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.

     

Vasodilator effects of adrenomedullin on small pulmonary arteries and veins in anaesthetized cats

1. This study was conducted to determine adrenomedullin (AM) action sites in the pulmonary vascular bed and the relation between its vasodilator effects and vascular tone. Moreover, an examination was made into whether calcitonin gene-related peptide (CGRP) receptors mediate pulmonary vasodilatations induced by AM. To this end, we directly measured internal diameter (i.d.) changes in small pulmonary arteries and veins (100–1100 μm i.d.) by use of an X-ray televison system on the in vivo cat lung.
2. Under control (resting vascular tone) conditions, AM injections into the left main pulmonary artery caused dose-related i.d. increases in both small arteries and veins. The mean i.d. increase of the 100–1100 μm arteries (4±1, 11±2, and 17±2% with 0.01, 0.1, and 1 nmol kg⁻¹ AM, respectively) was significantly larger than that for the veins (1±1, 5±2, and 7±2% with 0.01, 0.1 and 1 nmol kg⁻¹ AM, respectively) whatever the injected dose of AM.
3. When unilobar hypoxia (5% O₂) had decreased the i.d. of the 100–1100 μm arteries and veins by 16±3 and 6±3%, respectively, AM (0.1 nmol kg⁻¹) was able to induce significantly larger i.d. increases in the arteries (28±3%) and veins (11±3%) than those under control conditions.
4. The AM-induced i.d. response pattern in the serially connected pulmonary arteries was quite different from that induced by CGRP; AM caused a greater increase in smaller vessels (100–500 μm) than in larger vessels (500–1100 μm). In the case of CGRP, a greater increase was observed in the larger vessels.
5. CGRP_8–37 (100 nmol kg⁻¹, i.v., followed by a continuous infusion of 0.2 nmol kg⁻¹ min⁻¹) had no significant effect on the i.d. increase induced by AM (0.1 nmol kg⁻¹) in any serial segments of the arteries and veins.
6. The results indicate that, in the cat, AM induces greater vasodilatation in small pulmonary arteries and lesser vasodilatation in small veins, the maximum dilatation being in the more peripheral arterial segment (100–500 μm). The vasodilator effect of AM was enhanced when vascular tone was elevated. The data suggest that the AM-induced pulmonary vasodilatation is not mediated by CGRP receptors but by its own specific receptor.

     

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.

     

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.

     

Infarct size-reducing effect of heat stress and α1 adrenoceptors in rats

1. Noradrenaline (NA), which is abundantly released during heat stress (HS), is known to induce both delayed cardioprotection and heat stress protein (HSP) 72 expression by the mediation of α₁ adrenoceptors. Therefore, we have investigated the implication of α₁ adrenoceptors in HS-induced resistance to myocardial infarction, in the isolated rat heart model.
2. Rats were pretreated with prazosin (1 mg kg⁻¹, i.p., Praz) or 5-methylurapidil (3 mg kg⁻¹, i.v, 5MU) or chloroethylclonidine (3 mg kg⁻¹, i.v., CEC) or vehicle (V) in order to selectively antagonize α₁, α_1A and α_1B adrenoceptors. They were then either heat stressed (42°C for 15 min) or sham anaesthetized. Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30 min occlusion of the left coronary artery followed by 120 min of reperfusion.
3. Infarct-to-risk ratio was significantly reduced in HS+V (15.4±1.8%) compared to Sham+V (35.7±1.3%) hearts. This effect was abolished in Praz-treated (29.1±1.6% in HS+Praz vs 34.1±4.0% in Sham+Praz), 5MU-treated (34.5±2.2% in HS+5MU vs 31.2±2.0% in Sham+5MU) and CEC-treated (33.4±3.0% in HS+CEC vs 32.4±1.3% in Sham+CEC) groups. Western blot analysis of myocardial HSP72 showed an HS-induced increase of this protein, which was not modified by Praz, 5MU and CEC pretreatments.
4. We conclude that both α_1A and α_1B adrenoceptor subtypes appear to play a role in the heat stress-induced cardioprotection, independently of the HSP72 level. Further investigations are required to elucidate the precise role of HSPs in this adaptative response.

