Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle.

1. We investigated the characteristics of endothelin (ET)-induced contraction and changes in intracellular Ca2+ concentration ([Ca2+]i) using the fura-2-loaded and non-loaded rabbit iris dilator. ET-1 and ET-2 (3-100 nM) and ET-3 (30-100 nM) caused contraction in a concentration-dependent fashion. 2. The selective ETB-receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of potencies for the contraction (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL1620. 3. The contractile response to ET-3 was antagonized by pretreatment with BQ-123 (10 nM), a selective ETA receptor antagonist. The contractile responses to ET-1 and ET-2 were antagonized by pretreatment with BQ-123 (10 microM), but not at a concentration of 10 nM. 4. ETs increased [Ca2+]i and sustained muscle contraction. ET-1 (100 nM), ET-2 (100 nM), and ET-3 (1 microM) induced an elevation of [Ca2+]i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ETB receptor-selective agonist, IRL1620 (1 microM) and sarafotoxin S6c (1 microM) also induced a rapid and transient elevation of [Ca2+]i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5. ET-1 (100 nM) induced a transient increase in [Ca2+]i in a Ca(2+)-free, 2 mM EGTA-containing physiological saline solution (Ca(2+)-free PSS), and a small sustained contraction which was significantly different from that induced by ET-1 (100 nM) in normal PSS. The ET-1-induced increase in [Ca2+]i and sustained contraction were not affected by the voltage-dependent Ca2+ channel blocker, nicardipine (10 microM). The ET-1-induced transient increase in [Ca2+]i was significantly reduced by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (30 microM); however, the ET-1-induced sustained contraction was not affected by this agent. 6. The selective ETA receptor antagonist, BQ-123 (100 nM) reduced the ET-3 (100 nM)-induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca2+]i. However, this antagonist at 1 microM did not affect the ET-1 (100 nM)- and ET-2 (100 nM)-induced elevation of [Ca2+]i and contractile response, or the IRL1620-induced elevation of [Ca2+]i. 7. The selective ETB receptor antagonist, BQ-788 (1 microM) reduced the transient increase in [Ca2+]i induced by ET-1 (30 nM), ET-2 (30 nM), ET-3 (100 nM) and IRL1620 (1 microM), but did not affect the sustained elevation of [Ca2+]i and contractile responses produced by ET-1, ET-2 and ET-3. 8. Pretreatment with IRL1620 (1 microM) reduced the increase in [Ca2+]i induced by IRL1620 (1 microM) and sarafotoxin S6c (1 microM), as well as the ET-1 (100 nM)-, ET-2 (100 nM)- and ET-3 (1 microM)-induced elevation of [Ca2+]i, whereas in the presence of IRL1620, ET-1-, ET-2- and ET-3-induced contractions were unaltered. 9. These results suggest that ETA and ETB receptor subtypes exist in the rabbit iris dilator muscle, and that the ETA receptor is divided into: (1) BQ-123-sensitive ETA subtypes activated by ET-1, ET-2 and ET-3, and (2) BQ-123-insensitive ETA subtypes activated by ET-1 and ET-2, which cause the sustained increase of [Ca2+]i and contraction; in contrast, ETB receptor subtypes are activated by ET-1, ET-2, ET-3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca2+]i which is not able to contract the smooth muscle.     

Block of endothelin-1-induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ-788.

