Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals

Pretreatment with the novel neuropeptide nociceptin (20 μg kg⁻¹, i.p.) caused an inhibition of plasma extravasation evoked by antidromic stimulation of the saphenous nerve or by topical application of 1% mustard oil on the skin of the acutely denervated hindleg of the rat. In contrast, it did not affect non-neurogenic inflammation evoked by s.c. injection of bradykinin after chronic denervation. Release of substance P (SP) and calcitonin gene-related peptide (CGRP) from rat isolated tracheae in response to electrical field stimulation was diminished by nociceptin (100 nM). It is concluded that nociceptin inhibits the release of sensory neuropeptides from terminals of nociceptive neurones.     

Vascular actions of octreotide in the portal hypertensive rat

1. We have investigated the actions of the somatostatin analogue octreotide in the portal hypertensive Wistar rat in vivo and in rat small mesenteric artery and aorta in vitro.
2. In small mesenteric artery, octreotide (0.1–0.3 μM) failed to produce any direct contraction, nor did it affect contractions to noradrenaline (NA, 10 μM) or endothelium-dependent relaxations to acetylcholine.
3. In rat aorta, octreotide (0.3 μM) and somatostatin (1 μM) failed to affect contractions to NA (1 μM), or concentration-contractile response curves to NA.
4. In rat vas deferens, octreotide and somatostatin significantly reduced contractile responses to electrical stimulation with pD₂ values (−log IC₅₀) of 8.19±0.10 (n=4) and 8.16±0.26 (n=4), respectively. Hence, the lack of effect of these agents in aorta or mesenteric artery was not due to lack of efficacy or inappropriate choice of concentration.
5. In the anaesthetized portal hypertensive rat, intravenous injection of octreotide (1–100 μg kg⁻¹) did not significantly affect systemic blood pressure, nor did it affect mesenteric vascular conductance as measured by laser doppler flow probes. However, octreotide (100 μg kg⁻¹) significantly reduced vascular conductance to 74.2±7.7% of control (n=6) in porto-systemic shunt vessels as measured by laser doppler flow probes.
6. Phenylephrine (1 μg kg⁻¹) significantly raised blood pressure and significantly decreased vascular conductance in both mesenteric (66.6±3.7% of control) and porto-systemic shunt vessels (58.7±10.0% of control).
7. It was concluded that octreotide has selective effects on porto-systemic shunt vessles in vivo in the portal hypertensive rat.

     

A novel murine model of allergic inflammation to study the effect of dexamethasone on eosinophil recruitment

1. We have developed a novel model of allergen-induced eosinophil extravasation into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism(s) underlying the anti-inflammatory action of the glucocorticoid hormone dexamethasone (Dex).
2. Injection of 10 μg Ova into 6-day-old dorsal air-pouches of mice sensitized to the same antigen provoked an intense cell accumulation as early as 6 h post-challenge (0.08±0.03 and 4.0±1.0×10⁵ leucocytes in saline and Ova-treated air-pouches, respectively), maximal at 24 h (0.02±0.01 and 6.0±0.8×10⁵ leucocytes in saline and Ova-treated air-pouches, respectively) and persisted up to 48 h. At the 24 h time-point, the cellular infiltrate consisted of 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as assessed by histological examination. The same ratio of eosinophil/neutrophil was obtained by fluorescence-activated cell sorting (FACS) analysis, since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4 (VLA-4) expression.
3. Subcutaneous (s.c.) administration of Dex (50 or 100 μg per mouse, −1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P<0.05) and 75% (P<0.05), respectively, when compared to controls. Cell accumulation measured at 48 h after Ova injection was also significantly reduced (−75%) by Dex administration at the 24 h time-point (n=12, P<0.05).
4. Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eosinophil numbers in the bone marrow (naive mice: 2.7±0.3×10⁵; sensitized mice: 8.7±1.7×10⁵, P<0.05) and blood (naive mice: 0.5±0.2×10⁵; sensitized mice: 1.5±0.3×10⁵, P<0.01). However, 24 h following Ova challenge, the eosinophil numbers in the bone marrow had dropped (3.7±0.8×10⁵) with no change in the circulating pool, suggesting an equilibrium within the eosinophil pools had been reached.
5. Dex administration provoked a profound eosinopaenia in the blood of naive (5.2±1.5 to 0.9±0.6×10⁴) and sensitized mice (1.5±0.3 to 0.08±0.02×10⁵) at 4 h. This effect was reversed within 24 h. Dex also inhibited the release of eosinophils from the bone marrow in response to Ova challenge.
6. We show for the first time that eosinophils express the steroid-inducible protein lipocortin 1 (LC1). FACS analysis of eosinophils emigrated into the Ova challenged air-pouches revealed detectable LC1-like immunoreactivity (373×10³). These data were also substantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 μg) did not alter LC1 levels in blood eosinophils, such that 235±21×10³ LC1-like molecules per cell were measured after vehicle treatment (n=5), and 224±8×10³ LC1-like molecules per cell were associated with this cell type 1 h after steroid treatment (n=5, not significant). Finally, resident eosinophils (in the pleural cavity) were found to have much higher LC1 levels than that found in the blood circulation (2 fold increase, P<0.05).
7. Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modify the anti-migratory activity exerted by Dex towards eosinophil extravasation into Ova-challenged air-pouches. The steroid (50 μg s.c., −1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) and LCS3-treated mice (−56% and 59%, respectively, n=15–21, not significant).
8. In conclusion, the air-pouch provides a novel and convenient cavity to study allergen-induced cell recruitment which is sensitive to glucocorticoid hormone treatment. The effect of Dex on eosinophil distribution in these experimental conditions has been studied in detail and we failed to find an important role for endogenous LC1 in these actions of the steroid.

