β3肾上腺素能受体激动剂对离体人肺脏肺泡液体清除的作用

目的：制备离体人肺脏模型,探讨β3肾上腺素能受体激动剂CGP-12177对肺泡液体清除率（AFC）的作用及其机制。方法：以肺泡内Evans蓝标记的白蛋白浓度的变化测定AFC。首先将10^-4 mol/L到10^-7 mol/L的CGP-12177分别灌注到离体人肺脏肺泡腔内,测定AFC的变化。然后将10^-5 mol/L CGP-12177分别与α受体阻滞剂酚妥拉明、β1受体阻滞剂阿替洛尔、β2受体阻滞剂ICI-118551、β3受体阻滞剂SR59230A、钠通道阻断剂氨氯吡咪、Na^＋-K^＋-ATP酶阻断剂哇巴因混合后灌注到离体人肺脏肺泡腔内,测定AFC的变化。结果：10^-7 mol/L和10^-4 mol/L CGP-12177对离体人肺脏肺泡液体清除率没有影响,10^-6 mol/L和10^-5 mol/L的CGP-12177能够显著提高离体人肺脏肺泡液体清除率。CGP-12177增加离体人肺脏AFC的作用能够被SR59230A、氨氯吡咪和哇巴因抑制。结论：高选择性CGP-12177主要通过调节Na^＋-K^＋-ATP酶的活性提高离体人肺脏肺泡液体清除能力。     

Crosslinked hemoglobin inhibits endothelium-dependent relaxations in isolated canine arteries

• 1.1. Several previous in vivo studies demonstrated that crosslinked hemoglobin is a potent vasoconstrictor capable of significantly increasing arterial blood pressure following systemic administration. The precise mechanisms underlying the vascular effects of crosslinked hemoglobin are not clear. The present study was designed to determine the effect of crosslinked hemoglobin on the endothelial l-arginine-nitric oxide biosynthesis pathway in isolated canine arteries.
• 2.2. Isolated femoral and renal arteries were suspended in organ chambers for isometric tension recordings. Endothelium-dependent relaxations to acetylcholine and calcium ionophore A23187 were studied in the absence or in the presence of crosslinked hemoglobin or hemoglobin. A radioimmunoassay technique was used to determine levels of guanosine 3′,5′.cyclic monophosphate (cyclic GMP) and adenosine 3′,5′-cyclic monophosphate (cyclic AMP).
• 3.3. A nitric oxide synthase inhibitor l-NAME (10⁻⁴M) selectively inhibited endothelium-dependent relaxations to acetylcholine and calcium ionophore A23187. The inhibitory effect of l-NAME was reversed by l-arginine (3 × 10⁻⁴M). Crosslinked hemoglobin (10⁻⁷, 10⁻⁶ and 10⁻⁵M) inhibited endothelium-dependent relaxations to acetylcholine (10⁻⁹–10⁻⁵M) or A23187 (10⁻⁹–10⁻⁶M). In the same con. centration range, purified bovine hemoglobin exerted a similar inhibitory effect on relaxations mediated by activation of endothelial cells. Crosslinked hemoglobin (10⁻⁶M) significantly reduced basal production of cyclic GMP, but did not affect production of cyclic AMP. Acetylcholine (10⁻⁶M) stimulated production of cyclic GMP. This effect of acetylcholine was abolished in the presence of crosslinked hemoglobin.
• 4.4. These studies demonstrate that crosslinked hemoglobin impairs endothelium-dependent relaxations in isolated large conduit arteries. This effect appears to be mediated by the chemical antagonism of crosslinked hemoglobin against nitric oxide released from the endothelium. Inhibition of the endothelial l-arginine-nitric oxide biosynthesis pathway, with subsequent decrease of cyclic GMP in smooth muscle, may help to explain vasoconstrictor and pressor effects of crosslinked hemoglobin.