     

Local neurogenic regulation of rat hindlimb circulation: CO2-induced release of calcitonin gene-related peptide from sensory nerves

1. The mechanism of release of calcitonin gene-related peptide (CGRP) from sensory nerves in response to skeletal muscle contraction was investigated in the rat hindlimb in vivo and in vitro.
2. In the anaesthetized rat, sciatic nerve stimulation at 10 Hz for 1 min caused a hyperaemic response in the hindlimb. During the response, partial pressure of CO₂ in the venous blood effluent from the hindlimb significantly increased from 43±3 to 73±8 mmHg, whereas a small decrease in pH and no appreciable change in partial pressure of O₂ were observed.
3. An intra-arterial bolus injection of NaHCO₃ (titrated to pH 7.2 with HCl), which elevated PCO₂ of the venous blood, caused a sustained increase in regional blood flow of the iliac artery. Capsaicin (0.33 μmol kg⁻¹, i.a.) and a specific calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8–37), (100 nmol kg⁻¹ min⁻¹, i.v.) significantly suppressed the hyperaemic response to NaHCO₃. Neither ND_Ω-nitro-L-arginine methyl ester (1 μmol kg⁻¹ min⁻¹, i.v.) nor indomethacin (5 mg kg⁻¹, i.v.) affected the response.
4. The serum level of CGRP-like immunoreactivity in the venous blood was significantly increased by a bolus injection of NaHCO₃ (pH=7.2) from 50±4 to 196±16 fmol ml⁻¹.
5. In the isolated hindlimb perfused with Krebs-Ringer solution, a bolus injection of NaHCO₃ (pH=7.2) caused a decrease in perfusion pressure which was composed of two responses, i.e., an initial transient response and a slowly-developing long-lasting one. CGRP(8–37) significantly inhibited the latter response by 73%.
6. These results suggest that CO₂ liberated from exercising skeletal muscle activates capsaicin-sensitive perivascular sensory nerves locally, which results in the release of CGRP from their peripheral endings, and then the released peptide causes local vasodilatation.

     

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.

     

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.

     

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.

     

The role of tachykinin NK1 and NK2 receptors in atropine-resistant colonic propulsion in anaesthetized guinea-pigs

1. The role of endogenous tachykinins on guinea-pig colonic propulsion was investigated by using potent and selective tachykinin NK₁ and NK₂ receptor antagonists. Colonic propulsion and contractions were determined by means of a balloon-catheter device, inserted into the rectum of guanethidine (68 μmol kg⁻¹, s.c., 18 and 2 h before)-pretreated, urethane-anaesthetized guinea-pigs. Propulsion of the device (dynamic model) was determined by measuring the length of the catheter expelled during 60 min filling of the balloon (flow rate 5 μl  min⁻¹).
2. In control conditions the tachykinin NK₁ receptor antagonist SR 140333 (1 μmol kg⁻¹, i.v.) did not affect either colonic propulsion or the amplitude of contractions. The tachykinin NK₂ receptor antagonists MEN 10627 and MEN 11420 (1 μmol kg⁻¹, i.v.) increased colonic propulsion at 10 min (+120% and 150%, respectively) but at 60 min the effect was significant only for MEN 10627 (+84%). SR 48968 (1 μmol kg⁻¹, i.v.) did not significantly enhance the colonic propulsion. None of these tachykinin NK₂ receptor antagonists modified the amplitude of colonic contractions. In contrast, both atropine (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹) and hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished propulsion (81% and 87% inhibition, respectively) and decreased the amplitude of contractions (68% inhibition for either treatment).
3. In atropine-treated animals (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹), apamin (30 nmol kg⁻¹, i.v.) restored colonic propulsion (+416%) and increased the amplitude of contractions (+367% as compared to atropine alone). Hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished the apamin-induced, atropine-resistant colonic propulsion (97% inhibition) and reduced the amplitude of the atropine-resistant contractions (52% inhibition).
4. The apamin-induced, atropine-resistant colonic propulsion was inhibited by SR 140333 (−69% at 1 μmol kg⁻¹), SR 48968 (−78% at 1 μmol kg⁻¹), MEN 11420 (−59% at 1 μmol kg⁻¹) and MEN 10627 (−50% at 1 μmol kg⁻¹), although the latter effect was not statistically significant. The combined administration of SR 140,333 and MEN 10,627 (1 μmol kg⁻¹ for each antagonist) almost completely abolished colonic propulsion (90% inhibition). The amplitude of colonic contractions was also reduced by SR 140333 (−42%), SR 48968 (−29%), MEN 11420 (−45%) but not by MEN 10627 (−16%). The combined administration of SR 140333 and MEN 10,627 reduced the amplitude of contractions by 47%. SR 140603 (1 μmol kg⁻¹, i.v.), the less potent enantiomer of SR 140333, was inactive.
5. In control animals, apamin (30 nmol kg⁻¹, i.v.) enhanced colonic propulsion (+84%) and increased the amplitude of contractions (+68%), as compared to the vehicle. Hexamethonium (55 μmol kg⁻¹, i.v. plus infusion of 17 μmol h⁻¹) inhibited propulsion (86% inhibition) and decreased the amplitude of contractions (49% inhibition). SR 140333, SR 48968, MEN 11420, MEN 10627, or the coadministration of SR 140333 and MEN 10627 had no effect.
6. In a separate series of experiments, the mean amplitude of colonic contractions was also recorded under isovolumetric conditions through the balloon-catheter device kept in place at 75 mm from the anal sphincter (static model). In control conditions, neither SR 140333 nor MEN 11420 modified the amplitude of contractions. In atropine-pretreated guinea-pigs, SR 140333 and MEN 11420 (0.1–1 μmol kg⁻¹) dose-dependently decreased the amplitude of contractions. In apamin- and atropine-pretreated animals, only the highest (1 μmol kg⁻¹) dose of SR 140333 or MEN 11420 significantly decreased the amplitude of contractions. The inhibitory potency of atropine (0.3–1 μmol kg⁻¹) was similar in apamin-pretreated animals and in controls.
7. It was concluded that, in anaesthetized guinea-pigs, endogenous tachykinins, acting through both NK₁ and NK₂ receptors, act as non-cholinergic excitatory neurotransmitters in promoting an apamin-evoked reflex propulsive activity of the distal colon.

