Characterization and assessment of a novel poly(ethylene oxide)/polyurethane composite hydrogel (Aquavene) as a ureteral stent biomaterial

Although endothelium-derived hyperpolarizing factor (EDHF) activity has been demonstrated in arteries from various species, EDHF has not been chemically identified, nor its mechanism of action characterized. To elucidate this mechanism, we tested the effect of EDHF on large-conductance Ca2+-activated K+ (K(Ca)) channels in porcine coronary artery smooth muscle cells. By using a patch-clamp technique, single-channel currents were recorded in cultured smooth muscle cells; the organ bath also contained a strip of porcine coronary with endothelium, which served as the source of endothelium-derived relaxing factor(s) including EDHF. Exposure of endothelium to 10(-6) M bradykinin activated K(Ca) channels in cultured smooth muscle cells in cell-attached patches. When the experiment was performed in the presence of 10 microM indomethacin and 30 microM N(G)-nitro-L-arginine (L-NNA), which block the generation of prostaglandin I2 (PGI2) and NO, respectively, K(Ca) channel activity was stimulated by bradykinin, indicating the direct involvement of EDHF in K(Ca) channel stimulation. Neither 10 microM methylene blue nor 25 microM Rp-cAMPS inhibited bradykinin-induced K(Ca) channel activity. In inside-out patches, the addition of bradykinin to the solution was without effect on K(Ca) channel activation. However, in the presence of 0.5 mM guanosine triphosphate (GTP) and 1.0 mM adenosine triphosphate (ATP) in the bath solution, K(Ca) channels was activated by bradykinin. In outside-out patches, the addition of bradykinin also increased K(Ca) channel activity, when GTP and ATP were added to the pipette solution. The addition of GDP-beta-S (100 microM) in the cytosolic solution completely blocked the activation K(Ca) channels induced by bradykinin in inside-out and outside-out patches. Pretreatment with 30 microM quinacrine, a phospholipase A2 inhibitor, or 3 microM 17-octadecynoic acid (17-ODYA), a cytochrome P450 inhibitor, in addition to indomethacin and L-NNA, abolished bradykinin-stimulated K(Ca) channel activity in cell-attached patches. Both 14,15-epoxyeicosatrienoic acid (EET) and 11,12-EET increased the open probabilities of K(Ca) channels in cell-attached patches. These results suggest that EDHF, released from endothelial cells in response to bradykinin, hyperpolarizes smooth muscle cells by opening K(Ca) channels. Furthermore, our data suggest that EDHF is an endothelium-derived cytochrome P450 metabolite of arachidonic acid. The effect of EDHF on K(Ca) channels is not associated with an increase of cAMP and cGMP. The activation of K(Ca) channels appears to be due to the activation of GTP-binding protein.     

The structure of HLA-DM, the peptide exchange catalyst that loads antigen onto class II MHC molecules during antigen presentation

Endothelial cells produce C-type natriuretic peptide (CNP), which has been proposed as an endothelium-derived hyperpolarizing factor. In porcine coronary arteries, we investigated the vasodilatory effects of CNP and compared them with endothelium-dependent relaxations and hyperpolarizations to bradykinin. Isolated epicardial porcine coronary arteries were studied in organ chambers, and concentration-response curves to CNP and bradykinin were obtained. Membrane potential was measured in endothelial cells and smooth muscle of intact porcine coronary arteries during stimulation with CNP or bradykinin. In precontracted porcine coronary arteries with or without endothelium, CNP (10[-10]-10[-6] M) evoked relaxations (maximum, 42 +/- 4%) smaller than those evoked by bradykinin (100 +/- 1%), blunted in preparations contracted by KCl instead of U46619 (9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2alpha; p < 0.05) and unaffected by inhibition of NO synthase (NS). CNP evoked hyperpolarization of vascular smooth muscle of similar magnitude in endothelium-intact (-4.4 +/- 1 mV) and endothelium-denuded (-4.6 +/- 1 mV) porcine coronary arteries. Bradykinin (10[-10]-10[-6] M) evoked concentration-dependent relaxations in preparations with endothelium only. Although atrial natriuretic peptide-receptor antagonist HS-142-1 (25 microM) slightly reduced the sensitivity to bradykinin (log shift at IC50, twofold; p < 0.05), it had no effect on the maximal response to bradykinin. Inhibition of NO synthase partially attenuated, whereas high potassium chloride (30 mM) markedly inhibited relaxations to bradykinin (p < 0.05). Hyperpolarization to bradykinin was much more pronounced than that to CNP (-17 +/- 3 mV; p < 0.05 vs. CNP) and was observed in endothelium-intact preparations only and unaffected by HS-142-1. In conclusion, in contrast to bradykinin, CNP induces endothelium-independent and weaker relaxation and hyperpolarization of coronary artery vascular smooth muscle, suggesting that CNP is an unlikely mediator of endothelium-dependent hyperpolarization of porcine coronary arteries.     

