Complete in vivo reversal of P-glycoprotein pump function in the blood-brain barrier visualized with positron emission tomography

1. Homozygously mdr1a gene disrupted mice (mdr1a(−/−) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P-glycoprotein (P-gp) function imaging non-invasively and to study the effect of a P-gp reversal agent on its function in vivo.
2. [¹¹C]verapamil (0.1 mg/kg) was administered and the changes in tissue concentrations were determined ex vivo by organ extirpation and in vivo with PET. To block P-gp function, cyclosporin A was administered.
3. Biodistribution studies revealed 9.5-fold (P<0.001) and 3.4-fold (P<0.001) higher [¹¹C]verapamil in the brain and testes of mdr1a(−/−) mice than in mdr1a(+/+) mice. Cyclosporin A (25 mg/kg) increased [¹¹C]verapamil levels in the brain and testes of mdr1a(+/+) mice in both cases 3.3-fold (P<0.01 (brain); P<0.001 (testes)). Fifty mg/kg cyclosporin A increased [¹¹C]verapamil in the brain 10.6-fold (P<0.01) and in the testes 4.1-fold (P<0.001). No increases were found in the mdr1a(−/−) mice. This indicates complete inhibition of P-gp mediated [¹¹C]verapamil efflux.
4. Positron camera data showed lower [¹¹C]verapamil levels in the brain of mdr1a(+/+) mice compared to those in mdr1a(−/−) mice. [¹¹C]verapamil accumulation in the brain of mdr1a(+/+) mice was increased by cyclosporin A to levels comparable with those in mdr1a(−/−) mice, indicating that reversal of P-gp mediated efflux can be monitored by PET.
5. We conclude that cyclosporin A can fully block the P-gp function in the blood brain barrier and the testes and that PET enables the in vivo measurement of P-gp function and reversal of its function non-invasively.

     

Stereospecific effects of ketamine enantiomers on canine tracheal smooth muscle

1. Ketamine is a potent bronchodilator which relaxes airway smooth muscle (ASM). Clinically, ketamine is used as a 1:1 racemic mixture of enantiomers that differ in their analgesic and anaesthetic effects. The aim of this study was to determine whether there was a difference between the enantiomers in their ability to relax isolated ASM and to explore mechanisms responsible for any observed differences.
2. Canine tracheal smooth muscle strips were loaded with fura-2 and mounted in a photometric system to measure simultaneously force and [Ca²⁺]_i. Calcium influx was estimated by use of a manganese quenching technique.
3. In strips stimulated with 0.1 μM ACh (EC₅₀) R(−)-ketamine (1–100 μM) caused a significantly greater concentration-dependent decrease in force (P<0.0001) and [Ca²⁺]_i than S(+)-ketamine (1–100 μM) (P<0.0005). In contrast, there was no significant difference between the enantiomers in their ability to inhibit calcium influx (45% decrease in influx rate for R(−)-ketamine and 44% for S(+)-ketamine, P=0.782). In strips contracted with 24 mM isotonic KCl (which activates voltage-operated calcium channels), the enantiomers modestly decreased force and [Ca²⁺]_i; there was no significant difference between the enantiomers in their effects on force (P=0.425) or [Ca²⁺]_i (P=0.604).
4. The R(−)-enantiomer of ketamine is a more potent relaxant of ACh-induced ASM contraction than the S(+)-enantiomer. This difference appears to be caused by differential actions on receptor-operated calcium channels.

     

Contribution of K+ channels and ouabain-sensitive mechanisms to the endothelium-dependent relaxations of horse penile small arteries