     

Sarcolemmal beta-adrenoceptors determined in rat ventricular heart biopsies with (-)-[3H]CGP-12177

Binding of the hydrophilic beta-adrenoceptor ligand (-)-[H]CGP-12177 was investigated by incubating biopsies from rat hearts (left ventricle/interventricular septum) to elucidate the applicability of this approach in determining the content of cell membrane beta-adrenoceptors in heart biopsies. Binding of (-)-[3H]CGP-12177 at 1 nM and 37 degrees reached maximum after 4-10 hr and declined after 10 hr. Binding of (-)-[3H]CGP-12177 at nM and 4 degrees reached equilibrium at 24 hr and was stable up to 96 hr. Total and specific binding was independent of biopsy size in the weight range of 4-35 mg. Competition binding studies with (+)- and (-)-isoprenaline showed that binding was stereospecific. Non-specific binding, determined in the presence of 5 microM (-)-timolol, was 6-8% of total binding at 0.1 nM (at Kd) and 15% of total binding at 1 nM (-)-[3H]CGP-12177. The coefficient of variation for total binding was 5.1%. Dissociation initiated at equilibrium showed complete reversibility of specific binding and was monoexponential with half-life of 0.6 hr at 37 degrees and 30.1 hr at 4 degrees. Binding-saturation experiments at 4 degrees showed beta-adrenoceptor density of 7 fmol/mg wet weight and equilibrium dissociation constant of 0.1 nM. Kd calculated from the rate constants of association and dissociation was 0.15 nM. Rapid freezing of tissue in liquid nitrogen with subsequent thawing and binding at 4 degrees C reduced receptor density by 21%. Density of beta-adrenoceptors did not differ in hearts from lean and obese insulin resistant Zucker rats. The results show that the method allows direct determination of sarcolemmal beta-adrenoceptors in small myocardial biopsies at 4 degrees with a minimum of preparation and equipment, using (-)-[3H]CGP-12177 . The method may be useful for other hydrophilic ligands.     

Functional muscarinic cholinoceptors in the isolated canine ureter

The purpose of present study was to characterize the functional muscarinic cholinoceptor (mAChR) subtypes in the isolated canine ureter. Carbachol (CCh), a non-selective mAChR agonist, concentration-dependently increased the frequency of the rhythmic contractions in isolated spiral ureteral preparations, the pD(2) value being 5.78+/-0.12. We then evaluated the effects of subtype-selective mAChR antagonists on the CCh-induced rhythmic contractions. The rank order of antagonistic potencies (apparent pA(2)) was 4-diphenylacetoxy- N-methylpiperidinemethiodide (4-DAMP; M3-subtype selective; 9.31+/-0.06) >atropine (non-selective; 9.16+/-0.10) >himbacine (M4-subtype selective; 7.32+/-0.18) >pirenzepine (M1-subtype selective; 6.78+/-0.16) >methoctramine (M2-subtype selective; 5.51+/-0.43). In sharp contrast, CCh concentration-dependently reduced the 80 mM KCl-induced contraction in longitudinal ureteral preparations, the pD(2) value being 4.83+/-0.10. On this CCh-induced ureteral relaxation, the rank order of antagonistic potencies (apparent pA(2)) was atropine (8.56+/-0.09) >4-DAMP (7.63+/-0.21) >himbacine (7.46+/-0.09) >methoctramine (6.54+/-0.18) >pirenzepine (6.33+/-0.22). The nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 1 x 10(-4) M) had no effect on the CCh-induced ureteral relaxation. These data suggest that the CCh-induced rhythmic contraction in the spiral preparation was mediated via the M3-receptor, while the CCh-induced relaxation in the longitudinal preparation was probably mediated mainly via the M4-receptor.     

Comparison of the binding affinity of CGP-12177A at recombinant rat alpha(1D)-adrenoceptors expressed in BHK-21 cell membranes and alpha(1)-adrenoceptors present in rat cerebral cortex membranes