1. The present study characterizes the receptors responsible for endothelin-1-induced release of thromboxane A2 from the guinea pig lung and of endothelium-derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ-123, and a novel selective ETB receptor antagonist, BQ-788. 2. In the guinea pig perfused lung, endothelin-1 (ET-1) (5 nM) induced a marked increase of thromboxane A2 which was reduced by 17 +/- 5.0, 70 +/- 1.0 and 93 +/- 1.2% by BQ-788 infused at concentrations of 1, 5 and 10 nM respectively. In contrast, BQ-123 (0.1 and 1.0 microM) had little or no effect on the ET-1-induced release of thromboxane A2. 3. In the same perfused model, the selective ETB agonist, IRL 1620 (50 nM), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid-releasing properties of IRL 1620 were abolished by BQ-788 at 10 nM, yet were unaffected by BQ-123 (1 microM). 4. In the rabbit perfused kidney, BQ-788 (10 nM) potentiated the increase of perfusion pressure induced by endothelin-1 (1, 5 and 10 nM) by approximately 90%, but not that induced by angiotensin II (1 microM). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide. 5. In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L-NAME (100 microM), potentiated the pressor responses to both endothelin-1 and angiotensin II. Under L-NAME treatment, BQ-788 did not further potentiate the pressor response to endothelin-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium

Resveratrol causes endothelium dependent and independent relaxation of vascular smooth muscle. This study investigated the mechanisms behind the effect of resveratrol on vascular tone. Resveratrol (0.1 mM) inhibited KCl-stimulated contractions in endothelium-denuded rat aorta and this inhibition was not reversed by tetraethylammonium (TEA) (5 mM), glyburide (3 microM), ouabain (0.1 mM), thapsigargin (1 microM), or indomethacin (10 microM). KCl (90 mM) increased the intracellular free calcium concentration ([Ca2+]i) in the isolated smooth muscle cells from the rat aorta and resveratrol (0.1 mM) did not inhibit the KCl-stimulated [Ca2+]i increase. The CaCl2 (0.1-100 microM) stimulated contractions were inhibited by resveratrol (0.1 mM) in the Triton X-100 skinned smooth muscle of the aorta. In heart valve endothelium, resveratrol (0.1 mM) augmented the acetylcholine (10 microM) stimulated [Ca2+]i increase. Resveratrol-induced augmentation of the acetylcholine-stimulated [Ca2+]i elevation was reversed by glyburide (3 microM), but not by TEA (5 mM). The present study indicated that resveratrol affected vascular smooth muscle and endothelium in different ways. Resveratrol decreased the Ca2+ sensitivity but did not affect the KCl-stimulated [Ca2+]i increase in the vascular smooth muscle. In the endothelial cells, resveratrol enhanced the agonist-stimulated [Ca2+]i increase that might trigger nitric oxide synthesis from endothelial cells.     

Role of peptidase and cyclooxygenase inhibitors in the guinea-pig bronchial response to the synthetic endothelin ET(B) agonist IRL 1620 and antagonist BQ-788

In isolated guinea-pig bronchial preparations the selective endothelin ETB agonist, IRL 1620 caused a concentration-dependent contraction. The pD2 value (7.16 +/- 0.09, n = 6) was significantly increased in the presence of peptidase inhibitors (thiorfan 1 microM, captopril 1 microM, bestatin 1 microM) (pD2 = 7.75 +/- 0.09, n = 6). Indomethacin (5 microM) did not appear to influence the ETB-agonist pD2 value (6.92 + 0.11, n = 6) but potentiated its maximal response significantly (67.23 +/- 4.81% vs. 53.37 +/- 4.80%). The concentration-response curve for the contractile response to IRL 1620 (pD2=7.83 +/- 0.01, n=16); was reproducible, although not completely, since the second curve to this selective ETB agonist was shifted significantly to the right (pD2 = 7.34 +/- 0.09, n = 16) and a decrease in the maximal response was observed (20.0 +/- 2.0%). BQ 788, a selective antagonist for ETB receptors, employed in concentrations ranging from 1.5 to 150 nM, caused a dose-dependent shift to the right of the concentration-response curve to IRL 1620, with a pIC50 value of 8.11 +/- 0.03; this action was not influenced by adding enzyme inhibitors (pIC50 = 8.17 +/- 0.29). Our data show that IRL 1620 undergoes a hydrolytic metabolism in guinea-pig bronchial preparations, which could influence the calculation of the pD2. Pretreatment of the tissue with peptidase inhibitors and indomethacin is consequently significant in the evaluation of IRL 1620 activity, while it does not influence the action of the antagonist, BQ 788.     

delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells.