1. Penile small arteries (effective internal lumen diameter of 300–600 μm) were isolated from the horse corpus cavernosum and mounted in microvascular myographs in order to investigate the mechanisms underlying the endothelium-dependent relaxations to acetylcholine (ACh) and bradykinin (BK).
2. In arteries preconstricted with the thromboxane analogue U46619 (3–30 nM), ACh and BK elicited concentration-dependent relaxations, pD₂ and maximal responses being 7.71±0.09 and 91±1% (n=23), and 8.80±0.07 and 89±2% (n=24) for ACh and BK, respectively. These relaxations were abolished by mechanical endothelial cell removal, attenuated by the nitric oxide (NO) synthase (NOS) inhibitor, N^G-nitro-L-arginine (L-NOARG, 100 μM) and unchanged by indomethacin (3 μM). However, raising extracellular K⁺ to concentrations of 20–30 mM significantly inhibited the ACh and BK relaxant responses to 63±4% (P<0.01, n=7) and to 59±4% (P<0.01, n=6), respectively. ACh- and BK-elicited relaxations were abolished in arteries preconstricted with K⁺ in the presence of 100 μM L-NOARG.
3. In contrast to the inhibitor of ATP-sensitive K⁺ channels, the blockers of Ca²⁺-activated K⁺ (K_Ca) channels, charybdotoxin (30 nM) and apamin (0.3 μM), each induced slight but significant rightward shifts of the relaxations to ACh and BK without affecting the maximal responses. Combination of charybdotoxin and apamin did not cause further inhibition of the relaxations compared to either toxin alone. In the presence of L-NOARG (100 μM), combined application of the two toxins resulted in the most effective inhibition of the relaxations to both ACh and BK. Thus, pD₂ and maximal responses for ACh and BK were 7.65±0.08 and 98±1%, and 9.17±0.09 and 100±0%, respectively, in controls, and 5.87±0.09 (P<0.05, n=6) and 38±11% (P<0.05, n=6), and 8.09±0.14 (P<0.01, n=6) and 98±1% (n=6), respectively, after combined application of charybdotoxin plus apamin and L-NOARG.
4. The selective inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 μM) did not alter the maximal responses to either ACh or BK, but slightly decreased the sensitivity to both agonists, δpD₂ being 0.25±0.07 (P<0.05, n=6) and 0.62±0.12 (P<0.01, n=6) for ACh and BK, respectively. Combined application of ODQ and charybdotoxin plus apamin produced further inhibition of the sensitivity to both ACh (δpD₂=1.39±0.09, P<0.01, n=6) and BK (1.29±0.11, P<0.01, n=6), compared to either ODQ or charybdotoxin plus apamin alone.
5. Exogenous nitric oxide (NO) present in acidified solutions of sodium nitrite (NaNO₂) and S-nitroso-cysteine (SNC) both concentration-dependently relaxed penile resistance arteries, pD₂ and maximal responses being 4.84±0.06 and 82±3% (n=12), and 6.72±0.07 and 85±4% (n=19), respectively. Charybdotoxin displaced to the right the dose-relaxation curves for both NO (δpD₂ 0.38±0.06, P<0.01, n=6) and SNC (δpD₂ 0.50±0.10, P<0.01, n=5), whereas apamin only reduced sensitivity (δpD₂=0.35±0.12, P<0.05, n=5) and maximum response (65±9%, P<0.05, n=6) to SNC. ODQ shifted to the right the dose-relaxation curves to both NO and SNC. The relaxant responses to either NO or SNC were not further inhibited by a combination of ODQ and charybdotoxin or ODQ and charybdotoxin plus apamin, respectively, compared to either blocker alone.
6. In the presence of 3 μM phentolamine, 5 μM ouabain contracted penile resistance arteries by 50±6% (n=17) of K-PSS, but did not significantly change the relaxant responses to either ACh, BK or NO. However, in the presence of L-NOARG ouabain reduced the ACh- and BK-elicited relaxation from 94±3% to 16±5% (P<0.0001, n=6), and from 98±2% to 13±3% (P<0.0001, n=5), respectively. Combined application of ODQ and ouabain inhibited the relaxations to NO from 92±2% to 26±3% (P<0.0001, n=6).
7. The present results demonstrate that the endothelium-dependent relaxations of penile small arteries involve the release of NO and a non-NO non-prostanoid factor(s) which probably hyperpolarize(s) smooth muscle by two different mechanisms: an increased charybdotoxin and apamin-sensitive K⁺ conductance and an activation of the Na⁺-K⁺ATPase. These two mechanisms appear to be independent of guanylate cyclase stimulation, although NO itself can also activate charybdotoxin-sensitive K⁺ channels and the Na⁺-K⁺ pump through both cyclic GMP-dependent and independent mechanisms, respectively.

