Interactions between endothelium-derived relaxing factors in the rat hepatic artery: focus on regulation of EDHF

1. In rat isolated hepatic arteries contracted with phenylephrine, acetylcholine and the calcium ionophore A23187 each elicit endothelium-dependent relaxations, which involve both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). However, the contribution of prostanoids to these responses, and the potential interaction between EDHF and other endothelium-derived relaxing factors have not been examined.
2. In the presence of the NO synthase inhibitor N^G-nitro-L-arginine (L-NOARG, 0.3 mM) and a mixture of charybdotoxin (0.3 μM) and apamin (0.3 μM), inhibitors of the target potassium (K) channel(s) for EDHF, acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 each induced a concentration-dependent and almost complete relaxation, which was abolished in the additional presence of indomethacin (10 μM). Thus, in addition to EDHF and NO, a relaxing factor(s) generated by cyclo-oxygenase (COX) contributes to endothelium-dependent relaxation in the rat hepatic artery.
3. The resting membrane potentials of endothelium-intact and endothelium-denuded vascular segments were −57 mV and −52 mV, respectively (P>0.05). In intact arteries, the resting membrane potential was not affected by L-NOARG plus indomethacin, but reduced to −47 mV in the presence of charybdotoxin plus apamin. Acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (10 μM each) elicited a hyperpolarization of 13 mV and 15 mV, respectively. The hyperpolarization induced by these agents was not affected by L-NOARG plus indomethacin (12 mV and 14 mV, respectively), but reduced in the presence of charybdotoxin plus apamin (7 mV and 10 mV, respectively), and abolished in the combined presence of charybdotoxin, apamin and indomethacin.
4. The NO donor 3-morpholino-sydnonimine (SIN-1) induced a concentration-dependent relaxation, which was unaffected by charybdotoxin plus apamin, but abolished by the selective soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 μM). SIN-1 (10 μM) did not alter the resting membrane potential in endothelium-denuded vascular segments.
5. The COX-dependent relaxation induced by acetylcholine was abolished following exposure to 30 mM KCl, but unaffected by glibenclamide (10 μM). The prostacyclin analogue iloprost induced a concentration-dependent relaxation, which was also abolished in 30 mM KCl and unaffected by the combined treatment with glibenclamide, charybdotoxin and apamin. Iloprost (10 μM) induced a glibenclamide-resistant hyperpolarization (8 mV with and 9 mV without glibenclamide) in endothelium-denuded vascular segments.
6. Exposure to SIN-1 or iloprost did not affect the EDHF-mediated relaxation induced by acetylcholine (i.e. in the presence of L-NOARG and indomethacin). Replacement of L-NOARG with the NO scavenger oxyhaemoglobin (10 μM) or the soluble guanylate cyclase inhibitor ODQ (10 μM) or methylene blue (10 μM), which all significantly inhibited responses to endothelium-derived NO, did not affect the acetylcholine-induced relaxation in the presence of indomethacin, indicating that endogenous NO also does not suppress EDHF-mediated responses.
7. These results show that, in addition to EDHF and NO, an endothelium-derived hyperpolarizing factor(s) generated by COX contributes significantly to endothelium-dependent relaxation in the rat heptic artery. Neither this factor nor NO seems to regulate EDHF-mediated responses. Thus, EDHF does not serve simply as a `back-up'' system for NO and prostacyclin in this artery. However, whether EDHF modulates the NO and COX pathways remains to be determined.

     