Recent in vitro studies, performed in rat aorta, mesenteric and intrapulmonary arteries, and human pulmonary artery, demonstrated that the beta-adrenoceptor ligand CGP-12177A (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one) is also provided with antagonist or partial agonist properties at alpha(1)-adrenoceptors. These observations were supported by estimates of CGP-12177A binding affinity at alpha(1)-adrenoceptors, which have been always performed in rat cerebral cortex membranes, as a surrogate of vascular tissue. Since alpha(1D)-adrenoceptors are predominant in both rat aorta and mesenteric artery, in the present study, we measured, for the first time, the binding affinity of CGP-12177A at recombinant rat alpha(1D)-adrenoceptors expressed in BHK-21 cell membranes. CGP-12177A binding affinity was also determined in rat cerebral cortex membranes, where various alpha(1)-adrenoceptor subtypes are present. By means of [(3)H]prazosin binding competition experiments, we found that CGP-12177A bound to alpha(1D)-adrenoceptor-expressing BHK-21 cell membranes, with a binding affinity (pK(i)=5.39+/-0.27) almost identical to that measured in cerebral membranes (pK(i)=5.44+/-0.07), indicating that it is a non-subtype selective alpha(1)-adrenoceptor ligand. Moreover, CGP-12177A binding affinity was very close to its functional affinity evaluated in rat aorta in terms of antagonist potency against phenylephrine-induced contraction (pK(B)=5.65+/-0.07). In conclusion, our results demonstrate that, in order to evaluate CGP-12177A binding affinity at aorta and mesenteric artery alpha(1)-adrenoceptors, estimates in rat cerebral membranes are as reliable as those in recombinant rat alpha(1D)-adrenoceptors, since both values are very close to CGP-12177A functional affinities in isolated vessels.     

Comparison of the effects of nifedipine on ureter and coronary artery isolated from the dog

The effect of nifedipine (Adalat) on isolated dog ureter was compared with that on isolated coronary artery. Nifedipine at a concentration of 10(-8) mol/l significantly decreased the frequency of ureteral rhythmic contractions evoked by potassium, and suppressed the force of these contractions at a concentration of 3 X 10(-8) mol/l. In potassium-contracted coronary artery strips, nifedipine at concentrations more than 3 X 10(-9) mol/l produced significant relaxations in a concentration-dependent manner. The results indicate that nifedipine is able to act inhibitorily on ureteral contractions, and inhibition of pace making activities in ureteral smooth muscle was suggested as a mode of action of nifedipine.     

Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 μM), the neurokinin NK₁ receptor antagonist L-733060 (0.5 μM), the voltage-sensitive calcium channel blocker ruthenium red (100 μM), the TRPV1 receptor antagonist capsazepine (5 μM), the nitric oxide synthetase inhibitor N ^ω-nitro-l-arginine methyl ester HCl (l-NAME; 100 μM), the gap junction blocker 18α-glycyrrhetinic acid (10 μM), as well as the RhoA kinase inhibitor Y-27632 (1 μM). Further, we also used the K⁺ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 μM) + apamin (0.1 μM) and iberiotoxin (0.5 μM) + apamin (0.1 μM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) α-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK₁, NO, vanilloid receptors, voltage-sensitive calcium channels, K⁺ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.     

Comparison of the effects of norepinephrine and acetylcholine between intraarterial and extravascular administration to the isolated, blood-perfused canine atrium.

Effects of acetylcholine (ACh) and norepinephrine on atrial contractility and pacemaker activity were investigated in isolated and blood-perfused canine atrium preparations whith a support dog which were suspended in the blood-filled bath kept at 37 degrees C. The drug was given in two forms of administration, i.e., intraarterial injection into the cannulated sinus node artery or direct administration into the bath. ACh administered into the bath produced a significant decrease in the developed tension from a concentration of 10(-5) g/ml and norepinephrine produced a significant increase in the developed tension from 3 X 10(-6) g/ml. An injection via the sinus node artery resulted in 300 and 100 times greater response to ACh and norepinephrine, respectively, in the tension development. In atrial pacemaker activity, ACh given into the bath did not produce a dose-related decrease while norepinephrine produced a dose-related increase frequently accompanied by an irregularity of rhythmicity.     

Acetylcholine-induced endothelium-independent relaxations in monkey isolated superior and inferior caval veins.