     

Spatial heterogeneity of the effects of calcitonin gene-related peptide (CGRP) on the microvasculature of ligaments in the rabbit knee joint

1. Experiments were performed in anaesthetized rabbits to examine the effects of calcitonin gene-related peptide (CGRP) and the CGRP antagonist CGRP_8–37 on blood flow to the medial collateral ligament of the knee joint.
2. Topical application of CGRP (10⁻¹³ to 10⁻⁹  mol) to the exposed external surface of eight knee joints resulted in dose-dependent dilatation of vessels in both the ligament and the joint capsule. The magnitude of this response varied significantly in different regions of the medial collateral ligament, with the 10⁻⁹  mol dose of CGRP giving the maximum response (101.5±25.3% increase) at the femoral insertion site of the medial collateral ligament and lowest (23.1±8.8%) at the tibial insertion site.
3. Topical application of CGRP_8–37 (0.1, 1 and 10  nmol) produced dose-dependent constriction of vessels in the ligament and the joint capsule in five knees, with a trend towards the greatest effect occurring at the femoral insertion site (45.8±8.1% reduction in blood flow). With the 10  nmol dose, the vasoconstrictor response at the femoral insertion site differed significantly (P<0.05) from the responses obtained at the tibial insertion and joint capsule sites.
4. Topical application of CGRP_8–37 (0.1, 1 and 10  nmol) to four chronically denervated knees produced substantially smaller vasoconstrictor responses at all sites. At the femoral insertion site, where 10  nmol CGRP_8–37 normally produces a 45.8±8.1% reduction in blood flow (n=8), ten days following denervation this response was reduced to 6.5±6.1%, this difference being significant (P=0.01).
5. Adrenaline was applied topically to augment blood vessel tone, in order to establish how effectively co-administration of CGRP would offset this increase in tone. Adrenaline (10⁻¹⁰  mol) produced vasoconstriction at all sites (n=6). In the capsule this vasoconstriction was virtually abolished when CGRP (10⁻⁹  mol) was co-administered with adrenaline but in the ligament vasodilatation occurred at all sites. This vasodilatation was significantly greater at the femoral insertion site compared to the tibial insertion and mid ligament sites (P<0.05 for both) and the capsule (P<0.01).
6. Topical application of substance P (10⁻¹⁰ or 10⁻⁹  mol) failed to elicit dilatation of ligament blood vessels.
7. These results suggest that endogenous CGRP may play an important role in regulating blood flow to different structures in and around the knee joint.

     

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.

     

Effects of intracerebroventricular leptin administration on food intake,body weight gain and diencephalic nitric oxide synthase activity in the mouse