A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) by either carbachol (pEC₅₀ [negative logarithm of the EC₅₀]=6.19±0.01, R_max [maximum response]=93.2±0.4%; n=14) or calcium ionophore A23187 (pEC₅₀=6.46±0.02, R_max=83.6±3.6%; n=8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC₅₀=6.31±0.06, R_max=94.7±4.6%; n=10; with L-NAME: pEC₅₀=6.33±0.04, R_max=93.4±6.0%; n=4).
2. The selective cannabinoid receptor antagonist, SR 141716A (1 μM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 μM) greatly reduced the R_max values for EDHF-mediated relaxations to carbachol (control, 93.2±0.4%; SR 141716A, 10.7±2.5%; n=5; P<0.001) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control, 84.8±2.1%; SR 141716A, 3.5±2.3%; n=6; P<0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting R_max.
3. Precontraction with 60 mM KCl significantly reduced (P<0.01; n=4 for all) relaxations to 1 μM carbachol (control 68.8±5.6% versus 17.8±7.1%), {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control 71.4±6.1% versus 3.9±0.45%) and anandamide (control 71.1±7.0% versus 5.2±3.6%). Similar effects were seen in the presence of 25 mM K⁺. Incubation of vessels with pertussis toxin (PTX; 400 ng ml⁻¹, 2 h) also reduced (P<0.01; n=4 for all) relaxations to 1 μM carbachol (control 63.5±7.5% versus 9.0±3.2%), {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control 77.0±5.8% versus 16.2±7.1%) and anandamide (control 89.8±2.2% versus 17.6±8.7%).
4. Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 μM) significantly potentiated (P<0.01), to a similar extent (∼2 fold), relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (pEC₅₀: control, 6.45±0.04; PMSF, 6.74±0.10; n=4) and anandamide (pEC₅₀: control, 6.31±0.02; PMSF, 6.61±0.08; n=8). PMSF also potentiated carbachol responses both in the presence (pEC₅₀: control, 6.25±0.01; PMSF, 7.00±0.01; n=4; P<0.01) and absence (pEC₅₀: control, 6.41±0.04; PMSF, 6.88±0.04; n=4; P<0.001) of L-NAME. Responses to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC₅₀: control, 7.51±0.06; PMSF, 8.00±0.05, n=4, P<0.001).
5. EDHF-mediated relaxation to carbachol was significantly attenuated by the K⁺ channel blocker tetraethylammonium (TEA; 1 mM) (pEC₅₀: control, 6.19±0.01; TEA, 5.61±0.01; n=6; P<0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (pEC₅₀: control, 6.47±0.04; TEA, 6.41±0.02, n=4) or on anandamide (pEC₅₀: control, 6.28±0.06; TEA, 6.09±0.02; n=5). TEA (10 mM) significantly (P<0.01) reduced the R_max for anandamide (control, 94.3±4.0%; 10 mM TEA, 60.7±4.4%; n=5) but had no effect on the R_max to carbachol or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187.
6. BaCl₂ (100 μM), considered to be selective for blockade of inward rectifier K⁺ channels, had no significant effect on relaxations to carbachol or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, but caused a small shift in the anandamide concentration-response curve (pEC₅₀: control, 6.39±0.01; Ba²⁺, 6.20±0.01; n=4; P<0.01). BaCl₂ (1 mM; which causes non-selective block of K⁺ channels) significantly (P<0.01) attenuated relaxations to all three agents (pEC₅₀ values: carbachol, 5.65±0.02; {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, 5.84±0.04; anandamide, 5.95±0.02; n=4 for each).
7. Apamin (1 μM), a selective blocker of small conductance, Ca²⁺-activated, K⁺ channels (SK_Ca), 4-aminopyridine (1 mM), a blocker of delayed rectifier, voltage-dependent, K⁺ channels (K_v), and ciclazindol (10 μM), an inhibitor of K_v and adenosine 5′-triphosphate (ATP)-sensitive K⁺ channels (K_ATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or anandamide responses.
8. Glibenclamide (10 μM), a K_ATP inhibitor and charybdotoxin (100 or 300 nM), a blocker of several K⁺ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50 nM), an inhibitor of large conductance, Ca²⁺-activated, K⁺ channels (BK_Ca), had no significant effect on the relaxation responses, either alone or in combination with apamin (1 μM). Also, a combination of apamin (1 μM) with either glibenclamide (10 μM) or 4-aminopyridine (1 mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or anandamide.
9. A combination of apamin (1 μM) with charybdotoxin (100 nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Apamin (1 μM) and charybdotoxin (300 nM) together consistently inhibited the response to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, while apamin (1 μM) and ciclazindol (10 μM) together inhibited relaxations to both carbachol and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. None of these toxin combinations had any significant effect on relaxation to anandamide.
10. It was concluded that the differential sensitivity to K⁺ channel blockers of EDHF-mediated responses to carbachol and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX-sensitive G-proteins to activation of K⁺ conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K⁺ channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.

