Ang II.
5. The reflex response to stimulation of cardiopulmonary 5HT₃ afferents was also impaired by the Ang II type 1 receptor (AT₁) blocker ZD7155 but not by the type 2 (AT₂) blocker PD 123319.
6. Infusion of a volume load to stimulate cardiopulmonary baroreceptors induced a gradual decrease of RSNA which was impaired by exogenous Ang II (RSNA −26±6% in Ang II treated versus −47±6% in control rats after volume load, P<0.05, n=6 each) but unaffected by ACE inhibition.
7. The reflex control of RSNA by cardiopulmonary 5HT₃ receptors is enhanced by Ang II via AT₁ receptors. Thus, Ang II facilitates a chemosensitive cardiovascular reflex, in contrast to its inhibitory influences on mechanosensitive reflexes.

     

Investigation of the interaction between cholinergic and nitrergic neurotransmission in the pig gastric fundus

1. The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus.
2. In physiological salt solution containing 4×10⁻⁶ M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5–32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5–4 Hz, and large frequency-dependent contractions, sometimes followed by off-relaxations, at 8–32 Hz.
3. In the presence of L-N^G-nitroarginine methyl ester (L-NAME; 3×10⁻⁴ M) or physostigmine (10⁻⁶ M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L-NAME further enhanced contractions, while addition of L-NAME to physostigmine had no additional effect. Off-relaxations were enhanced in the presence of L-NAME and physostigmine. L-NAME and physostigmine consistently increased basal tone.
4. Tissues contracted by 5-hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L-NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies.
5. Off-relaxations in the presence of L-NAME were partially reduced by α-chymotrypsin (10 U ml⁻¹).
6. In the absence of physostigmine, the concentration-response curve to exogenous acetylcholine was not influenced by L-NAME.
7. Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10⁻⁵ M), depending upon the degree of relaxation by SNP.
8. These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.

     

Role of endothelin receptors,calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz), under different conditions, at 37°C and during cooling (30°C).
2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30°C, but not at 37°C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43±7% to 190±25% for 8 Hz).
3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ET_A receptors BQ-123 (10 μM) but not by the antagonist for endothelin ET_B receptors BQ-788 (10 μM). The agonist for endothelin ET_B receptors IRL-1620 (0.1 μM) did not modify the contraction to electrical stimulation.
4. The blocker of L-type Ca²⁺ channels verapamil (10 μM l⁻¹) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca²⁺-channels NiCl₂ (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30°C.
5. Inhibition of nitric oxide synthesis with N^G-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30°C more than at 37°C (for 8 Hz, this increment was 297±118% at 30°C, and 66±15% at 37°C). Endothelium removal increased the contraction to electrical stimulation at 30°C (about 91% for 8 Hz) but not at 37°C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30°C.
6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca²⁺ entry after activation of endothelin ET_A receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.