Intracellular divalent cations block smooth muscle K+ channels

The patch-clamp technique was used to examine the sensitivity of delayed rectifier K+ channels to changes in intracellular divalent cations (Mg2+ and Ca2+). During voltage-step and ramp depolarizations, a delayed rectifier K+ current (IK(dr)) was identified in renal, pulmonary, coronary, and colonic smooth muscle cells as a low-noise outward current that activated near -40 mV, was sensitive to 4-aminopyridine (4-AP), and was insensitive to charybdotoxin. During whole-cell voltage-clamp experiments in each of the cell types, the 4-AP-sensitive IK(dr) was significantly less in cells dialyzed with 10 mM Mg2+ as compared with cells in which no Mg2+ was added to the internal dialysis solution (P < or = .05, n > or = 4). In coronary artery cells, 100 microM 2-(2-aminoethyl)pyridine (an H1 receptor agonist) or 10 microM ryanodine, agents that cause an increase in [Ca2+]i, also caused a significant reduction of the 4-AP-sensitive IK(dr) similar to that produced by Mg2+. 4-AP (5 mM) significantly depolarized single renal arterial cells that were dialyzed with Mg(2+)-free solution but not those dialyzed with 10 mM Mg2+ (P < .01, n = 4). In inside-out patches of renal arterial smooth muscle cells, with 200 nM charybdotoxin in the patch pipette to block large conductance Ca(2+)-activated K+ channels, a 59 +/- 10-picosiemen K+ channel that was sensitive to cytoplasmic Mg2+ was identified. In Mg(2+)-free solution, channel open probability was 0.028 +/- 0.012 (n = 8) and 0.095 +/- 0.011 (n = 8) at +40 and +80 mV, respectively. When the bath solution was changed to one containing 5 or 15 mM Mg2+, channel open probability was significantly reduced by 66% and 68% (+40 mV) or 93% and 96% (+80 mV), respectively. This decrease in the open probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. At +40 mV, time constants for the open time distribution were significantly decreased from 5.5 +/- 0.52 to 1.2 +/- 0.14 milliseconds, whereas the closed time constant was significantly increased from 634 +/- 11.1 to 820 +/- 14.4 milliseconds (P < .01, n = 4). It is concluded that a 4-AP-sensitive delayed rectifier K+ channel in both vascular and visceral smooth muscle cells is modulated by changes in intracellular Ca2+ and Mg2+ that may alter membrane potential and the contractile state of smooth muscle.     

Peptides that mimic the group B streptococcal type III capsular polysaccharide antigen

In some but not all arterial beds, smooth muscle cell calcium-activated K+ channels (KCa channels) play a central role in the mediation of the vasodilator response to nitric oxide (NO) and other nitrates. We investigated the effect of nitrates on KCa channels in the relaxation of human coronary arteries by means of isometric contraction experiments in arterial rings. We also measured whole-cell currents in freshly isolated human coronary artery vascular smooth muscle cells via the patch-clamp technique. Sodium nitroprusside, diethylamine-nitric oxide complex sodium salt and isosorbide mononitratre completely relaxed rings preconstricted with 5 microM serotonin and produced dose-dependent relaxations of 5 microM serotonin-preconstricted human rings. The relaxations were inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-oxyl 3-oxide (10 microM), which neutralizes nitric oxide. The KCa channel blockers iberiotoxin (100 nM) and tetraethylammonium ions (1 mM) significantly inhibited SNP-induced relaxations of human coronary arteries. Moreover, in the patch-clamp experiments, SNP (1 microM) stimulated KCa currents and spontaneous transient outward K+ currents carried by Ca spark activated KCa channels. The SNP-induced (1 microM) KCa current was strongly inhibited by iberiotoxin (100 nM). These data show that activation of KCa channels in smooth muscle cells contributes to the vasodilating actions of nitrates and nitric oxide in human coronary arteries. This finding may have unique clinical significance for the development of antianginal and antihypertensive drugs that selectively target K+ channels and Ca sparks.     