     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03–10 μM) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.03–10 μM), and nitric oxide (NO), applied either as authentic NO (0.01–10 μM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1–10 μM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1–10 μM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1–3 μM; mean contraction and depolarization, 45.8±5.3 mV and 31.5±3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively.
2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75–80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4–7 in each case; P<0.01). The responses which persisted to ACh and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-N^G-nitroarginine methyl ester (L-NAME; 100 μM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM; 10 min; n=4 in each case; P<0.01).
3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3–5; P<0.01).
4. The potassium channel blockers glibenclamide (10 μM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and authentic NO each by 30% (n=4–8; P<0.01). Application of ODQ (10 μM; 10 min) abolished the ChTX-insensitive responses to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and authentic NO (n=4 in each case; P<0.01
5. When the concentration of phenylephrine was reduced (to 0.3–0.5 μM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1–3 μM phenylephrine (51.2±3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2±6.3%, n=4; P<0.01).
6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.

     

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

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

     

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

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

     

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine,barium, ouabain and 6-hydroxydopamine in chromaffin cells

1. Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154, did not enhance LDH release. Both lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (0.3–10 μM) decreased the veratridine-induced LDH release.
2. Penfluridol did not increase LDH release at concentrations 0.003–1 μM; 3–10 μM increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μM) did not protect against the cytotoxic effects of veratridine; at 1 μM, 15% protection was produced. Higher concentrations (3–10 μM) enhanced the cytotoxic effects of veratridine.
3. Ba²⁺ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μM lubeluzole and fully counteracted by 1 μM penfluridol. {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba²⁺.
4. Ouabain (10 μM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μM) and the lower concentrations of penfluridol (0.003–0.3 μM) prevented the ouabain cytotoxic effects. At higher concentrations (3 μM), penfluridol increased drastically the ouabain cytotoxic effects.
5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μM) had no cytoprotective effects against 6-OHDA.
6. Lubeluzole (3 μM), {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (3 μM) and penfluridol (1 μM) blocked the current through Na⁺ channels in voltage-clamped chromaffin cells (I_Na) by around 20–30%. Ca²⁺ current through Ca²⁺ channels (I_Ca) was inhibited 57% by lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 were readily reversible.
7. Lubeluzole (3 μM) induced reversible blockade of the oscillations of the cytosolic Ca²⁺, [Ca²⁺]_i, in fura-2-loaded cells exposed to 30 or 100 μM veratridine. Penfluridol (1 μM) inhibited those oscillations in an irreversible manner.
8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na⁺ and Ca²⁺ entry, as well as the [Ca²⁺]_i oscillations. The Ba²⁺ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na⁺/Ca²⁺ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.

     

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

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

     

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

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

     

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

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

     

ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia

1. ATP (10–100 μM), but not glutamate (100  μM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 μM) nor glutamate (100 μM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 μM), which is metabolized in the cytosol to BAPTA (an intracellular Ca²⁺ chelator), completely inhibited the plasminogen release evoked by ATP (100 μM). The Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 induced plasminogen release in a concentration-dependent manner (0.3 μM to 10 μM).
2. ATP induced a transient increase in the intracellular calcium concentration ([Ca²⁺]_i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 μM) had no effect on [Ca²⁺]_i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μM) stimulated an increase in [Ca²⁺]_i similar to that of the first application (21 out of 21 cells).
3. The ATP-evoked increase in [Ca²⁺]_i was totally dependent on extracellular Ca²⁺, 2-Methylthio ATP was active (7 out of 7 cells), but α,β-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca²⁺]_i. Suramin (100 μM) was shown not to inhibit the ATP-evoked increase in [Ca²⁺]_i (20 out of 20 cells). 2′- and 3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), a selective agonist of P2X₇ receptors, evoked a long-lasting increase in [Ca²⁺]_i even at 1 μM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′-triphosphate-2′, 3′-dialdehyde (oxidized ATP, 100 μM), a selective antagonist of P2X₇ receptors, blocked the increase in [Ca²⁺]_i induced by ATP (10 and 100 μM).
4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca²⁺]_i via the ionotropic P2X₇ receptor which stimulates the release of plasminogen from the microglia.