     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

1. The receptors involved in mediating the haemodynamic effects of three 5-HT_1B/D receptor agonists were investigated in pentobarbitone anaesthetized rats (n=6–17 per group).
2. Cumulative intravenous (i.v.) infusions of rizatriptan and sumatriptan (from 0.63 to 2500 μg kg⁻¹; each dose over 5 min) induced dose-dependent and marked hypotension (−42±6 and −34±4 mmHg at the highest dose, respectively; both P<0.05 vs vehicle: +5±3 mmHg) and bradycardia (−85±16 and −44±12 beats min⁻¹ at the highest dose, respectively; both P<0.05 vs vehicle: +16±6 beats min⁻¹). Zolmitriptan evoked only moderate hypotension at the highest dose (−19±9 mmHg; P<0.05 vs vehicle).
3. A high dose of the 5-HT_1B/D receptor antagonist, GR 127935 (0.63 mg kg⁻¹, i.v.), failed to antagonize the hypotension and bradycardia evoked by sumatriptan (−35±6 mmHg and −52±19 beats min⁻¹, respectively; both not significant vs sumatriptan in untreated rats), but moderately reduced the hypotension and bradycardia evoked by rizatriptan (−20±5 mmHg and −30±17 beats min⁻¹, respectively; both P<0.05 vs vehicle and vs rizatriptan in untreated rats).
4. The selective 5-HT_1A receptor antagonist, WAY 100635 (0.16 and 0.63 mg kg⁻¹, i.v.), dose-dependently attenuated the haemodynamic responses evoked by rizatriptan and sumatriptan, which were almost abolished by the higher dose of WAY 100635 (−4±3 mmHg and −15±8 beats min⁻¹; both not significant vs vehicle and P<0.05 vs rizatriptan in untreated rats). A slight but statistically significant reduction in mean arterial pressure (MAP) persisted at the highest dose of sumatriptan (−13±4 mmHg following the higher dose of WAY 100635; P<0.05 vs vehicle).
5. In pithed rats with MAP normalized by angiotensin II, rizatriptan failed to induce hypotension or bradycardia (+5±4 mmHg and −6±16 beats min⁻¹, respectively; both NS vs vehicle and P<0.05 vs rizatriptan in untreated rats). Similarly, sumatriptan failed to induce bradycardia in pithed rats (+5±6 beats min⁻¹; not significant vs vehicle and P<0.05 vs sumatriptan in untreated rats), whereas a slight but statistically significant reduction in MAP, compared to controls, occurred at the highest dose (−9±9 mmHg; P<0.05 vs both vehicle and sumatriptan in untreated rats).
6. In bilaterally vagotomized and atropine-treated (1 mg kg⁻¹, i.v.) rats, the reductions in MAP and heart rate evoked by rizatriptan (−31±4 mmHg and −64 ±9 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs rizatriptan in controls) and sumatriptan (−47±8 mmHg and −56±10 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs sumatriptan in controls) were not statistically significantly different from those observed in controls.
7. In conclusion, the 5-HT_1B/D receptor agonists, rizatriptan and sumatriptan, elicit hypotension and bradycardia in the normotensive anaesthetized rat predominantly via activation of central 5-HT_1A receptors, and a consequent reduction in sympathetic outflow.

     

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.

     

Role of Ca2+-activated K+ channel in epithelium-dependent relaxation of human bronchial smooth muscle

1. To elucidate whether K⁺ channels play a role in the action of epithelium-dependent bronchodilatation, we studied responses in human bronchial strips in the presence of indomethacin and N^G-nitro-L-arginine methylester under isometric conditions, in vitro.
2. Mechanical removal of the epithelium increased the contractile responses to acetylcholine; the pD₂ values increased from 5.0±0.2 to 5.9±0.3 (P<0.001). This potentiation was abolished by iberiotoxin but not by apamin or glibenclamide.
3. In cascade bioassay, application of the bathing medium from dispersed, bronchial epithelial cells to epithelium-denuded bronchial strips decreased acetylcholine-induced contraction by 44±6%. This effect was reduced to 10±3% (P<0.01) when the epithelial cells were pretreated with iberiotoxin, and to 4±1% (P<0.001) when the epithelial cells were incubated with Ca²⁺-free medium containing [1,2-bis (2) aminophenoxy] ethane N,N,N′,N′-tetraacetic acid-acetomethoxy ester.
4. In contrast, the bronchodilator effect of the medium bathing epithelial cells was not altered by the direct addition of iberiotoxin to epithelium-denuded tissues.
5. These results suggest that the Ca²⁺-activated K⁺ channel may play a role in the synthesis and/or release of smooth muscle relaxing factor, which is neither nitric oxide nor a cyclo-oxygenase product, from airway epithelial cells.