     

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.

     

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.

     

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.

     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious,hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3–4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 μg kg⁻¹ h⁻¹, i.v.)
2. The early (1–2 h) hypotension in SD rats (−11±3 mmHg; n=7) was significantly less than that in TG rats (−35±3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108±3 mmHg; 24 h LPS, 112±4 mmHg; TG rats: baseline, 171±2 mmHg; 24 h LPS, 169±3 mmHg). At this stage in the SD rats there was a renal vasodilatation (Δ vascular conductance, 29±10 [kHz mmHg⁻¹]10³) but not in TG rats (Δ vascular conductance 2±3[kHz mmHg⁻¹]10³).
3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 μg kg⁻¹ bolus, 600 μg kg⁻¹ h⁻¹) between 24 and 31 h in SD rats caused a fall in MAP of 16±2 mmHg accompanied by hindquarters vasodilatation (Δ vascular conductance 11±3 (kHz mmHg⁻¹)10³). In TG rats, under the same conditions, the fall in MAP was −60±6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (Δ vascular conductance, 23±5, 32±7, and 14±4 (kHz mmHg⁻¹)10³, respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats.
4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was −17±4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5±3, −4±5, and 12±4 (kHz mmHg⁻¹)10³, respectively.
5. Administration of the angiotensin (AT₁)-receptor antagonist, losartan (10 mg kg⁻¹, i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (−12±3 and −14±4 mmHg, respectively).
6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.

     

Systemic anti-inflammatory effect induced by counter-irritation through a local release of somatostatin from nociceptors