1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM-1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. In the presence of 1 microM or 100 microM carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol. 6. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml-1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i. 7. The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml-1, 16 h). This treatment did not significantly affect the response of the cells to carbachol. 8. The opioids appeared to elevate [Ca2+]i by mobilizing Ca2+ from intracellular stores. Both DPDPE and DAMGO continued to elevate [Ca2+]i when applied in nominally Ca(2+)-free external buffer or when applied in a buffer containing a cocktail of Ca2+ entry inhibitors. Thapsigargin (100 nM), an agent which discharges intracellular Ca2+ stores, also blocked the opioid elevations of [Ca2+]i. 9. delta and mu Opioids did not appear to mobilize intracellular Ca2+ by modulating the activity of protein kinases. The application of H-89 (10 microM), an inhibitor of protein kinase A, H-7 (100 microM), an inhibitor of protein kinase C, protein kinase A and cyclic GMP-dependent protein kinase, or Bis I, an inhibitor of protein kinase C, did not alter the opioid mobilization of [Ca2+]i. 10. Thus, in SH-SY5Y cells, opioids can mobilize Ca2+ from intracellular stores but they require ongoing muscarinic receptor activation. Opioids do not elevate [Ca2+]i when applied alone.     

Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.

1. The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 microM, respectively. 2. Atropine (10 microM) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3. AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 119 and high affinity for 4-DAMP in antagonizing this response. 4. The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mM EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mM) in the continued presence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-dependent difference in the relationship between cytosolic Ca2+ level and release of vascular relaxing factors in the endothelium of rabbit aortic valve

The correlation between changes in cytosolic Ca2+ levels ([Ca2+]i) and the release of vascular relaxing factor(s) was investigated in the endothelium of rabbit aortic valve. ATP, carbachol and thapsigargin increased endothelial [Ca2+]i in rabbit aortic valve loaded with a leakage resistant, fluorescent Ca2+ indicator, fura-PE3. Release of relaxing factors was bioassayed using the 'sandwich' preparation in which contraction was measured in the endothelium-denuded rabbit aorta attached to the endothelial surface of the valve. Addition of ATP, carbachol and thapsigargin induced sustained relaxation of the phenylephrine-induced contraction of the aorta in the 'sandwich' preparation. N(G)-monomethyl-L-arginine (L-NMMA) greatly attenuated the relaxation induced by carbachol, and combined treatment with tetra-n-butylammonium completely inhibited the relaxation. These results suggest that the endothelial relaxing factors released from aortic valve are nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). When the increase in endothelial [Ca2+]i was plotted against the relaxation, the carbachol-induced increase in [Ca2+]i elicited greater relaxation than did ATP or thapsigargin at a given [Ca2+]i. This suggests that various agonists differently modulate the relationship between [Ca2+]i and release of NO.     

Pharmacological characterization of endothelin receptors-mediated contraction in the mouse isolated proximal and distal colon

The study investigated the role of endothelin (ET) and the ET receptor subtypes ET(A) and ET(B) in mediating longitudinal contraction in the mouse proximal and distal colon. Cumulative concentration-response curves to a range of ET agonists (ET-1, ET-2, ET-3, (Ala(1,3,11,13)) ET and IRL 1620) were established by administering concentrations ranging from 0.01 nM to 0.3 microM. Concentration-response curves to ET-1, which exhibits a high affinity for both ET(A) and ET(B) receptor subtypes, were also established in the presence of the ET(A) antagonist BMS 182874 and the ET(B) antagonist IRL1038. The addition of the selective ET(A) receptor antagonist BMS 182874 caused a rightward shift of the concentration-response curve to ET-1 in both sections of the colon. The ET(B) receptor antagonist IRL1038 (0.3-1 microM) did not significantly effect the response to ET-1 in the proximal colon but caused a significant decrease in response towards higher concentrations ranges (>or=3 nM) in the distal colon. A comparison of the concentration-response curves to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1>or=ET-2>ET-3 in the proximal colon and ET-1>or=ET-2>or=ET-3 in the distal colon. The selective ET(B) receptor agonists, (Ala(1,3,11,13)) ET and IRL 1620 did not produce any response in the proximal sections of the colon but produced a smaller contraction in the distal segments. The data indicate that ET can contract the proximal tissues of the mouse colon predominantly via ET(A) receptors and in the distal tissues via ET(A) and ET(B) receptors.     