     

Signalling pathways in bradykinin- and nitric oxide-induced hypotension in the normotensive rat; role of K+-channels

1. Bradykinin and nitric oxide (NO) are potent hypotensive agents. In the present study, the role of K⁺-channels in the signalling pathways responsible for their hypotensive action was investigated in normotensive, anaesthetized rats. The rats were treated with ion-channel inhibitors before administration of bradykinin (2.8, 5.6, 28 and 56 nmol kg⁻¹, i.v.) followed in some of the protocols by nitroprusside (1.1, 3.5, 7, 14, and 28 nmol kg⁻¹, i.v.).
2. No attenuation of the hypotensive response to bradykinin was detected for inhibitors of the Na-K-Cl-cotransporter (30 μmol kg⁻¹ furosemide), the ATP-sensitive K⁺-channel (40 μmol kg⁻¹ glibenclamide), high conductance Ca²⁺-activated K⁺-channel (180 μmol kg⁻¹ tetraethylammonium, 54 μmol kg⁻¹ tetrabutylammonium, 35 nmol kg⁻¹ iberiotoxin, 35 nmol kg⁻¹ charybdotoxin) or the low conductance Ca²⁺-activated K⁺-channel (74 nmol kg⁻¹ apamin).
3. However, the voltage-sensitive K⁺-channel (I_A) inhibitor 4-aminopyridine (4.05–40.5 μmol kg⁻¹) induced a concentration-dependent (P<0.0001) attenuation of the hypotensive response (P<0.0001). Bradykinin had no effect on heart rate in anaesthetized rats and this observation was not altered by pretreatment with 4-aminopyridine.
4. 4-Aminopyridine (53 μmol kg⁻¹) also significantly attenuated the hypotensive response to nitroprusside (P<0.0003) without altering the heart rate concentration-response curve. Of the two Ca²⁺-activated K⁺-channel inhibitors tested on nitroprusside-induced hypotension, tetrabutylammonium induced a slight attenuation (P<0.0101), whereas iberiotoxin had no effect.
5. We therefore concluded that, although the acute hypotensive response to bradykinin in the normotensive rat is not mediated through nitric oxide synthesis, the hypotensive response to both agents was mediated through opening of voltage-sensitive K⁺-channels (I_A), resulting in a decrease in peripheral vascular resistance.

     

Sex differences in the relative contributions of nitric oxide and EDHF to agonist-stimulated endothelium-dependent relaxations in the rat isolated mesenteric arterial bed

1. We have used the isolated, buffer-perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxations to carbachol.
2. Carbachol caused dose-related relaxations of methoxamine-induced tone in mesenteric vascular beds from male rats described by an ED_50(M) of 0.43±0.15 nmol and a maximum relaxation (R_max(M) of 89.6±1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED_50(F)=0.72±0.19 nmol and R_max(F)=90.7±0.9%; n=22).
3. In the males, the addition of 100 μM N^G-nitro-L-arginine methyl ester (L-NAME) caused the dose-response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED_50(M)=6.45±3.53 nmol) and significantly (P<0.01) reduced R_max(M) (79.7±2.8%, n=13). By contrast, L-NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED_50(F)=0.89±0.19 nmol, R_max(F)=86.9±2.3%, n=9).
4. Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (R_max(M)=40.3±9.2%, n=4; P<0.001) and female rats by 1.5 fold (R_max(F)=55.3±3.3%, n=6; P<0.001), compared with methoxamine-induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED_50(M)=0.87±0.26 nmol; P<0.01) but not the females (ED_50(F)=4.04±1.46 nmol). In the presence of both 60 mM KCl and L-NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups.
5. The cannabinoid receptor antagonist SR141716A (1 μM), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED_50(M)=1.41±0.74 nmol, R_max(M)=89.4±2.5%, n=7; ED_50(F)=2.17±0.95 nmol, R_max(F)=89.9±1.8%, n=9). In mesenteries from male rats, in the presence of 100 μM L-NAME, SR141716A significantly (P<0.05) shifted the dose-response curve to carbachol 8 fold further to the right than that seen in the presence of L-NAME alone (ED_50(M)=53.8±36.8 nmol) without affecting R_max(M) (72.4±4.8%, n=10). In mesenteries from female rats, the combined presence of L-NAME and SR141716A, significantly (P<0.01) shifted the dose-response curve to carbachol 7.5 fold, (ED_50(F)=6.66±2.46 nmol), as compared to L-NAME alone and significantly (P<0.001) decreased R_max(F) (70.1±5.5%, n=8).
6. Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP-sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds.
7. The continuous presence of sodium nitroprusside (SNP; 20–60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L-NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED_50(M)=40.9±19.6 nmol, R_max(M)=79.4±2.5%, n=11). Similarly in mesenteries from female rats, the ED_50(F) was also significantly (P<0.01) increased 7 fold (6.24±2.02 nmol), while the R_max(F) was unaffected (81.9±11.0%; n=4).
8. The results of the present investigation demonstrate that the relative contributions of agonist-stimulated NO and EDHF to endothelium-dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium-dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functionally more important in females than males.