MSX-1 gene expression and regulation in embryonic palatal tissue

1. During cardiac surgery, the heart is arrested and protected by hyperkalaemic cardioplegia. The coronary endothelium may be damaged by ischaemia-reperfusion and cardioplegia. Subsequently, this may affect cardiac function immediately after cardiac surgery and cause mortality and morbidity. 2. We investigated coronary endothelium-smooth muscle interaction after exposure to depolarizing (hyperkalaemic; K+ 20 or 50 mmol/L) and hyperpolarizing (the K+ channel opener aprikalim) cardioplegia and organ preservation solution (University of Wisconsin (UW) solution). Endothelium-dependent relaxation and hyperpolarization of the coronary smooth muscle were studied in the porcine and human large conductance and micro-coronary arteries. Intracellular free calcium concentration in endothelial cells was also measured. 3. The endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation to A23187, bradykinin, and substance P in arteries contracted by either U46619 (10 nmol/L) or K+ (25 mmol/L) was reduced after exposure to either high K+ or UW solution, but was maximally preserved after exposure to aprikalim. The hyperpolarization of the membrane potential in response to the above endothelium-derived relaxing factor stimuli was also reduced by exposure to depolarizing cardioplegia. Studies in microcoronary arteries are in accordance with findings in large arteries. The intracellular free calcium concentration remained unchanged after exposure to hyperkalaemia. 4. We concluded that: (i) during cardiac surgery, the function of coronary circulation may be changed due to exposure to depolarizing cardioplegia or preservation solutions; (ii) the functional change in the coronary circulation is related to the altered interaction between the endothelium and smooth muscle; (iii) depolarizing (hyperkalaemia) cardioplegia or hyperkalaemic organ preservation solutions affect endothelium-smooth muscle interaction through the EDHF pathway; (iv) EDHF relaxes the porcine large and microcoronary arteries through multiple K+ channels; and (v) that hyperpolarizing vasodilators (K+ channel openers) may protect EDHF-mediated endothelial function when used as cardioplegia.     

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.     

Perfusion-induced changes in cardiac contractility and oxygen consumption are not endothelium-dependent

OBJECTIVE: Are substances released from rat coronary endothelial cells responsible for the increase in contractility and oxygen consumption (Gregg phenomenon) seen with an increase in cardiac perfusion? METHODS: In an isovolumically contracting, Langendorff, crystalloid perfused rat heart (n = 6) at 27 degrees C, coronary flow was changed (from 4.4 to 15.4 ml.min-1.gww(-1)) before and after the endothelium was made dysfunctional by Triton X-100. Vascular endothelium and smooth muscle function were tested with bradykinin (BK, 1 microM, an endothelium-dependent dilator) and papaverine (PAP, 1 microM, an endothelium-independent dilator) in a preconstricted vascular bed (vasopressin, VP, 3 nM). RESULTS: Before Triton X-100, coronary resistance (at constant flow) decreased significantly in response to BK and to PAP. After Triton X-100 treatment the dilatory response to BK was abolished while the PAP response was still present, suggesting endothelial dysfunction with intact smooth muscle function. Due to Triton X-100 treatment, coronary resistance increased significantly. Therefore coronary flow changes were also applied during a similar increase in coronary resistance induced by VP infusion (3 nM) before Triton X-100 treatment. During control, developed left ventricular pressure (dev Plv) increased with 68 +/- 21% and oxygen consumption (VO2) increased with 122 +/- 25% in response to the maximal increase in coronary flow. During increased coronary resistance with and without functional endothelium, dev Plv increased by 57 +/- 16 and 64 +/- 22%, respectively, and VO2 increased by 126 +/- 21 and 103 +/- 20%, respectively, in response to the maximal increase in flow. These changes were not significantly different from control. CONCLUSION: The results suggest that the arterial endothelium is not involved in the Gregg phenomenon.     