1. Intracranial administration of leptin reduces both food intake and body weight gain in the mouse. Inhibitors of nitric oxide (NO) synthase produce similar effects.
2. To investigate the role of the brain L-arginine/NO pathway in mediating this effect of leptin, we have evaluated food intake and body weight gain after daily (5 days) intracerebroventricular (i.c.v.) administration of leptin (0.5–2 μg) alone or in association with L-arginine (10 μg). Moreover, we measured diencephalic nitric oxide synthase (NOS) activity after a single i.c.v. leptin (0.25–2 μg) injection and after consecutive doses of leptin (0.25–2 μg) over 5 days. The time course of the effect of leptin on NOS activity was also evaluated.
3. I.c.v. injected leptin (1 and 2 μg) significantly and dose-dependently reduced food intake and body weight gain with respect to vehicle (food intake: 5.97±0.16 g 24 h⁻¹ and 4.27±0.18 g 24 h⁻¹, respectively, vs 8.05±0.34 g 24 h⁻¹, P<0.001, n=6 for each group; body weight gain: −10.7±0.46% and −15.2±0.65%, respectively, vs 5.14±0.38%, P<0.001, n=6 for each group). This effect was antagonized by L-arginine (food intake: 7.90±0.37 g 24 h; body weight gain: 5.11±0.31%, n=6). Diencephalic NOS activity was significantly reduced by the highest doses of leptin with respect to vehicle (vehicle: 0.90±0.04 nmol citrulline min⁻¹ g⁻¹ tissue; leptin 1 μg: 0.62±0.03 nmol citrulline min⁻¹ g⁻¹ tissue, P<0.001; leptin 2 μg: 0.44±0.03 nmol citrulline min⁻¹ g⁻¹ tissue, P<0.001, n=6 for each group). Similar results were obtained in animals treated with daily consecutive doses of leptin. The inhibitory effect appeared rapidly (within 30 min) and was long lasting (up to 12 h).
4. Our results suggest that the brain L-arginine/NO pathway may be involved in the central effect of leptin on feeding behaviour and body weight gain in mice.

     

Effects of prostaglandins and nitric oxide on the renal effects of angiotensin II in the anaesthetized rat

1. The potential influences of nitric oxide (NO) and prostaglandins on the renal effects of angiotensin II (Ang II) have been investigated in the captopril-treated anaesthetized rat by examining the effect of indomethacin or the NO synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), on the renal responses obtained during infusion of Ang II directly into the renal circulation.
2. Intrarenal artery (i.r.a.) infusion of Ang II (1–30 ng kg⁻¹ min⁻¹) elicited a dose-dependent decrease in renal vascular conductance (RVC; −38±3% at 30 ng kg⁻¹ min⁻¹; P<0.01) and increase in filtration fraction (FF; +49±8%; P<0.05) in the absence of any change in carotid mean arterial blood pressure (MBP). Urine output (Uv), absolute (UNaV) and fractional sodium excretion (FENa), and glomerular filtration rate (GFR) were unchanged during infusion of Ang II 1–30 ng kg⁻¹ min⁻¹ (+6±17%, +11±17%, +22±23%, and −5±9%, respectively, at 30 ng kg⁻¹ min⁻¹). At higher doses, Ang II (100 and 300 ng kg⁻¹ min⁻¹) induced further decreases in RVC, but with associated increases in MBP, Uv and UNaV.
3. Pretreatment with indomethacin (10 mg kg⁻¹ i.v.) had no significant effect on basal renal function, or on the Ang II-induced reduction in RVC (−25±7% vs −38±3% at Ang II 30 ng kg⁻¹ min⁻¹). In the presence of indomethacin, Ang II tended to cause a dose-dependent decrease in GFR (−38±10% at 30 ng kg⁻¹ min⁻¹); however, this effect was not statistically significant (P=0.078) when evaluated over the dose range of 1–30 ng kg⁻¹ min⁻¹, and was not accompanied by any significant changes in Uv, UNaV or FENa (−21±12%, −18±16% and +36±38%, respectively).
4. Pretreatment with L-NAME (10 μg kg⁻¹ min⁻¹ i.v.) tended to reduce basal RVC (control −11.8±1.4, +L-NAME −7.9±1.8 ml min⁻¹ mmHg⁻¹×10⁻²), and significantly increased basal FF (control +15.9±0.8, +L-NAME +31.0±3.7%). In the presence of L-NAME, renal vasoconstrictor responses to Ang II were not significantly modified (−38±3% vs −35±13% at 30 ng kg⁻¹ min⁻¹), but Ang II now induced dose-dependent decreases in GFR, Uv and UNaV (−51±11%, −41±14% and −31±17%, respectively, at an infusion rate of Ang II, 30 ng kg⁻¹ min⁻¹). When evaluated over the range of 1–30 ng kg⁻¹ min⁻¹, the effect of Ang II on GFR and Uv were statistically significant (P<0.05), but on UNaV did not quite achieve statistical significance (P=0.066). However, there was no associated change in FENa observed, suggesting a non-tubular site of interaction between Ang II and NO.
5. In contrast to its effects after pretreatment with L-NAME alone, Ang II (1–30 ng kg⁻¹ min⁻¹) failed to reduce renal vascular conductance in rats pretreated with the combination of L-NAME and the selective angiotensin AT₁ receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR117289","term_id":"238364164","term_text":"GR117289"}}GR117289 (1 mg kg⁻¹ i.v.). This suggests that the renal vascular effects of Ang II are mediated through AT₁ receptors. Over the same dose range, Ang II also failed to significantly reduce GFR or Uv.
6. In conclusion, the renal haemodynamic effects of Ang II in the rat kidney appear to be modulated by cyclooxygenase-derived prostaglandins and NO. The precise site(s) of such an interaction cannot be determined from the present data, but the data suggest complex interactions at the level of the glomerulus.