     

Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP).
2. Using 10 μM of each donor compound, mean±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min⁻¹ (for SNP) to 3.15±0.29 nmol min⁻¹ (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 10⁸ platelets (for SNP) to 2.67±0.31 pmol per 10⁸ platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC).
3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34).
4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8-bromo cyclic GMP).
5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu⁺) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO.
6. Cyclic GMP accumulation in response to GSNO (10⁻⁹–10⁻⁵M) was undetectable following treatment of platelets with ODQ (100 μM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu⁺ ions were required for the cyclic GMP-independent pathway to operate.
7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP.
8. The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (CMPS) produced a concentration-dependent inhibition of aggregation of ODQ-treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP-independent inhibition of platelet aggregation.
9. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP-dependent and -independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP-independent pathway may depend upon a cellular system which involves both copper (I) (Cu⁺) ions and surface membrane thiol groups. The potent anti-platelet action of GSNO results from its ability to exploit this cyclic GMP-independent mechanism.

     

The stimulation of human neutrophil migration by angiotensin II: its dependence on Ca2+ and the involvement of cyclic GMP

1. Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10⁻¹⁰ M it caused a maximal increase of migration; migration increased from 47.2±2.1 μm in the absence of angiotensin II, to 73.1±2.2 μm with 10⁻¹⁰ M angiotensin II present in the lower compartment of the Boyden chamber (n=5, P<0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10⁻⁸ M and higher inhibited chemotactic peptide-stimulated chemotaxis.
2. The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca²⁺. In the presence of 1 mM Ca²⁺, angiotensin II stimulated migration to 76.1±1.7 μm, while migration in the absence of Ca²⁺ was 42.2±1.9 μm (n=4, P<0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II-activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca²⁺ than in electroporated cells. This supports the view that there is an influx of Ca²⁺ through the plasma membrane, and a requirement of calcium for an intracellular target.
3. Angiotensin II-stimulated migration was inhibited by pertussis toxin; from 71.6±2.0 μm in the absence, to 43.6±1.5 μm in the presence of pertussis toxin (n=4, P<0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTPγS. This suggests that one or more G-proteins are involved in the activating effect of angiotensin II.
4. Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP-dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP-dependent kinase.

     

Role of endothelium in regulation of smooth muscle membrane potential and tone in the rabbit middle cerebral artery

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA).
2. In endothelium-intact strips, histamine (His, 3–10 μM) and high K⁺ (20–80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 μM), 5-hydroxytryptamine (10 μM) and 9,11-epithio-11, 12-methano-thromboxane A₂ (10 nM) each produced only a small contraction (less than 5% of the maximum K⁺-induced contraction).
3. N^G-nitro-L-arginine (L-NOARG, 100 μM), but not indomethacin (10 μM), greatly enhanced the phasic and the tonic contractions induced by His (1–10 μM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3–3 μM) caused concentration-dependent relaxation (maximum relaxation by 89.7±7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 μM), but not glibenclamide (3 μM), abolished the relaxation induced by ACh (0.3–3 μM) in L-NOARG-treated strips (n=4, P<0.05).
4. In endothelium-intact tissues, His (3 μM) depolarized the smooth muscle membrane potential (by 4.4±1.8 mV, n=12, P<0.05) whereas ACh (3 μM) caused membrane hyperpolarization (−20.9±3.0 mV, n=25, P<0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (−23.5±5.9 mV, n=8, P<0.05) or glibenclamide (−20.6±5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to −5.8±3.2 mV, n=3, P<0.05).
5. Sodium nitroprusside (0.1–10 μM) did not hyperpolarize the smooth muscle cell membrane potential (0.2±0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n=4, P<0.05).
6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.