     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists.
2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (−log EC₅₀) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18).
3. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (1–3 μM) and BQ-123 (1 μM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 μM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 μM) had no additional effect. RES-701-1 (0.1–1 μM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM).
4. Modulation of the Ca²⁺ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca²⁺ concentrations decreased. Verapamil (30 μM) abolished rhythmic motility induced by ET-1 or SX6c. Ni²⁺ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 μM) completely inhibited ET-1-induced contractions.
5. We conclude that there are two types of ET-receptors, excitatory ET_A- and ET_B-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca²⁺ channels (ROCs) and partly by opening T-type Ca²⁺ channels, and mediate rhythmic motility by opening L-type Ca²⁺ channels.

     

Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium-dependent relaxation to bradykinin in human small resistance-like coronary arteries

1. The effects of the nitric oxide (NO) synthase inhibitor, N^G-nitro-L-arginine (L-NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K⁺ upon endothelium-dependent relaxation to bradykinin were investigated in human isolated small coronary arteries.
2. Endothelium-dependent relaxations to bradykinin were compared in vessels contracted to ∼50% of their maximum contraction to 124 mM KCl Krebs solution, regardless of treatments, with the thromboxane A₂ mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force.
3. L-NOARG (100 μM) caused a small but significant, 12% (P<0.01), decrease in the maximum relaxation (R_max: 91.5±5.4%) to bradykinin but did not significantly affect the sensitivity (pEC₅₀: 8.08±0.17). Increasing the concentration of L-NOARG to 300 μM had no further effect on the pEC₅₀ or R_max to bradykinin. HbO (20 μM) and a combination of HbO (20 μM) and L-NOARG (100 μM) reduced R_max to bradykinin by 58% (P<0.05) and 54% (P<0.05), respectively. HbO (20 μM) and L-NOARG (100 μM, combined but not HbO (20 μM) alone, caused a significant 11 fold (P<0.05) decrease in sensitivitiy to bradykinin. HbO (20 μM) decreased the sensitivity to the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), approximately 17 fold (P<0.05).
4. Raising the extracellular concentration of K⁺ isotonically to 30 mM, reduced the R_max to bradykinin from 96.6±3.1% to 43.9±10.1% (P<0.01) with no significant change in sensitivity. A combination of HbO, L-NOARG and high K⁺ (30 mM) abolished the response to bradykinin. High K⁺ did not change either the sensitivity or maximum relaxation to SNAP.
5. In conclusion, L-NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non-NO -dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K⁺-sensitive vasodilator mechanism, possibly endothelium-derived hyperpolarizing factor (EDHF).

     

The effects of bradykinin on K+ currents in NG108-15 cells treated with U73122, a phospholipase C inhibitor,or neomycin

1. Bradykinin has multiple effects on differentiated NG108-15 neuroblastoma×glioma cells: it increases Ins(1,4,5)P₃ production and intracellular Ca²⁺ concentration [Ca²⁺]_i, evokes a Ca²⁺ activated K⁺ current (I_K(Ca)) and inhibits M current (I_M). We studied the effect of the aminosteroid {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and the antibiotic neomycin, both putative blockers of phospholipase C (PLC), on these four bradykinin effects.
2. Preincubation with 1 or 5 μM {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 for 15 min partly suppressed Ins(1,4,5)P₃ generation and the increase in [Ca²⁺]_i induced by 1 μM bradykinin. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 10 μM caused total and irreversible inhibition. The inactive analogue {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 was without effect.
3. Resting levels of Ins(1,4,5)P₃ were not affected. However, resting [Ca²⁺]_i was increased by 10 μM {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, but not by {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343. Individual cells responded to 10 μM {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 with a small increase in [Ca²⁺]_i, followed in some cells by a large further rise.
4. Pretreatment of whole-cell clamped cells with 1 μM {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 for 30 min reduced the bradykinin-induced I_K(Ca) to a fifth of its normal size. To suppress it totally, a 7–12 min pretreatment with 5 μM {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 was required. Again, {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 was without effect.
5. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 at concentrations of 5–10 μM irreversibly decreased the holding current (I_h) which at a holding potential of −30 or −20 mV mainly flows through open M channels. The decrease was often preceded by a transient increase.
6. M current (I_M) measured with 1 s pulses, was also decreased by 5–10 μM {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343, but short applications of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 could cause a small increase. The bradykinin-induced inhibition of I_M was not affected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.
7. Preincubation with 1 or 3 mM neomycin for 15 min did not affect Ins(1,4,5)P₃ generation and the increase in [Ca²⁺]_i induced by bradykinin. Pretreatment with 3 mM neomycin for about 20 min diminished the bradykinin-induced I_K(Ca) to a fifth of its normal size.
8. The four main conclusions drawn from the results are: (a) {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 suppresses bradykinin-induced PLC activation and I_K(Ca), but not I_M inhibition. (b) This indicates that the transient outward current I_K(Ca), but not the decrease of I_M in response to bradykinin, is mediated by PLC. (c) {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 itself inhibits I_M and mobilizes Ca²⁺ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC-mediated signalling pathways in NG108-15 cells.