1. We examined the effects of acetylcholine (ACh), isoprenaline (Isop) and Ca-ionophore, A23187 on monkey isolated superior (SCV) and inferior caval veins (ICV) with and without intact endothelium, which had been partially contracted by 2 x 10(-6)-5 x 10(-6) M prostaglandin F2 alpha (PGF2 alpha). 2. Low concentrations of ACh (10(-10)-10(-9) M) produced a dose-dependent relaxation in the precontracted venous segments with endothelium. ACh at concentrations more than 10(-7) M elicited a transient contraction followed by a relaxation in these segments. 3. An addition of 5 x 10(-7) M A 23187 induced about 60% of maximum relaxation produced by 10(-5) M sodium nitroprusside (SNP) in each venous segment with endothelium. 4. Isop (10(-10)-10(-5) M) caused a dose-related relaxation in the precontracted caval veins with intact endothelium. 5. Removal of endothelium caused no significant effect on the ACh-induced dual responses but a significant inhibition of the A23187-induced relaxation. 6. Pretreatment with atropine antagonized competitively the ACh-induced relaxations in the endothelium-intact and endothelium-denuded caval veins. The Schild plot analysis showed that the pA2 values of the segments with and without endothelium were 9.72 +/- 0.14 (n = 5) and 10.01 +/- 0.23 (n = 6) in the ICV; and 9.95 +/- 0.20 (n = 5) and 9.70 +/- 0.10 (n = 5) in the SCV, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

NG-monomethyl-L-arginine and NG-nitro-L-arginine inhibit endothelium-dependent relaxations in human isolated omental arteries.

The L-arginine analogues NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) and NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), which specifically inhibit the synthesis of nitric oxide from L-arginine, significantly reduced acetylcholine-induced endothelium-dependent relaxations in rings of human omental arteries. The inhibitory potency of L-NMMA and L-NAME was similar. Addition of L-NMMA or L-NAME to the organ bath did not induce any significant changes in the resting tension of the tissues. The effects of L-NMMA were reversed by L-arginine (3 x 10(-4) M). The L-NMMA enantiomer, D-NMMA (10(-4) M), did not influence either the basal tone of the preparation or the relaxing effects of acetylcholine. Arterial relaxations induced by sodium nitroprusside (10(-6) M) were not influenced by incubation with L-NMMA or L-NAME. These results suggest that endothelium-dependent relaxations in human omental arteries are mediated by the endogenous and substrate-specific generation of nitric oxide from L-arginine.     

Heterogeneity of neuronal and smooth muscle receptors involved in the VIP- and PACAP-induced relaxations of the pig intravesical ureter

1. The mechanisms and receptors involved in the vasoactive intestinal peptide (VIP)- and pituitary adenylate cyclase-activating polypeptide (PACAP)-induced relaxations of the pig intravesical ureter were investigated. 2. VIP, PACAP 38 and PACAP 27 concentration-dependently relaxed U46619-contracted ureteral strips with a similar potency. [Ala(11,22,28)]-VIP, a VPAC(1) agonist, showed inconsistent relaxations. 3. The neuronal voltage-gated Ca(2+) channel inhibitor, omega-conotoxin GVIA (omega-CgTX, 1 microm), reduced the VIP relaxations. Urothelium removal or blockade of capsaicin-sensitive primary afferents, nitric oxide (NO) synthase and guanylate cyclase with capsaicin (10 microm), N(G)-nitro-l-arginine (l-NOARG, 100 microm) and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microm), respectively, did not change the VIP relaxations. However, the PACAP 38 relaxations were reduced by omega-CgTX, capsaicin, l-NOARG and ODQ. 4. The VIP and VIP/PACAP receptor antagonists, [Lys(1), Pro(2,5), Arg(3,4), Tyr(6)]-VIP (1 microm) and PACAP (6-38) (0.4 microm), inhibited VIP and VIP and PACAP 38, respectively, relaxations. 5. The nonselective and large-conductance Ca(2)-activated K(+) channel blockers, tetraethylammonium (3 mm) and charybdotoxin (0.1 microm), respectively, and neuropeptide Y (0.1 microm) did not modify the VIP relaxations. The small-conductance Ca(2)-activated K(+) channel blocker apamin (1 microm) did not change the PACAP 27 relaxations. 6. The cAMP-dependent protein kinase A (PKA) blocker, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate (Rp-8-CPT-cAMPS, 100 microm), reduced VIP relaxations. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin relaxed ureteral preparations. The rolipram relaxations were reduced by Rp-8-CPT-cAMPS. Forskolin (30 nm) evoked a potentiation of VIP relaxations. 7. These results suggest that VIP and PACAP relax the pig ureter through smooth muscle receptors, probably of the VPAC(2) subtype, linked to a cAMP-PKA pathway. Neuronal VPAC receptors localized at motor nerves and PAC(1) receptors placed at sensory nerves and coupled to NO release, seem also to be involved in the VIP and PACAP 38 relaxations.     