     

Central role of intracellular calcium stores in acute flow- and agonist-evoked endothelial nitric oxide release

1. We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of Ca²⁺ release from endothelial intracellular stores to nitric oxide (NO) production evoked by increases in shear stress and by acetylcholine in rabbit aorta.
2. Experiments were performed before and following incubation with either the endoplasmic reticulum Ca²⁺-ATPase inhibitors cyclopiazonic acid (CPA, 10 μM) and thapsigargin (TSG, 1 μM) or ryanodine (30, 100 μM) which binds to a specific endoplasmic reticulum Ca²⁺-release channel.
3. In cascade bioassay all three agents induced relaxations of the recipient ring (CPA, 24.4±3.8%; TSG, 51.5±10.6%; ryanodine, 17.4±1.6%) which were significantly attenuated by preincubation of the donor with 100 μM N^G-nitro-L-arginine methyl ester (L-NAME). However, in isolated rings, only CPA and TSG induced L-NAME-sensitive relaxations (CPA 52.7±6.5%; TSG 61.3±7%).
4. Addition of superoxide dismutase (SOD) to the donor perfusate evoked relaxations of the recipient ring in cascade bioassay (13.3±1.4%, n=22). Prior administration of SOD attenuated relaxations to TSG (23.2±3.8%, n=4) and ryanodine (1.7±0.8%, n=4), and pre-incubation with TSG and ryanodine blunted SOD-induced responses (4±1.5%, n=4 and 8.9±1.1%, n=4, respectively). By contrast, no interaction was observed between the relaxations evoked by SOD and CPA. In isolated rings, SOD exerted no direct relaxant action and did not modulate relaxations to CPA, TSG or ryanodine.
5. In cascade bioassay studies time-averaged shear stress was manipulated with dextran (1–4% w/v, 80000 MW) to increase perfusate viscosity. NO-dependent relaxation of the recipient ring induced by increased perfusate viscosity was significantly attenuated by CPA (P<0.01; n=6) and TSG (P<0.05; n=7), but not by ryanodine (n=6).
6. Endothelium-dependent relaxations to acetylcholine (0.1–30 μM) in cascade bioassay and in isolated aortic ring preparations were markedly attenuated by pretreatment with CPA and TSG, but were unaffected by ryanodine. Ryanodine and CPA caused only a small attenuation of endothelium-independent relaxations to sodium nitroprusside (0.001–10 μM), whereas TSG had no effect.
7. We conclude that release of Ca²⁺ from CPA- and TSG-sensitive endothelial stores is necessary for NO release evoked by acute flow changes and agonists in rabbit abdominal aorta. Ca²⁺-induced Ca²⁺ release via the ryanodine-sensitive release channel plays no direct role in these responses. Free radical interactions may complicate the interpretation of findings in cascade bioassay compared with isolated ring preparations.

     

Altered active but not passive properties of mesenteric resistance arteries from the vitamin E-deprived rat

1. We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deprivation would alter active and passive mechanical properties in resistance arteries of the rat. Specifically, we hypothesized that vascular tone in isolated mesenteric arteries of the vitamin E-deprived rats would be altered due to impaired endothelial influences of nitric oxide and/or prostaglandins.
2. Lumen diameter and wall thickness were measured in pressurized arteries (≈amp;250 μm diameter) from control (n=9) and vitamin E deprived (n=9) Sprague-Dawley female rats by use of a dimension analysing system.
3. Treatment with a cyclo-oxygenase inhibitor (meclofenamate) did not affect the basal vascular tone in either group. Treatment with a nitric oxide synthase inhibitor (N^G-methyl-L-arginine) caused a significant increase in basal tone only in the vitamin E-deprived rats (% tone: 6.2±1.1 vs 1.2±0.3%; P<0.05). When tone was induced to 25% of the initial diameter with phenylephrine, treatment with the nitric oxide synthase inhibitor resulted in a greater potentiated tone in the vitamin E-deprived rats compared to the controls (26.5±2.7 vs 16.4±3.4%; P<0.05); suggesting a greater nitric oxide affect in the vessels from the vitamin E-deprived rats. Meclofenamate treatment in the induced tone arteries significantly relaxed (−17.4±4.0%; P<0.05) only the arteries from the vitamin E-deprived rats, indicating that a vasoconstrictor was modifying tone. The passive characteristics of distensibility and stress-strain relationship were not different between the two groups of rats.
4. In summary, vitamin E deprivation in the rat enhanced the modulation of vascular tone by both the nitric oxide and cyclo-oxygenase pathways but did not alter passive characteristics of mesenteric arteries.