     

Modelling of the pharmacodynamic interaction between phenytoin and sodium valproate

1. Treatment of epilepsy with a combination of antiepileptic drugs remains the therapeutic choice when monotherapy fails. In this study, we apply pharmacokinetic-pharmacodynamic modelling to characterize the interaction between phenytoin (PHT) and sodium valproate (VPA).
2. Male Wistar rats received a 40 mg kg⁻¹ intravenous dose of PHT over 5 min either alone or in combination with an infusion of VPA resulting in a steady-state concentration of 115.5±4.9 μg ml⁻¹. A control group received only the infusion of VPA. The increase in the threshold for generalized seizure activity (ΔTGS) was used as measure of the anticonvulsant effect.
3. PHT pharmacokinetics was described by a pharmacokinetic model with Michaelis-Menten elimination. The concentration-time course and plasma protein binding of PHT were not altered by VPA. The pharmacokinetic parameters V_max and K_m were, respectively, 294±63 μg min⁻¹ and 7.8±2.4 μg ml⁻¹ in the absence of VPA and 562±40 μg min⁻¹ and 15.6±0.9 μg ml⁻¹ upon administration in combination with VPA.
4. A delay of the onset of the effect relative to plasma concentrations of PHT was observed. The assessment of PHT concentrations at the effect site was based on the effect-compartment model, yielding mean k_e0 values of 0.128 and 0.107 min⁻¹ in the presence and absence of VPA, respectively.
5. A nonlinear relationship between effect-site concentration and the increase in the TGS was observed. The concentration that causes an increase of 50% in the baseline TGS (EC_50%TGS) was used to compare drug potency. A shift of EC_50%TGS from 13.27±3.55 to 4.32±0.52 μg ml⁻¹ was observed upon combination with VPA (P<0.01).
6. It is concluded that there is a synergistic pharmacodynamic interaction between PHT and VPA in vivo.

     

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.

     

Tachykinin regulation of basal synovial blood flow

1. Experiments were performed to investigate the role of endogenously released tachykinins in the regulation of blood flow to the rat knee joint. Synovial perfusion was assessed by laser Doppler perfusion imaging, which permitted spatial measurement of relative changes in perfusion from control (pre drug administration), expressed as the percentage change. Most experiments were performed on the exposed medial aspect of the knee joint capsule.
2. Neither the selective tachykinin NK₁ receptor antagonist, FK888, nor the selective tachykinin NK₂ receptor antagonist, SR48968, significantly influenced synovial blood flow at doses of 10⁻¹², 10⁻¹⁰ and 10⁻⁸ mol. However, topical co-administration of these agents produced significant dose-dependent reductions in basal synovial perfusion of 6.3±4.6, 12.0±3.4 and 19.9±2.6%, respectively; n=29. The non-selective tachykinin NK₁/NK₂ receptor antagonist, FK224, also produced significant (at 10⁻¹⁰ and 10⁻⁸ mol), but less potent, reductions in perfusion of 5.3±4.0, 8.4±2.2 and 5.9±2.8%, respectively; n=25.
3. Topical administration of the α₁-, α₂-adrenoceptor antagonist phenoxybenzamine elicited a 31.3±6.2% increase in blood flow which was substantially reduced to 10.4±3.8% by co-administration of the FK888 and SR48968 (both at 10⁻⁸ mol; n=8–13), suggesting that normally there is sympathetic vasoconstrictor ‘tone'' which is opposed by the vasodilator action of endogenous tachykinins.
4. One week after surgical interruption of the nerve supply to the knee joint, co-administration of FK888 and SR48968 (both at 10⁻⁸ mol) now produced slight vasodilatation (6.7±4.6%; n=9) which did not differ significantly from vehicle treatment. Depletion of tachykinins from sensory nerve fibres by systemic capsaicin administration also resulted in abolition of the vasoconstrictor effect of FK888 and SR48968 (both at 10⁻⁸ mol), with these agents only producing a slight vasodilatation (2.5±5.3%; n=6).
5. By use of a near infra-red laser source it was possible to image knee joint perfusion transcutaneously, the overlying skin being left intact. In this more physiological situation, close intra-arterial injection of the combination of FK888 and SR48968 (both at 10⁻⁸ mol) again elicited vasoconstriction (48.8±16.2% reduction in blood flow; n=4).
6. These results indicate that endogenous tachykinins may be continuously released from sensory fibres innervating the joint. Basal release of tachykinins could therefore be an important physiological influence opposing sympathetic vasoconstrictor tone.