Effects of endothelin on the mechanical activity and cytosolic calcium level of various types of smooth muscle.

1. Effects of porcine/human endothelin (endothelin-1), a novel vasoconstrictor peptide, on various smooth muscles were examined. 2. In rat aorta, endothelin (1 pM-30nM) induced contraction in a concentration-dependent manner. Removal of endothelium shifted the concentration-response curve to the left. When added during the sustained contraction induced by 0.1 microM noradrenaline, endothelin (1 nM) induced a relaxation that was inhibited by removing endothelium or by methylene blue. 3. In rat aorta without endothelium, endothelin (1-30 nM) increased cytosolic Ca2+ level [( Ca2+]cyt) followed by contraction. Endothelin induced less contraction than high K+ at a given [Ca2+] cyt when the concentration of endothelin was lower (1-3nm) and/or during the early phase of the contraction (less than 10 min). In contrast, endothelin induced a greater contraction than KCl after prolonged exposure to high concentrations (greater than 10 nM). 4. The increase in [Ca2+]cyt due to endothelin was strongly inhibited by 10 microM verapamil or 0.3 microM nicardipine although muscle contraction was only partially inhibited. 5.In Ca2+ -free solution, endothelin (30 nM) induced a transient increase in [Ca2+] cyt and a slow increase in muscle tension. After a prolonged incubation in Ca2+-free solution, endothelin (30 nM) still induced a slow increase in tension without changing [Ca2+]cyt. This contraction was inhibited by 1 microM sodium nitropusside or 10 microM forskolin. 6. In canine trachea and guinea-pig uterus, endothelin (30 nM) induced sustained contraction with an increase in [Ca2+]cyt. In the absence of external Ca2+, endothelin (30 nM) induced a sustained contraction in canine trachea without changing [Ca2+]cyt. In guinea-pig vas deferens, taenia caeci and ileal longitudinal muscle, endothelin induced small increases in [Ca2+]cyt and tension. 7. In permeabilized smooth muscles, endothelin (30 nM) did not change the muscle tone. 8. These results suggest that endothelin acts on the endothelium and increases the synthesis or release of endothelin-derived relaxing factor (EDRF). These results also suggest that endothelin acts directly on smooth muscle and increases [Ca2+]cyt by releasing Ca2+ from sites and increasing Ca2+ influx through the verapamil- and 1,4-dihydropyridine-sensitive pathway. Endothelin seems to decrease Ca2+ -sensitivity of contractile elements at lower concentrations and/or during the early phase of the contraction, whereas it increases Ca2+ -sensitivity at higher concentrations during the sustained phase of the contraction. Furthermore, endothelin induces a contraction that is not dependent on [Ca2+]cyt.     

Group 1 mGlu receptors elevate [Ca2+]i in rat cultured cortical type 2 astrocytes: [Ca2+]i synergy with adenosine A1 receptors.