1. Neurogenic plasma extravasation evoked by topical application of 1% vv⁻¹ mustard oil on the skin of the acutely denervated rat hindleg (primary reaction) inhibited the development of a subsequent oil-induced plasma extravasation induced in the skin of the contralateral hindleg by 49.3±7.06% (n=9) and in the conjunctival mucosa due to 0.1% wv⁻¹ capsaicin instillation by 33.5±10.05% (n=6). The primary reaction also inhibited the non-neurogenic hindpaw oedema evoked by s.c. injection of 5% wv⁻¹ dextran into the chronically denervated hindpaw by 48.0±4.6% (n=5).
2. Capsaicin injection (100 μg ml⁻¹ in 50 μl, s.c.) into the acutely denervated hindleg caused 56.5±4.0% (n=5) inhibition in the intensity of plasma extravasation elicited by 1% vv⁻¹ mustard oil smearing on the contralateral side. After chronic denervation, subplantar injection of 5% wv⁻¹ dextran elicited a non-neurogenic inflammatory response with intensive tissue oedema without causing any systemic anti-inflammatory effect. Bilateral adrenalectomy did not inhibit the mustard oil-induced anti-inflammatory effect in the contralateral hindleg.
3. Pretreating the rats with polyclonal somatostatin antiserum (0.5 ml rat⁻¹, i.v.) or with the somatostatin depleting agent cysteamine (280 mg kg⁻¹, s.c.) prevented the inhibitory action of mustard oil-induced inflammation on subsequent neurogenic plasma extravasation and strongly diminished the inhibition of non-neurogenic oedema formation evoked by dextran.
4. Exogenous somatostatin (10 μg kg⁻¹, i.p.) caused a 30.3±8.3% (n=6) inhibition of plasma extravasation caused by mustard oil smearing on the acutely denervated hindleg and this inhibitory effect was abolished by somatostatin antiserum (0.5 ml rat⁻¹, i.v.). The plasma level of somatostatin-like immunoreactivity (SST-LI) increased by 40.03±6.8% (n=6) 10 min after topical application of 1% vv⁻¹ mustard oil on the acutely denervated hindpaws compared to the paraffin oil treated control group. Chronic denervation of the hindlegs or cysteamine (280 mg kg⁻¹, s.c.) pretreatment prevented the mustard oil-induced elevation of SST-LI in plasma.
5. It is concluded that chemical excitation of the capsaicin-sensitive sensory receptors not only induces local neurogenic plasma extravasation but also inhibits the development of a subsequent inflammatory reaction at remote sites of the body in the rat. A role for somatostatin in this systemic anti-inflammatory effect is suggested.

     

Local neurogenic regulation of rat hindlimb circulation: role of calcitonin gene-related peptide in vasodilatation after skeletal muscle contraction

1. The mechanism of neurogenic regulation of skeletal muscle circulation was studied in the hindlimb of anaesthetized rats in vivo. Regional blood flow (RBF) of the hindlimb was recorded with a pulsed Doppler flow probe positioned in the iliac artery.
2. A short period (1 min) of sciatic nerve stimulation at 10 Hz caused a sustained increase in RBF (from 2.0±0.2 to 3.7±0.2 kHz at the peak), but no appreciable change in either MBP or HR, suggesting that the nerve stimulation produced local vasodilatation of the peripheral vasculature. The hyperaemic response reached a peak within 15 s and characteristically remained above the basal level for more than 5 min after the cessation of nerve stimulation. The response was regarded as a secondary response brought about by the contraction of skeletal muscles since (+)-tubocurarine (0.73 μmol kg⁻¹, i.a.) almost abolished it.
3. Lignocaine (43 μmol kg⁻¹, i.a.) and capsaicin (0.33 μmol kg⁻¹, i.a.) significantly suppressed the hyperaemic response to skeletal muscle contraction, suggesting that capsaicin-sensitive sensory nerves contribute to the hyperaemia. In contrast, an inhibitor of NO synthase, N_ω-nitro-L-arginine methyl ester (1 μmol kg⁻¹ min⁻¹, i.v.), did not affect the hyperaemic response.
4. Serum levels of calcitonin gene-related peptide (CGRP) in iliac venous effluent significantly increased from 51±4 to 77±5 fmol ml⁻¹ during the hyperaemic response to skeletal muscle contraction. A bolus injection of CGRP (300 pmol kg⁻¹, i.a.) induced a long-lasting increase in RBF of the hindlimb. Moreover, CGRP(8–37) (100 nmol kg⁻¹ min⁻¹, i.v.), a specific CGRP₁ receptor antagonist, significantly suppressed the hyperaemic response, especially the sustained phase of the response which was almost abolished by this antagonist.
5. These results suggest that CGRP, which is released from peripheral endings of capsaicin-sensitive sensory nerves, partly mediates the hyperaemia evoked by skeletal muscle contraction of the rat hindlimb.