     

The signalling pathway which causes contraction via P2-purinoceptors in rat urinary bladder smooth muscle

1. The signalling pathway which causes contractions to adenosine 5′-O-2-thiodiphosphate (ADPβS) and α,β-methylene adenosine 5′-diphosphate (α,β-Me ADP) was investigated in rat urinary bladder smooth muscle by measuring isotonic tension.
2. The responses to 10 μM α,β-methylene adenosine 5′-triphosphate (α,β-Me ATP) in 0 and 3.6 mM Ca²⁺ were 5.9±1.3 (n=10) and 122.2±6.4 (n=8) % respectively of those obtained in 1.8 mM Ca²⁺, whereas those to 100 μM ADPβS were 34.6±3.3 (n=8) and 96.8±7.2 (n=8) %, in 0 and 3.6 mM Ca²⁺, respectively. In both experimental conditions, the responses to the two agonists expressed as % of the control responses were significantly different (P<0.01).
3. Indomethacin at high concentrations (>1 μM) decreased the responses to α,β-Me ATP (10 μM), ADPβS (100 μM) and α,β-Me ADP (100 μM). However, no significant difference was obtained between the responses to all the agonists at 30 μM indomethacin.
4. 2-Nitro-4-carboxphenyl n,n-diphenylcarbamate (NCDC) at concentrations between 1 μM and 100 μM concentration-dependently decreased the responses to ADPβS (100 μM) and α,β-Me ADP (100 μM) and almost completely inhibited them at 100 μM. Although the responses to α,β-Me ATP (10 μM) were also inhibited by the drug, at 50 and 100 μM NCDC the responses to α,β-Me ATP were significantly larger than those to ADPβS and α,β-Me ADP (P<0.01).
5. NCDC 100 μM significantly inhibited the KCl-induced contraction to 65.9±4.9% (n=6) of the control (P<0.01).
6. It is suggested that the contraction via ADPβS-sensitive receptors in the rat urinary bladder smooth muscle mainly depends on Ca²⁺ ions liberated from intracellular Ca²⁺ stores, though the contribution of Ca²⁺ ions from the extracellular space cannot be neglected. The release of Ca²⁺ ions from stores is mainly mediated by the production of inositol trisphosphate (IP₃) via the activation of phospholipase C.

     

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

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

     

Aminoguanidine – effects on endoneurial vasoactive nitric oxide and on motor nerve conduction velocity in control and streptozotocin-diabetic rats

1. The effects of aminoguanidine (AG) treatment on reductions in motor nerve conduction velocity (MNCV) and sciatic nerve blood flow, indexed by laser Doppler flux (LDF), were investigated in rats with experimental diabetes (streptozotocin-induced; 8–10 weeks duration). The contribution of endoneurial vasoactive nitric oxide to the LDF of these animals was also investigated by the direct micro-injection of N^G-nitro-L-arginine methyl ester (L-NAME; 1 nmol in 1 μl), followed by L-arginine (100 nmol in 1 μl), into the sciatic nerve endoneurium.
2. The MNCV (m s⁻¹, mean±1 s.d.) of diabetic rats (38.2±1.5) was lower (P<0.01) than that of age-matched controls (47.2±4.2). AG treatment (50 mg kg⁻¹ day⁻¹, i.p.) attenuated the diabetes-induced deficits in MNCV (43.4±5.9; P<0.01), but had no effect in controls (48.8±3.8) or, if administered via drinking water (1 g l⁻¹), diabetics (37.4±4.1).
3. L-NAME markedly reduced the resting LDF (arbitrary units; mean±s.e.mean) of controls (209±13 to 120±18; P<0.005), an effect reversed by subsequent L-arginine (to 206±27). In diabetic rats the LDF reduction following L-NAME was much smaller (111±11 to 84±6; P<0.05), but the change with L-arginine was significantly increased (to 145±12; P<0.001).
4. AG treatment increased the resting LDF of control (265±34) and diabetic rats (133±14 for daily injection and 119±13 for drinking water). The responses to L-NAME and L-arginine were not changed markedly by AG treatment. However, L-arginine appeared to be less effective.
5. In conclusion, these data suggest that AG treatment may affect nitric oxide production in the vasa nervorum of peripheral nerves. However, the effects of AG-treatment are not consistent with the prevention of a diabetes-associated reduction in endoneurial nitric oxide production. The mechanisms by which AG attenuates nerve conduction slowing in streptozotocin-diabetic rats therefore remain unclear.