     

Differential effects of the neuroprotectant lubeluzole on bovine and mouse chromaffin cell calcium channel subtypes

1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (−) enantiomer {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 on whole-cell currents through Ca²⁺ channels, with 10 mM Ba²⁺ as charge carrier (I_Ba), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of −80 mV, at 10 s intervals had an average magnitude of 1 nA.
2. Lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 blocked the peak I_Ba of bovine chromaffin cells in a time and concentration-dependent manner; their IC₅₀s were 1.94 μM for lubeluzole and 2.54 μM for {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154. In a current-voltage protocol, lubeluzole (3 μM) inhibited peak I_Ba at all test potentials. However, no obvious shifts of the I-V curve were detected.
3. After 10 min exposure to 3 μM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered.
4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at −110 mV and 87% at −50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at −110 mV, decreased at −80 mV and did not occur at −50 mV.
5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 μM lubeluzole caused neither blockade nor inactivation of I_Ba. The external application of 3 μM lubeluzole to those dialysed cells produced inhibition as well as inactivation of I_Ba.
6. The effects of lubeluzole (3 μM) on I_Ba in mouse chromaffin cells were similar to those in bovine chromaffin cells. At −80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late I_Ba (66%) as compared with peak I_Ba (46% after 10 min superfusion with lubeluzole).
7. In mouse chromaffin cells approximately half of the whole-cell I_Ba was sensitive to 3 μM nifedipine (L-type Ca²⁺ channels) and the other half to 3 μM ω-conotoxin MVIIC (non-L-type Ca²⁺ channels). In ω-conotoxin MVIIC-treated cells, 3 μM lubeluzole caused little blockade and inactivation of I_Ba. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of I_Ba that reversed upon wash out of the compound.
8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca²⁺ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L-type Ca²⁺ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca²⁺ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca²⁺ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.

     

No evidence for a significant non-nitrergic,hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli.
2. Phenylephrine (0.1–100 μM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 μM phenylephrine (EC₂₆) was used to raise tone in subsequent experiments.
3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-N^G-nitro-arginine (L-NOARG; 100 μM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 μM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01–5 μM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 μM).
4. Nifedipine (1 μM) reduced the contractile response to 0.2 μM phenylephrine by 38%; however, it had no effect on NANC relaxations.
5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.

     

Dopamine stimulation of cardiac β-adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393

1. Mechanisms underlying β-adrenoceptor stimulation by dopamine were examined on guinea-pig Langendorff-perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L-type Ca²⁺ current (I_Ca,L) in the presence of prazosin as indicators of β-adrenoceptor stimulation. Dopamine-induced overflow of noradrenaline (NA) concentrations was measured by high-performance liquid chromatography.
2. Dopamine caused positive chronotropic effects with an EC₅₀ of 2.5 μM and induced NA overflow with a similar EC₅₀ (1.3 μM). The chronotropic effect of dopamine was abolished by bisoprolol (1 μM).
3. The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 μM) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine.
4. Dopamine (3 μM)-induced NA release was not affected by tetrodotoxin, ω-conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC₅₀ ∼1 μM). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system.
5. {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393, a D₁ receptor partial agonist, potently inhibited the 3 μM dopamine-induced release of NA (IC₅₀ ∼0.1 μM). D₁ receptors are not involved in the DA-induced release of NA, since {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (3 μM), a potent D₁ antagonist, inhibited the NA release only slightly, and dihydrexidine (1 μM) and chloro-APB (1 μM), full D₁ agonists, caused no significant NA release.
6. {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 inhibited tyramine-induced overflow of NA, and potentiated the field stimulation-induced NA release. {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 and desipramine retarded the decay of the stimulation-induced tachycardia in a similar manner. These results indicate that {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly-acting sympathomimetic agonists in the heart. In the presence of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 (10 μM), dopamine at 1 μM showed no chronotropic effect.
7. Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC₅₀ for I_Ca,L stimulation by dopamine was high (13 μM). As a result, dopamine at 1 μM did not affect I_Ca,L. Bisoprolol abolished the stimulation of I_Ca,L by dopamine (30 μM), and dihydrexidine (1 μM) did not affect I_Ca,L.
8. It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (<1 μM) result almost exclusively from the indirect effect of β adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of β adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D₁ receptors seem negligible. The desipramine- and {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-sensitive monoamine transporter mediates the release of NA.