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.

     

The role of nitric oxide in serotonin-induced relaxations in the canine terminal ileum and ileocolonic junction

Summary The role of nitric oxide (NO) in 5-HT-induced non-adrenergic non-cholinergic (NANC) relaxations was studied on circular muscle strips of the canine ileocolonic junction (ICJ) and terminal ileum. During an acetylcholine-induced contraction, NO (10^–5 M) evoked a transient relaxation, whereas 5-HT (10^–4 M) caused an initial NANC relaxation followed by a contraction. This initial relaxation to 5-HT, but not the relaxation to NO, was significantly inhibited by the stereospecific inhibitors of the NO biosynthesis N^G-monomethyl-Larginine (L-NMMA) and N^G-nitro-Irarginine (L-NNA). Larginine, but not D-arginine, prevented the inhibitory effect of L-NMMA and LNNA. The enantiomer of L-NMMA, D-NMMA, had no effect. Hemoglobin abolished the NO-induced relaxations and significantly inhibited the relaxation to 5-HT.From these experiments it is concluded that the 5-HT-induced NANC relaxation is mediated by NO or a NO releasing substance.Research Assistant of the National Fund for Scientific Research Belgium (NFWO) Send offprint requests to Y.M. Van Maercke at the above address     

Pharmacology of pentoxifylline in isolated canine arteries and veins

Pentoxifylline possesses vasodilator properties, but little information is available on the mechanism of action explaining this vasodilator effect. The present experiments were designed to determine the effects of the compound on vascular smooth muscle, endothelium, and adrenergic nerves in rings of isolated canine blood vessels. Pentoxifylline did not affect basal tension in coronary and femoral arteries or in saphenous and mesenteric veins; it did not alter the rhythmic activity of the latter, but did cause endothelium-independent relaxations of unstimulated basilar arteries. In coronary arteries and saphenous veins, but not femoral arteries contracted with prostaglandin F2 alpha, the compound caused relaxations which were not affected by propranolol or by removal of the endothelium. Pentoxifylline inhibited the endothelium-dependent contractions to the Ca2(+)-ionophore A23187 in the basilar artery. In saphenous veins (with endothelium), pentoxifylline did not inhibit responses to high K+, electrical stimulation of the adrenergic nerves, or exogenous norepinephrine (NE); it reduced contractions evoked by xylazine and hypoxia. In the basilar artery and the saphenous vein, the inhibitory effect of pentoxifylline was prevented by inhibitors of cyclooxygenase and thromboxane synthetase. These experiments suggest that the dilator properties of pentoxifylline in isolated canine blood vessels are primarily at the level of the vascular smooth muscle and may involve decreased production of, or reduced responsiveness to, endogenous thromboxanes.     

Flunarizine and alpha-adrenergic responses in isolated canine saphenous veins

Experiments were designed to determine how flunarizine affects contractions of cutaneous veins to alpha-adrenergic activation. Rings of canine saphenous vein were mounted at optimal length for isometric tension recording in organ chambers filled with physiological salt solution. At concentrations higher than those needed to inhibit KCl-induced contractions, flunarizine inhibited the contractile responses evoked by alpha 2-adrenergic agonists (B-HT 920, xylazine), partial (St-587) and full (cirazoline, phenylephrine) alpha 1-adrenergic agonists and the combined alpha 1-/alpha 2-adrenergic agonist, norepinephrine. The inhibitory effect of flunarizine against alpha 2-adrenergic responses was similar to that produced by other calcium-antagonists and results presumably from inhibition of the influx of extracellular calcium. The inhibitory effect of flunarizine against alpha 1-adrenergic responses was greater than expected and appears to result from competitive antagonism of alpha 1-adrenoceptors (pA2 = 5.79). Therefore, flunarizine can decrease adrenergic contractile responses by depressing the influx of extracellular calcium and by blocking postjunctional alpha 1-adrenoceptors.     