     

Carrier-dependent and Ca2+-dependent 5-HT and dopamine release induced by (+)-amphetamine, 3,4-methylendioxy-methamphetamine,p-chloroamphetamine and (+)-fenfluramine

1. The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the ³H neurotransmitters.
2. Their rank order of potency for [³H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCA⩾MDMA⩾(+)-Fen>>(+)-Amph). Similarly, their rank order as [³H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph>>pCA=MDMA>>(+)-Fen). We also confirmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine).
3. [³H]-5-HT and/or [³H]-dopamine release induced by all these compounds was partially (31–80%), but significantly Ca²⁺-dependent. Lack of extracellular Ca²⁺ did not alter uptake mechanisms nor did it modify the carrier-dependent dopamine-induced [³H]-dopamine release. (+)-Amph-induced [³H]-dopamine release and pCA- and MDMA-induced [³H]-5-HT release were significantly inhibited by ω-agatoxin-IVA, a specific blocker of P-type voltage-operated Ca²⁺-channels, similar to the previous results on (+)-Fen-induced [³H]-5-HT release.
4. Methiothepin inhibited the Ca²⁺-dependent component of (+)-Amph-induced [³H]-dopamine release with high potency (70 nM), as previously found with (+)-Fen-induced [³H]-5-HT release. The inhibitory effect of methiothepin was not due to its effects as a transporter inhibitor or Ca²⁺-channel blocker and is unlikely to be due to its antagonist properties on 5-HT_1/2, dopamine or any other extracellular receptor.
5. These results indicate that the release induced by these compounds is both ‘carrier-mediated'' and Ca²⁺-dependent (possibly exocytotic-like), with the specific carrier allowing the amphetamines to enter the synaptosome. The Ca²⁺-dependent release is mediated by Ca²⁺-influx (mainly through P-type Ca²⁺-channels), possibly triggered by the drug interacting with an unknown intracellular target, affected by methiothepin, common to both 5-HT and dopamine synaptosomes.

     

Platelets,neutrophils, and vasoconstriction after arterial injury by angioplasty in pigs: effects of MK-886, a leukotriene biosynthesis inhibitor

1. Leukotrienes constitute a class of potent bioactive mediators known to play a pivotal role in inflammation. Since their biosynthesis has been shown to be enhanced by platelet-neutrophil interactions, leukotrienes may be involved in these interactions and the arterial response to injury. Therefore, we investigated the effects of the selective leukotriene biosynthesis inhibitor 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886) on the acute thrombotic and vasomotor responses after arterial injury by angioplasty.
2. Carotid arterial injury was produced by balloon dilatation in control (molecusol vehicle; n=10) and treated (MK-886, 10 mg kg⁻¹, i.v.; n=9) pigs. The acute thrombotic reaction following deep arterial wall injury was quantified with ⁵¹Cr labelled platelets and ¹¹¹In labelled neutrophils, and the vasomotor response was determined angiographically.
3. Platelet deposition at the site of deep arterial wall injury averaged 56.4±11.0×10⁶ platelets cm⁻² in the control group, and was significantly reduced to 18.2±3.8×10⁶ platelets cm⁻² (P<0.005) by treatment with MK-886. Neutrophil deposition was also decreased by MK-886, from 242.8±36.8 to 120.9±20.3×10³ neutrophils cm⁻² (P<0.01). MK-886-treated animals had a significant decrease in the postangioplasty vasoconstrictive response at the site of endothelial injury distally, from 37.5±3.1% in the control group to 13.5±2.5% (P<0.001).
4. The effects of MK-886 were associated with a profound inhibition of ex vivo leukotriene B₄ (LTB₄) synthesis in blood stimulated by the calcium ionophore A23187 and a significant reduction of neutrophil aggregation in whole blood (P<0.01), whereas neutrophil superoxide anion production, serum thromboxane B₂ and platelet aggregation in whole blood were not influenced.
5. The relevant effects of MK-886 are primarily related to inhibition of neutrophil function and suggest an important modulatory role for leukotrienes in the pathophysiological response associated with platelet and neutrophil interactions following arterial injury in vivo.