     

Epoxyeicosatrienoic acids,potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

1. Using intracellular microelectrodes, we investigated the effects of 17-octadecynoic acid (17-ODYA) on the endothelium-dependent hyperpolarization induced by acetylcholine in the guinea-pig isolated internal carotid artery with endothelium.
2. In the presence of N^ω-nitro-L-arginine (L-NOARG, 100 μM) and indomethacin (5 μM) to inhibit nitric oxide synthase and cyclo-oxygenase, acetylcholine (1 μM) evoked an endothelium-dependent hyperpolarization which averaged −16.4 mV starting from a resting membrane potential of −56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential.
3. The acetylcholine-induced endothelium-dependent hyperpolarization was not altered by charybdotoxin (0.1 μM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 μM) to −12.8±1.2 mV (n=10) or the combination of apamin plus iberiotoxin (−14.3±3.4 mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+7.1±3.7 mV, n=8).
4. 17-ODYA (10 μM) produced a significant hyperpolarization of the resting membrane potential which averaged −59.6 mV and a partial but significant inhibition of the acetylcholine-induced endothelium-dependent hyperpolarization (−10.9 mV).
5. Apamin did not modify the effects of 17-ODYA but in the presence of charybdotoxin or iberiotoxin, 17-ODYA no longer influenced the resting membrane potential or the acetylcholine-induced hyperpolarization.
6. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6-, 8,9-, 11,12- and 14,15-EpETrE, 3 μM) did not affect the cell membrane potential and did not relax the guinea-pig isolated internal carotid artery.
7. These results indicate that, in the guinea-pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P₄₅₀ pathway in endothelium-dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium-derived hyperpolarizing factor (EDHF) is probably not due to the opening of BK_Ca channels.

     

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

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

     

Noradrenaline release and the effect of endogenous activation of the phospholipase C/protein kinase C signalling pathway in rat atria

1. It has been proposed that protein kinase C (PKC) in sympathetic nerves is activated during action-potential evoked release of noradrenaline and helps maintain transmitter output. We studied this phenomenon further in rat atria radiolabelled with [³H]-noradrenaline.
2. Noradrenaline release was elevated by continuous electrical stimulation of the atria for 10 min at either 5 or 10 Hz. Two inhibitors of PKC, polymyxin B (21 μM) and Ro 318220 (3 μM), markedly inhibited the release of noradrenaline but only at the higher stimulation frequency.
3. Further experiments were conducted with 10 Hz stimulation but for shorter train durations. In this case polymyxin B inhibited noradrenaline release during a 10 or 15 s train of impulses but not during a 5 s train. This suggests that PKC effects are induced during the stimulation train by some process.
4. The diacylglycerol kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 (10 μM), which prevents the breakdown of diacylglycerol, enhanced noradrenaline release elicited by stimulation at 10 Hz for 10 or 15 s. This effect was not seen if polymyxin B was present and suggests that diacylglycerol is the endogenous activator of PKC.
5. The source of the diacylglycerol may be through phospholipase C pathways, since the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (3 μM) inhibited noradrenaline release at 10 Hz for 10 s and the effect was not seen if polymyxin B was also present.
6. It is unlikely that phospholipase D is the source of diacylglycerol. Although the phospholipase D inhibitor wortmannin (1 μM) inhibited noradrenaline release, this effect was still observed in the presence of polymyxin B. Furthermore ethanol, which inhibits diacylglycerol formation by phospholipase D, had no effect on noradrenaline release.
7. We therefore suggest that during a train of high frequency pulses phospholipase C is activated and this results in the production of diacylglycerol which in turn activates PKC. This enables the neurones to maintain transmitter release at a high level.