Brain macroglia are known to express a diverse array of neurotransmitter receptors whose signal transduction pathways may be subject to heteroreceptor 'cross-talk'. In the current study we have examined group 1 mGlu receptor-evoked [Ca2+]i signalling, and possible heteroreceptor cross-talk, in cultured type 2 astrocytes. The selective group 1 metabotropic glutamate (mGlu) receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elevated [Ca2+]i (EC50 = 1.7 +/- 0.6 microM); an effect reversed by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 52.7 +/- 8.7 microM). DHPG-evoked [Ca2+]i responses were abolished by (1) thapsigargin (100 nM), implicating the involvement of internal Ca2+ stores in group 1 mGlu [Ca2+]i responses and (2) the removal of extracellular Ca2+. When applied alone, the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA, 100 nM) failed to influence [Ca2+]i. However, in the presence of 1 microM DHPG, CPA potently (EC50 = 12.3 +/- 1.9 nM) increased [Ca2+]i responses. In the presence of 100 nM CPA, the efficacy of DHPG was doubled without any significant change in the DHPG EC50 value. This effect was reversed by either the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (IC50 = 50.3 +/- 19.9 nM) or overnight incubation with Pertussis toxin (100 ng/ml). We conclude that (1) type 2 astrocytes contain group 1 mGlu receptors coupled to [Ca2+]i signalling and (2) co-activation of adenosine A1 receptors enhances group 1 mGlu-evoked [Ca2+]i responses in these cells via a Gi/o G protein-mediated mechanism.     

Characterization of the binding of endothelin ETB selective ligands in human and rat heart.

1. We determined competition binding characteristics of endothelin ETB receptor selective ligands in human left ventricle and compared these values to those obtained with rat left ventricle. Sarafotoxin S6c, ET-3, BQ788 and IRL2500 competed against [125I]-PD151242 (ETA selective radioligand) with low affinity in human left ventricle, confirming the ETB selectivity of these compounds. 2. ET-3 competed with moderate selectivity for ETB over ETA receptors in human left ventricle and with slightly higher selectivity in rat left ventricle (460 and 1,400 fold, respectively). There was a small difference in the affinity of ETA receptors for ET-3 (KD ETA in human left ventricle = 0.07 +/- 0.02 microM; KD ETA in rat left ventricle = 0.27 +/- 0.08 microM; P = 0.05) but no difference in the affinity of ETB receptors for this ligand (KD ETB in human left ventricle = 0.15 +/- 0.06 nM; KD ETB in rat left ventricle = 0.19 +/- 0.03 nM). 3. The selectivity of sarafotoxin S6c for ETB over ETA receptors in human left ventricle was 5,900 fold compared with 59,400 fold in rat left ventricle. The affinity of ETA receptors for sarafotoxin S6c was higher in human than in rat left ventricle (KD ETA = 2.00 +/- 0.20 microM and 3.50 +/- 0.26 microM, respectively; P = 0.03), while the affinity of ETB receptors for this ligand was higher in rat left ventricle (KD ETB = 0.06 +/- 0.02 nM) than in human left ventricle (KD ETB = 0.34 +/- 0.13 nM) (P = 0.02). The affinity of ETB receptors for sarafotoxin S6c in rat left ventricle determined in the absence or presence of GTP was the same indicating that differing affinity states of ETB receptors in human and rat left ventricle do not account for the variation observed between species. 4. There was no difference in the affinity of ETA receptors for BQ788 (KD ETA = 1.01 +/- 0.20 microM and KD ETA = 1.39 +/- 0.35 microM) or for the novel ETB selective antagonist. IRL2500 (KD ETA = 30.0 +/- 20.8 microM and KD ETA = 55.6 +/- 9.93 microM) in human and rat left ventricle, respectively. ETB receptors had a significantly higher affinity for BQ788 (KD ETB = 9.8 +/- 1.3 nM and KD ETB = 31.0 +/- 5.4 nM; P = 0.02) and IRL2500 (KD ETB = 78.2 +/- 9.7 nM and KD ETB = 300.0 +/- 75.1 nM; P = 0.03) in human and rat left ventricle, respectively. The synthetically synthesized ETB selective antagonist RES-701-1 (0.1 -3 microM) failed to inhibit [125I]-ET-1 binding in either tissue. 5. In conclusion, we have compared equilibrium dissociation constants for a number of ETB selective compounds in human and rat heart. The affinity of ETB receptors for sarafotoxin S6c, BQ788 and IRL2500 differed in human and rat left ventricle. No difference in affinity was detected for ET-3 binding at ETB receptors. Sarafotoxin S6c binding was unaffected by GTP indicating that the different receptor affinities in human and rat heart cannot be explained by differing ETB receptor affinity states. This study highlights the need to consider differences in binding characteristics that may arise from the use of tissues obtained from different species.     