     

Haemodynamic effects of a selective adenosine A2A receptor agonist,CGS 21680, in chronic heart failure in anaesthetized rats

1. Recently we demonstrated that the administration of an A_2A adenosine receptor agonist, CGS 21680, to anaesthetized rats with acute heart failure (1 h post-coronary artery ligation) resulted in an increase in cardiac output. In the present investigation, the effects of CGS 21680 on cardiac output, vascular resistance, heart rate, blood pressure and mean circulatory filling pressure (Pmcf) were investigated in anaesthetized rats with chronic heart failure (8 weeks post-coronary artery ligation).
2. Experiments were conducted in five groups (n=6) of animals: sham-operated vehicle-treated (0.9% NaCl; 0.037 mL kg⁻¹ min⁻¹) animals in which the occluder was placed but not pulled to ligate the coronary artery; coronary artery-ligated vehicle-treated animals; and coronary artery-ligated CGS 21680-treated (0.1, 0.3 or 1.0 μg kg⁻¹ min⁻¹) animals.
3. Baseline blood pressure, cardiac output and rate of rise in left ventricular pressure (+dP/dt) were significantly reduced in animals with coronary artery ligation when compared to sham-operated animals. Coronary artery ligation resulted in a significant increase in left ventricular end-diastolic pressure, Pmcf and venous resistance when compared to sham-operated animals.
4. Administration of CGS 21680 at 0.3 and 1.0 μg kg⁻¹ min⁻¹ significantly (n=6; P<0.05) increased cardiac output by 19±4% and 39±5%, and heart rate by 14±2% and 15±1%, respectively, when compared to vehicle treatment in coronary artery-ligated animals. Administration of CGS 21680 also significantly reduced blood pressure and arterial resistance when compared to coronary artery-ligated vehicle-treated animals. Infusion of CGS 21680 also significantly reduced venous resistance when compared to vehicle-treated coronary artery-ligated animals.
5. The results show that heart failure is characterized by reduced cardiac output, and increased left ventricular end-diastolic pressure, venous resistance and Pmcf. Acute treatment with CGS 21680 in animals with chronic heart failure decreased left ventricular end-diastolic pressure and increased cardiac output. This increase in cardiac output was the result of reduced arterial and venous resistances and increased heart rate.

     

Modulation of airway hyperresponsiveness and eosinophilia by selective histamine and 5-HT receptor antagonists in a mouse model of allergic asthma

1. Since both histamine and 5-hydroxytryptamine (5-HT) can be released by murine mast cells, we investigated the possible role of these autacoids on airway hyperresponsiveness (AHR), eosinophil infiltration and serum-IgE levels in a murine model of allergic asthma.
2. Ovalbumin-sensitized mice were exposed to either ovalbumin (2 mg ml⁻¹) or saline aerosols on 8 consecutive days. Starting one day before the challenge, animals were injected i.p. twice a day with a 5-HT-type 1 (5-HT₁) or type 2 (5-HT₂) receptor antagonist (methiotepine, 1.25 or 2.0 mg kg⁻¹ and ketanserin, 12 mg kg⁻¹, respectively) or a histamine-type 1 (H₁) or type 2 (H₂) receptor antagonist (mepyramine, 12 or 20 mg kg⁻¹ and cimetidine, 10 or 25 mg kg⁻¹, respectively). Furthermore, animals were injected with a combination of cimetidine and ketanserin or with an α-adrenoceptor antagonist (phentolamine, 5 mg kg⁻¹).
3. In vehicle-treated ovalbumin-challenged animals airway responsiveness to intravenous injections of methacholine in vivo was significantly (9 fold increase, P<0.01) increased when compared to vehicle-treated saline-challenged animals. Furthermore, ovalbumin challenge of vehicle-treated animals induced a significant increase in both eosinophil numbers in bronchoalveolar lavage (BAL) fluid (0±0, vehicle/saline and 15.0±5.9×10⁴ cells vehicle/ovalbumin, P<0.05) and ovalbumin-specific IgE levels in serum (157±69 and 617±171 units ml⁻¹, respectively, P<0.05) compared to saline-challenged mice. Virtually no eosinophils could be detected in saline-challenged animals after all different treatments.
4. Treatment with ketanserin or cimetidine resulted in a partial but significant decrease of the ovalbumin-induced AHR compared to ovalbumin-challenged controls (P<0.05) and reduced eosinophil infiltration after ovalbumin challenge by 60% and 58%, respectively. The combination of cimetidine and ketanserin almost completely abolished AHR whereas eosinophilia was decreased by 49%. No effects of these antagonists were observed on IL-16 levels in BAL fluid or on serum antigen-specific IgE levels. Treatment with either the H₁-receptor, the 5-HT₁-receptor or the α-adrenoceptor antagonist, did not decrease the observed ovalbumin-induced airway responsiveness or eosinophilia in vehicle-treated animals. Higher doses of either methiotepine (2.0 mg kg⁻¹) or mepyramine (20 mg kg⁻¹) did decrease ovalbumin-induced eosinophil infiltration (by 67%, P<0.05 and 73%, respectively), whereas no effects of these antagonists were observed on ovalbumin-specific IgE levels in serum.
5. From these data it can be concluded that both histamine and 5-HT play a role in antigen-induced AHR and eosinophilia in the mouse.