Alpha2-adrenoceptor antagonists evoke endothelium-dependent and -independent relaxations in the isolated rat aorta.

This study was designed to determine whether the alpha2-adrenoceptor antagonists idazoxan, yohimbine, and rauwolscine cause endothelium-dependent and -independent responses in the rat aorta. Rings of rat aorta, with and without endothelium, were suspended for the measurement of isometric force in modified Krebs-Ringer bicarbonate solution (37 degrees C; aerated with 95% O2 and 5% CO2). The alpha2-adrenoceptor antagonists, in the concentration range of 10(-8)-10(-6) M, relaxed phenylephrine-contracted rings with, but not those without endothelium. alpha2-Adrenoceptor antagonists (3 x 10(-6) M for 1 min) increased the accumulation of cyclic guanosine monophosphate (cGMP) about twofold in the aortas with endothelium. The relaxation and the increased cGMP induced by alpha2-antagonists were attenuated by methylene blue (10(-6) M) and N(G)-nitro-L-arginine (L-NA, 3 x 10(-5) M), whereas propranolol (10(-6) M) did not affect the relaxation. In concentrations >10(-6) M, alpha2-adrenoceptor antagonists relaxed the rat aorta without endothelium. The endothelium-independent relaxation by alpha2-adrenoceptor antagonists was abolished by increased external K+ and reduced significantly by tetraethylammonium (TEA, 10(-2) M, a Ca2+-dependent K+ channel blocker), but not inhibited by glibenclamide (10(-5) M, an ATP-sensitive K+ channel blocker). In the rabbit aorta, only endothelium-independent relaxations were observed with alpha2-adrenoceptor antagonists in the concentration range of 10(-8)-10(-5) M, and these relaxations were not antagonized by TEA. These results suggest that alpha2-adrenoceptor antagonists relax the rat aorta through endothelium-dependent mechanism at lower concentrations and endothelium-independent mechanisms at higher concentrations. The endothelium-dependent relaxations are likely to be mediated by the endothelium-derived relaxing factor (EDRF)/NO pathway because they are associated with the accumulation of cGMP, whereas the endothelium-independent relaxations may be caused by the opening of potassium channels in the vascular smooth muscle.     

Mechanism underlying relaxations caused by prostaglandins and thromboxane A2 analog in isolated dog arteries

In helical strips of dog cerebral, coronary, mesenteric, and renal arteries treated with ONO3708, an inhibitor of vasoconstricting prostaglandin (PG) receptors, and previously contracted with serotonin, PGF2 alpha, PGD2 and epithio-methano thromboxane A2 (sTxA2), a TxA2 analog, caused a relaxation. The cerebral arterial relaxation was suppressed by treatment with indomethacin and abolished by diphloretin phosphate (DPP), a PG antagonist. On the other hand, the relaxation of mesenteric arteries was not influenced by indomethacin but was markedly attenuated by DPP. Removal of endothelium did not alter the relaxation. Relaxations of coronary and renal arteries by PGF2 alpha were suppressed by indomethacin and DPP, whereas the PGD2-induced relaxation was not affected by indomethacin but was abolished by DPP. Concentration--relaxation curves for PGI2 were shifted to the right by treatment with DPP. It is concluded that after ablation of the constrictor response, dog cerebral arteries relax in response to PGs and TxA2, probably due mainly to the release of PGI2-like substance from the arterial wall and to the action of PGI2 receptive sites, whereas the mesenteric arterial relaxation appears to be associated with their action on PGI2 receptors in smooth muscle cells. PGF2 alpha-induced relaxations in coronary and renal arteries may result from the release of PGI2, and relaxations by PGD2 from the action on PGI2 receptors.