     

In vitro and in vivo characterization of NK3 receptors in the rabbit eye by use of selective non-peptide NK3 receptor antagonists

1. Inhibition of NK₃ receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK₃ receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK₃ receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists.
2. In vitro senktide (succinyl-[Asp⁹,MePhe⁸]-substance P (6-11) and [MePhe⁷]-neurokinin B ([MePhe⁷]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD₂=9.03±0.06 and mean n_H=1.2±0.02 (n=14). In contrast, [MePhe⁷]-NKB produced shallow log concentration-effect curves which often appeared biphasic (n_H=0.54±0.04, n=8), preventing the accurate determination of pD₂ values.
3. The contractile responses to the NK₃ receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((−)-(S)-N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide; 3–30 nM, pA₂=8.4, slope=1.8±0.3, n=4), SB 222200 ((−)-(S)-N-(α-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide; 30–300 nM, pA₂=7.9, slope=1.4±0.06, n=4) and SB 218795 ((−)-(R)-N-(α-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 μM apparent pK_B=7.4±0.06, n=6).
4. Contractile responses to the NK₃ receptor agonist [MePhe⁷]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 μM, n=8). In contrast, SB 223412 (30 and 300 μM, n=4) and SB 222200 (0.3 and 3 μM, n=4) inhibited responses to low concentrations (⩽1 nM), to a greater extent than higher concentrations (>1 nM) of [MePhe⁷]-NKB. Furthermore, log concentration-effect curves to [MePhe⁷]-NKB became steeper and monophasic in the presence of each antagonist.
5. SB 218795 (3 μM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK₃ receptors over NK₁ receptors in the rabbit.
6. In vivo, senktide (1, 10 and 25 μg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25±0.25 mm; basal pupillary diameter 7.75±0.48 mm; n=4). The onset of miosis was within 2–5 min of application of senktide and responses lasted up to 30 min. Responses to two i.v. administrations of 25 μg senktide given 30 min apart revealed no evidence of tachyphylaxis. Topical administration of atropine (1%) to the eye enhanced pupillary responses to 25 μg senktide. This was probably due to the mydriatic effect of atropine since it significantly increased baseline pupillary diameter from 7.0±0.4 mm to 9.0±0.7 mm (n=4), thereby increasing the maximum capacity for miosis. Senktide-induced miosis was inhibited by SB 222200 (1 and 2 mg kg⁻¹, i.v., i.e. 2.63 and 5.26 μmol kg⁻¹; maximum inhibition 100%; n=3–4), SB 223412 (0.5 and 1 mg kg⁻¹, i.v., i.e. 1.31 and 2.61 μmol kg⁻¹; maximum inhibition 100%; n=3), SB 218795 (0.5 and 1 mg kg−1, i.v., i.e. 1.26 and 2.52 μmol kg−1; maximum inhibition 78%; n=3), and the structurally distinct NK₃ receptor antagonist SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylepipiperidin-4-yl)-N-methylacetamide; 1.5 mg kg⁻¹, i.v., i.e. 2.47 μmol kg⁻¹, maximum inhibition 92%; n=3).
7. Topical administration of senktide (25 μg; 29.7 nmol) to the eye induced unilateral miosis in the treated eye only. At this dose there was no significant difference (P<0.05) between pupillary constriction obtained by topical or i.v. senktide, and topically administered atropine had no significant effect on responses to topical senktide (n=4).
8. [MePhe⁷]-NKB (125, 250 and 500 μg, i.v., i.e. 98.31, 196.62 and 393.24 nmol, respectively) also induced bilateral miosis in conscious rabbits (maximum pupillary constriction=4.13±0.30 mm; n=4), but in contrast to in vitro studies this agonist was approximately 100 fold less potent than senktide. [MePhe⁷]-NKB-induced miosis was inhibited by SB 222200 (5 mg kg⁻¹, i.v., i.e. 13.14 μmol kg⁻¹; maximum inhibition 69%; n=3).
9. In summary, SB 223412, SB 222200 and SB 218795 are potent and selective antagonists of NK₃ receptor-mediated contraction in the rabbit isolated iris sphincter muscle. In addition, NK₃ receptor agonist-induced miosis in conscious rabbits is a good in vivo correlate of the in vitro rabbit iris sphincter muscle preparation and appears to be a useful model for characterizing the pharmacodynamic profile and efficacy of structurally distinct NK₃ receptor antagonists, such as SB 222200, SB 223412, SB 218795 and SR 142801.

     

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.

     

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.

     

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.