     

Systemic and renal effects of an ETA receptor subtype-specific antagonist in healthy subjects

1. Endothelins (ETs) might play a pathophysiological role in a variety of vascular diseases. The aim of the present study was to characterize the effects of BQ-123, a specific ET_A receptor antagonist on systemic and renal haemodynamics in healthy subjects. This was done at baseline and during infusion of exogenous ET-1.
2. The study was performed in a balanced, randomized, placebo-controlled, double blind 4 way cross-over design in 10 healthy male subjects. Subjects received co-infusions of ET-1 (2.5 ng kg⁻¹ min⁻¹ for 120 min) or placebo and BQ-123 (15 μg min⁻¹ for 60 min and subsequently 60 μg min⁻¹ for 60 min) or placebo. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were assessed by the para-aminohippurate (PAH) and the inulin plasma clearance method, respectively.
3. BQ-123 alone had no renal or systemic haemodynamic effect. ET-1 significantly reduced RPF (−24%, P<0.001) and GFR (−12%, P=0.034). These effects were abolished by co-infusion of either dose of BQ-123 (RPF: P=0.0012; GFR: P=0.020).
4. BQ-123 reversed the renal haemodynamic effects induced by exogenous ET-1 in vivo. This indicates that vasoconstriction in the kidney provoked by ET-1 is predominantly mediated by the ET_A receptor subtype.

     

Imbalance between the endothelial cell-derived contracting factors prostacyclin and angiotensin II and nitric oxide/cyclic GMP in human primary varicosis

1. The role of the endothelium in the vasomotor control of human veins in the lower extremity is little understood. We tested the hypothesis that the production of relaxing and contracting factors is altered in endothelial cells from varicose saphenous veins which may predispose to the decreased vessel tone observed in primary varicosis.
2. We determined the intracellular accumulation of guanosine 3′:5′-cyclic monophosphate cyclic GMP; a measure of nitric oxide production) and the release of endothelin and prostacyclin (measured as its stable metabolite 6-keto-prostaglandin F_1α) from cultured cells derived from the long saphenous veins of patients with primary varicosis (Varicose saphena group, n=27) or from patients undergoing coronary artery bypass surgery (Healthy saphena group, n=22). In addition, levels of endothelin, angiotensin II, bradykinin, cyclic GMP and cyclic AMP in plasma from patients with primary varicosis and healthy volunteers (n=8–11 in each group) were determined.
3. Although basal cyclic GMP levels were similar, more cyclic GMP accumulated in response to histamine (1–100 μmol l⁻¹) in cells from varicose saphenous veins (0.75±0.1 pmol per well) than in cells from veins without varicosis (0.27±0.05 pmol per well). Furthermore, the relaxant potency of nitroprusside (1 nmol l⁻¹–300 μmol l⁻¹) in vitro was higher for varicose veins (mean EC₅₀=5.9 μmol l⁻¹; n=8) than healthy veins (mean EC₅₀=20.0 μmol l⁻¹; n=7).
4. The production of prostacyclin was significantly less in cells from varicose than healthy saphenous veins (66±8.7 and 121±20.1 nmol g⁻¹ protein), but the production of endothelin was similar in both groups. Prostacyclin (3 nmol l⁻¹–30 μmol l⁻¹) consistently contracted rings of varicose saphenous vein in vitro with a mean EC₅₀ value of 10–20 μmol l⁻¹ (n=7); the maximum tension generated was ∼50% of that of a completely depolarizing solution of K⁺ (120 mmol l⁻¹).
5. In plasma from patients with varicose veins, levels of cyclic GMP were higher than in healthy controls (9.2±0.03 and 7.2±0.02 nmol l⁻¹), levels of angiotensin II were lower (81±11.5 and 147±21.7 pmol l⁻¹), and levels of endothelin, cyclic AMP, and bradykinin were not different.
6. It is concluded that endothelial cells from diseased saphenous veins secrete less constrictor mediators than cells from healthy veins and that in diseased veins the nitric oxide/cyclic GMP system is up-regulated which may shift the balance of vasoactive factors towards vasodilatation and contribute to the development of primary varicosis.