Neurokinin A receptors are coupled to calcium mobilization in transfected fibroblasts.

Neurokinin A (NKA) mediated a concentration dependent increase in the intracellular free Ca2+ concentration, [Ca2+]i, in B82 fibroblasts transfected with the neurokinin 2 (NK2) receptor. The EC50 value of this response was 24 nM. A selective NK2 antagonist, MEN 10207, at a concentration of 1 microM completely inhibited the [Ca2+]i rise to 0.1 microM NKA. These results suggest that activation of NK2 receptors expressed in the transfected fibroblasts are functionally coupled to intracellular calcium mobilization.     

Statins rise cytoplasmic calcium level [Ca2+]i in cultured endothelial cells

Recently, we have shown that some HMG-CoA reductase inhibitors (statins) induce immediate pleiotropic effects in vascular endothelium both in vivo and in vitro, to mention only PGI2-mediated thrombolysis in rats and NO-mediated endothelium-dependent vasodilation in guinea pig coronary circulation. Here we look whether immediate endothelial effect of statins is associated with mobilization of intracellular calcium ions [Ca2+]i in cultured bovine aortic endothelial cells (BAEC). We analyzed the effects of various statins (atorvastatin, cerivastatin, simvastatin, lovastatin and pravastatin at concentration of 10-30 microM) on [Ca2+]i in BAEC in comparison to responses induced by bradykinin (Bk) (10 nM), adenosine diphosphate (1 microM), acetylcholine (100 nM), adrenaline (10 microM), serotonin (10 microM) or calcium ionophore A 23187 (0.1 microM) using FURA-2 according to fluorimetric method of Grynkiewicz et al. Basal [Ca2+]i level in BAEC was between 60 and 100 nM. Bk was the most potent to induce [Ca2+]i response. Delta[Ca2+]i induced by Bk was 331.9 +/- 19.49 nM (n = 36). Delta[Ca2+]i induced by statins (30 microM), i.e. atorvastatin, cerivastatin, simvastatin, lovastatin and pravastatin were 66.4 +/- 7.38% (n = 6), 54.8 +/- 10.12% (n = 5), 58.8 +/- 13.9% (n = 8), 27.7 +/- 7.19% (n = 5) and 0% (n = 5) of the response induced by Bk (10 nM), respectively. In summary, all statins tested, except pravastatin, induce immediate increase in [Ca2+]i in endothelium. This pleiotropic activity of statins in endothelium, most likely not related to the inhibition of HMG-CoA reductase, may represent an intracellular correlate for the immediate release of NO and PGI2 by these drugs that was reported by us previously.     

Contractile and relaxant effects of phorbol ester in the intestinal smooth muscle of guinea-pig taenia caeci.

1. Effects of phorbol esters on the cytosolic Ca2+ level ([Ca2+]i) and muscle tension in the intestinal smooth muscle of guinea-pig taenia caeci were examined. 2. 12-Deoxyphorbol 13-isobutyrate (DPB, 1 microM) did not change the [Ca2+]i and tension in resting muscle. 3. In high K(+)-stimulated muscle, 1 microM DPB transiently augmented the contraction and decreased [Ca2+]i. 12-Deoxyphorbol 13-isobutyrate 20-acetate (1 microM) and phorbol 12, 13-dibutyrate (1 microM) showed similar effects to DPB whereas phorbol 12-myristate 13-acetate (1 microM) and phorbol 12, 13-didecanoate (1 microM) were ineffective. 4. DPB (1 microM) inhibited both [Ca2+]i and tension stimulated by 300 nM carbachol or 3 microM histamine. In the presence of a higher concentration of carbachol (1 microM), DPB decreased [Ca2+]i and transiently increased muscle tension. 5. In the muscle strips permeabilized with bacterial alpha-toxin, 1 microM DPB shifted the Ca(2+)-tension curve to the left. An inhibitor of protein kinase C, H-7 (30 microM), inhibited the effect of DPB. 6. DPB did not change the high K(+)-induced contraction in the muscle strips pretreated with 3 microM phorbol 12-myristate 13-acetate for 24 h. 7. These results suggest that activation of protein kinase C has dual effects; it augments contraction by increasing the Ca2+ sensitivity of the contractile elements and it inhibits contraction by decreasing [Ca2+]i.     