     

Dose-dependent effects of chronic ethanol on mouse adipose tissue lipase activity and cyclic AMP accumulation

1. The effects of two chronic ethanol treatment schedules, which produce different plasma ethanol concentrations, on the specific activities of adipose tissue lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) have been investigated in brown and white fat.
2. Mice provided with 20% ethanol solution as sole drinking fluid for 28 days consumed between 13 and 15 g ethanol kg⁻¹ body weight day⁻¹ over days 22–28. The mean plasma ethanol concentration was 4.94±1.4 mM (n=8) at 09 h 00 min on day 28 when the lipase assays were performed. Mice given ethanol in a liquid diet for 7 days consumed between 15 and 18 g ethanol day⁻¹ over days 3–7. The mean plasma ethanol concentration was 15.9±4.7 mM (n=8) at 09 h 00 min on day 7. These concentrations of ethanol had no effect on the activity of either LPL or HSL in vitro.
3. LPL activity in white and brown fat (expressed as nmol fatty acids released h⁻¹ mg⁻¹ acetone powder) was unaltered 60 min following an acute injection of ethanol (2.5 g kg⁻¹, i.p.) which produced a mean blood ethanol level of 37.5±6.7 mM. HSL activity in white fat (expressed as nmol fatty acid released h⁻¹ mg⁻¹ protein) was also unaffected by this acute dose of ethanol, but the activity in brown fat was significantly reduced: 3.07±0.30 (n=8) after ethanol compared to 4.36±0.25 (n=12) in controls (P<0.01).
4. LPL activity in white fat was little altered by either of the chronic ethanol treatment schedules whilst LPL activity in the brown fat from the same animals was significantly increased compared to the respective control values: 0.27±0.03 (ethanol drinking), control: 0.16±0.01; 0.79±0.14 (ethanol liquid diet), control: 0.39±0.05.
5. HSL activity in white fat was significantly increased by the chronic drinking treatment (7.7±0.5; control: 3.78±0.17, n=8) at the same time that the activity in brown fat was reduced (3.76±0.2; control: 4.74±0.16). The ethanol liquid diet also reduced HSL activity in brown fat but had negligible effect in white fat.
6. The effects of the two chronic ethanol treatments on adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) accumulation in brown and white fat were very similar, both qualitatively and quantitatively, to the effects on HSL.
7. It has been shown that brown and white adipose tissues respond differently to the presence of chronic ethanol and that the response is dependent both upon the concentration of ethanol and the nature of the diet with which the ethanol is administered. The effects of ethanol on adipose tissue HSL activity appear to be mediated via changes in the tissue cyclic AMP level and, in this respect, brown fat is more sensitive to ethanol than white fat.

     

Differential effects of cyclosporine A after acute antigen challenge in sensitized cats in vivo and ex vivo

1. We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (R_L) in vivo, and tracheal smooth muscle (TSM) contraction ex vivo after antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen-induced responses of TSM in vitro.
2. Cats (n=10) were sensitized by i.m. injection of Ascaris suum antigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin-2 secretion from feline peripheral blood mononuclear cells ex vivo.
3. Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542±74 pg ml⁻¹ post AA for CsA+ and from 74±19 pg ml⁻¹ at baseline, to 970±180 pg ml⁻¹ post AA CsA− (P<0.05; P=NS vs CsA+).
4. In excised TSM, active tension elicited by exposure to AA in vitro was 107±38% KCl in the CsA+ group vs 144±56% KCl in the CsA− group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre-incubated with 1 μM CsA in vitro (8±8% KCl, P<0.05 vs CsA+ and CsA−). This was associated with inhibited release of 5-hydroxytryptamine into the organ bath fluid of tissues treated with CsA in vitro only.
5. We demonstrated that CsA treatment in vivo does not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell function ex vivo do not reflect lack of effects of CsA on mast cell function in vivo in this animal model of atopic asthma.

     

Pharmacokinetic-pharmacodynamic modelling of the anti-lipolytic and anti-ketotic effects of the adenosine A1-receptor agonist N6-(p-sulphophenyl)adenosine in rats

1. The purpose of this study was to develop and validate an integrated pharmacokinetic-pharmacodynamic model for the anti-lipolytic effects of the adenosine A₁-receptor agonist N⁶-(p-sulphophenyl)adenosine (SPA). Tissue selectivity of SPA was investigated by quantification of haemodynamic and anti-lipolytic effects in individual animals.
2. After intravenous infusion of SPA to conscious normotensive Wistar rats, arterial blood samples were drawn for determination of blood SPA concentrations, plasma non-esterified fatty acid (NEFA) and β-hydroxybutyrate levels. Blood pressure and heart rate were monitored continuously.
3. The relationship between the SPA concentrations and the NEFA lowering effect was described by the indirect suppression model. Administration of SPA at different rates and doses (60 μg kg⁻¹ in 5 min and 15 min, and 120 μg kg⁻¹ in 60 min) led to uniform pharmacodynamic parameter estimates. The averaged parameters (mean±s.e., n=19) were E_max: −80±2% (% change from baseline), EC₅₀: 22±2 ng ml⁻¹, and Hill factor: 2.2±0.2.
4. In another group, given 400 μg kg⁻¹ SPA in 15 min, pharmacodynamic parameters for both heart rate and anti-lipolytic effect were derived within the same animal. The reduction in heart rate was directly related to blood concentration on the basis of the sigmoidal E_max model. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The EC₅₀ values (mean±s.e., n=6) were 131±31 ng ml⁻¹ and 20±3 ng ml⁻¹ for heart rate and NEFA lowering effect, respectively.
5. In conclusion, the relationship between blood SPA concentrations and anti-lipolytic effect was adequately described by the indirect suppression model. For SPA a 6 fold difference in potency was observed between the effects on heart rate and NEFAs, indicating some degree of tissue selectivity in vivo.

     

Catecholamine modulatory effects of nepicastat (RS-25560-197), a novel,potent and selective inhibitor of dopamine-β-hydroxylase

1. Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine-β-hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study.
2. Nepicastat produced concentration-dependent inhibition of bovine (IC₅₀=8.5±0.8 nM) and human (IC₅₀=9.0±0.8  nM)dopamine-β-hydroxylase. The corresponding R-enantiomer (RS-25560-198) was approximately 2–3 fold less potent than nepicastat. Nepicastat had negligible affinity (>10 μM) for twelve other enzymes and thirteen neurotransmitter receptors.
3. Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg⁻¹, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg⁻¹, p.o.; b.i.d., for 5 days) produced dose-dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noradrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg⁻¹, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R-enantiomer (RS-25560-198), in the mesenteric artery and left ventricle.
4. Administration of nepicastat (2 mg kg⁻¹, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day-6 and day-7 of dosing, respectively.
5. The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine-β-hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over-activation of the sympathetic nervous system, such as congestive heart failure.

     

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.

     

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1. The functional role of the nitric oxide (NO)/guanosine 3′:5′-cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts.
2. Rat isolated hearts were perfused at constant pressure with Krebs-Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min⁻¹ for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored.
3. At baseline, heart rate was 287±12 beats min⁻¹, coronary flow was 12.8±0.6 ml min⁻¹, left ventricular developed pressure (LVDevP) was 105±4 mmHg and left ventricular end-diastolic pressure 4.6±0.2 mmHg in vehicle-treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05).
4. In normoxic perfused hearts, 1 μM N^G-nitro-L-arginine (L-NOARG) significantly reduced coronary flow from 13.5±0.2 to 12.1±0.1 ml min⁻¹ (10%) and LVDevP from 97±1 to 92±1 mmHg (5%; P<0.05, n=5).
5. Ischaemic contracture was 46±2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 μM) but reduced to ∼30 mmHg (∼25%) at higher concentrations (100 or 1000 μM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 μM to 26±3 mmHg (23%), but increased it to 63±4 mmHg (59%) at 1000 μM (n=6). Dobutamine (10 μM) exacerbated ischaemic contracture (81±3 mmHg; n=7) and the cyclic GMP analogue Sp-8-(4-p-chlorophenylthio)-3′,5′-monophosphorothioate (Sp-8-pCPT-cGMPS; 10 μM) blocked this effect (63±1 mmHg; P<0.05 vs dobutamine alone, n=5).
6. At the end of reperfusion, LVDevP was 58±5 mmHg, i.e. 55% of pre-ischaemic value in control hearts, significantly increased to ∼80% by high concentrations of nitroprusside (100 or 1000 μM) or L-NOARG at 1 μM, while a high concentration of L-NOARG (1000 μM) reduced LVDevP to ∼35% (P<0.05 vs control; n=6).
7. Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 μM had no sustained effect, but increased cyclic GMP ∼6 fold at 1000 μM; L-NOARG (1 or 1000 μM) was without effect (n=6). Nitroprusside (1 or 1000 μM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6).
8. In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.