Endothelin receptor antagonist activity of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy)acetamide hydrochloride (PABSA) in rat aortic smooth muscle cells and isolated rat thoracic aorta

Antagonist activities of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy) acetamide hydrochloride (CAS 188710-94-3, PABSA), a novel endothelin (ET) receptor antagonist, for ETA and ETB receptors were evaluated using rat aortic smooth muscle A7r5 cells and isolated rat thoracic aorta. PABSA concentration-dependently inhibited the ET-1-induced increase in intracellular calcium concentration ([Ca2+]i) mediated via ETA receptors in A7r5 cells with an IC50 of 0.17 nmol/l. PABSA antagonized the ETA receptor-mediated contraction induced by ET-1 in endothelium-denuded rat aorta with a Kb of 0.74 nmol/l. The potency of PABSA in inhibiting ETA receptor-mediated vasocontraction was approximately 40- and 100-fold greater than those of BQ-123, a selective ETA antagonist, and bosentan, a mixed ETA/ETB receptor antagonist, respectively. ETB receptor-mediated endothelium-dependent vasorelaxation induced by ET-3 in the aorta was also antagonized by PABSA, with a Kb of 9.8 nmol/l. In contrast, PABSA affected neither the vasocontraction induced by KCl or norepinephrine nor the vasorelaxation induced by acetylcholine or prostaglandin I2 in the aorta. These results suggest that PABSA is a highly potent and selective ETA receptor antagonist.     

Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells.

1. Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin-induced changes in [Ca2+]i by dynamic video imaging. 2. Bradykinin (10 pM-10 microM)-induced an increase in [Ca2+]i over basal levels (69 +/- 2 nM; n = 353) which was concentration-dependent (log EC50 = -8.7 M) in the presence of extracellular calcium ions (2 mM). The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]- bradykinin, produced a parallel shift to the right of the bradykinin concentration-response curve (log EC50 = -7.1 M and -5.8 M in the presence of 1 microM and 10 microM antagonist respectively) yielding an apparent KD of 26 nM. 3. In the absence of extracellular calcium ions (with 0.1 mM EGTA), bradykinin (10 pM-10 microM) produced a uniform increase in [Ca2+]i from a basal level of 33 +/- 2 nM (n = 140) to approximately 180 nM in BTSM cells indicating an 'all-or-nothing' release of intracellular calcium ions. In the presence of 10 microM D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin no responses could be induced by bradykinin at concentrations below 100 nM. However, at 100 nM and 1 microM bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed. 4. In both the absence or presence of D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin, there was a concentration-dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium-rich or calcium-free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quisqualate and carbachol-induced increases in intrasynaptosomal free calcium are mediated by different products of phospholipid hydrolysis

The mechanisms by which quisqualate and carbachol increase intrasynaptosomal free calcium ([Ca2+]i) were studied in rat cortical synaptosomes. Quisqualate (0.01-100 microM) and carbachol (100-1000 microM) increased [Ca2+]i in Fura-2 acetoxymethyl ester (Fura-2 AM)-loaded synaptosomes. The resting level of [Ca2+]i was 118 nM. The maximum increase (55%) was produced by 10 microM quisqualate which had an EC50 of 0.2 microM. The maximum increase (28%) elicited by carbachol occurred at 1000 microM and the EC50 was 30 microM. The stimulatory effects of quisqualate on [Ca2+]i were blocked by heparin (100 I.U.) but not by staurosporine (1 microM), nifedipine (1 microM) or omega-conotoxin fraction GVIA (omega-CgTx) (0.5 microM). On the other hand, the effects of carbachol on [Ca2+]i were abolished by staurosporine, nifedipine or omega-CgTx but not by heparin. Carbachol (100 microM) also significantly increased 45Ca accumulation into either resting or K+ (30 mM)-depolarised synaptosomes and these effects were inhibited by staurosporine and nifedipine. Quisqualate (10 microM) had no effect on 45Ca accumulation under resting or depolarised conditions. When quisqualate and carbachol were used in combination, there were apparently additive effects on [Ca2+]i but not on 45Ca accumulation. It is concluded that carbachol increases [Ca2+]i by facilitating Ca2+ entry through L-type Ca2+ channels via a 1,2-diacylglycerol (DAG)-protein kinase C (PKC)-dependent pathway while quisqualate mobilizes Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores.     

The induction of a biphasic bronchospasm by the ETB agonist, IRL 1620, due to thromboxane A2 generation and endothelin-1 release in guinea-pigs.

1. IRL 1620 (0.01-0.1 mg kg-1, i.v.), a selective endothelin B (ETB) receptor agonist, induced a dose-dependent biphasic increase in total lung resistance and a decrease in dynamic compliance in anaesthetized and artificially ventilated guinea-pigs. After intravenous injection of IRL 1620 (0.03 mg kg-1), the first phase was observed within 2 min whereas the second phase started between 5 and 10 min after injection and was long lasting. 2. In order to characterize which endothelin receptors are involved in both phases of bronchoconstriction, we studied the effect of ETA and ETB receptor antagonists (BQ 123 and BQ 788, respectively). BQ 788 (0.1-1 mg kg-1, i.v.) inhibited, in a dose-dependent manner, both phases of bronchoconstriction. BQ 123 (3 mg kg-1, i.v.) markedly inhibited (by 76%) the second phase of bronchoconstriction but had no effect on the early component of the response. 3. The effect of atropine, neurokinin-I (NK1) and neurokinin-2 (NK2) receptor antagonists (SR140333 and SR48968, respectively) were tested to investigate the possible involvement of cholinergic and sensory nerve activation, respectively, in the response to IRL 1620. Likewise, the role of arachidonic acid metabolites (leukotriene D4 antagonist, ONO-1078 and thromboxane A2 (TXA2) inhibitor, OKY-046) in this response was also investigated. OKY-046 (1 mg kg-1, i.v.) and atropine (1 mg kg-1, i.v.) partially inhibited the first phase (by 80% and 20%, respectively) without affecting the late phase of bronchoconstriction. Neither ONO-1078 (1 mg kg-1, i.v.) nor the combination of SR140333 (0.2 mg kg-1, i.v.) and SR 48968 (0.2 mg kg-1, i.v.) modified IRL 1620-induced bronchoconstriction. 4. A low dose of IRL 1620 (0.005 mg kg-1, i.v.) induced a monophasic bronchoconstriction. Pretreatment by phosphoramidon (100 mumol kg-1, i.v.) restored the second phase of bronchoconstriction. In this condition, BQ 123 (3 mg kg-1, i.v.) was able to inhibit partially the second phase of bronchoconstriction. 5. These results suggest that both phases of bronchoconstriction induced by IRL 1620 were mediated primarily by ETB receptor activation, the first phase being a consequence of TXA2 and acetylcholine release. The inhibition by an ETA receptor antagonist and the restoration by a neutral endopeptidase (NEP) inhibitor of the second phase of bronchoconstriction suggests that primary activation of ETB receptors leads to autocrine/paracrine endothelin-1 (ET-1) release that would subsequently cause profound bronchoconstriction through both ETA and ETB receptor activation.