Effects of timolol and betaxolol on choroidal blood flow in the rabbit

Endothelium-dependent hyperpolarization of vascular smooth muscle cells (VSMCs) plays a crucial role in regulating vascular tone, especially in resistance vessels. It has been proposed that metabolites of arachidonic acid (AA), formed by cytochrome P-450 monooxygenase (P450), are endothelium-derived hyperpolarizing factors (EDHFs). These metabolites have been reported to mediate dilation to endogenous vasoactive compounds, such as bradykinin and acetylcholine. However, it is not known whether these metabolites of AA contribute to dilation of human resistance vessels. This is important since it has been proposed that EDHF serves as a compensatory mechanism to maintain dilation in disease states. Therefore, we studied the effect of AA on vessel diameter and VSMC membrane potential in isolated human coronary microvessels. Arterioles (81+/-5 microm, n=70) were dissected from right atrial appendages at the time of cardiac surgery and cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in internal diameter were recorded with videomicroscopy. Some vessels were impaled with glass microelectrodes to measure membrane potential of VSMCs while internal diameters were simultaneously recorded. After constriction (47+/-2%) with endothelin-1, AA (10(-10)to 10(-5)mol/L) induced substantial dilation of human coronary microvessels, which was abolished by removal of the endothelium. Treatment with 17-octadecynoic acid (17-ODYA, 10(-5) mol/L; a P450 inhibitor) attenuated maximal dilation to AA (49+/-9% versus 91+/-4% [control]; P<0.05 versus control), whereas indomethacin (INDO, 10(-5) mol/L; a cyclooxygenase inhibitor) and N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L; a NO synthase inhibitor) were without effect. Both 17-ODYA and miconazole (10(-5) mol/L, a chemically distinct P450 inhibitor) further reduced the dilation to AA in the presence of INDO. The presence of 40 mmol/L KCl or charybdotoxin (10(-8) mol/L, a blocker of large-conductance Ca2+-activated K+ channels) impaired dilation to AA (19+/-9% [KCI] versus 76+/-5% [control] and 47+/-6% [charybdotoxin] versus 91+/-3% [control]; P<0.05 for both). After depolarization with endothelin-1 (-26+/-1 mV from -48+/-3 mV [before endothelin]), AA (10(-5)mol/L) in the presence of INDO and L-NAME induced hyperpolarization of VSMCs (-57+/-5 mV). In the presence of 17-ODYA together with INDO and L-NAME, endothelin produced similar depolarization (-26+/-2 mV from - 48+/- 3 mV), but hyperpolarization to AA was reduced (-33+/-2 mV; P<0.05 versus absence of 17-ODYA). AA metabolites formed primarily by P450 produce potent endothelium-dependent dilation of human coronary arterioles via opening of Ca2+-activated K+ channels and hyperpolarization of VSMCs. These findings support an important role for P450 metabolites in the regulation of human coronary arteriolar tone.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Different populations of progesterone receptor-steroid complexes in binding to specific DNA sequences: effects of salts on kinetics and specificity

The endothelium plays an obligatory role in a number of relaxations of isolated arteries. These endothelium-dependent relaxations are due to the release by the endothelial cells of potent vasodilator substances [endothelium-derived relaxing factors (EDRF)]. The best characterized EDRF is nitric oxide (NO). Nitric oxide is formed by the metabolism of L-arginine by the constitutive NO synthase of endothelial cells. In arterial smooth muscle, the relaxations evoked by EDRF are explained best by the stimulation by NO of soluble guanylate cyclase that leads to the accumulation of cyclic GMP. The endothelial cells also release an unidentified substance that causes hyperpolarization of the cell membrane (endothelium-derived hyperpolarizing factor, EDHF). The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive (alpha2-adrenergic activation, serotonin, thrombin, aggregating platelets) and insensitive (adenosine diphosphate, bradykinin) G-proteins. In blood vessels from animals with regenerated endothelium, and/or atherosclerosis, there is a selective loss of the pertussis-toxin sensitive mechanism of EDRF-release which favors the occurrence of vasospasm, thrombosis and cellular growth.     

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 NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) by either carbachol (pEC50 [negative logarithm of the EC50] = 6.19 +/- 0.01, Rmax [maximum response] = 93.2 +/- 0.4%; n = 14) or calcium ionophore A23187 (pEC50 = 6.46 +/- 0.02, Rmax = 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, Rmax = 94.7 +/- 4.6%; n = 10; with L-NAME: pEC50 = 6.33 +/- 0.04, Rmax = 93.4 +/- 6.0%; n = 4). 2. The selective cannabinoid receptor antagonist, SR 141716A (1 microM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and 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 microM) greatly reduced the Rmax values for EDHF-mediated relaxations to carbachol (control, 93.2 +/- 0.4%; SR 141716A, 10.7 +/- 2.5%; n = 5; P < 0.001) and 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 Rmax. 3. Precontraction with 60 mM KCl significantly reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 68.8 +/- 5.6% versus 17.8 +/- 7.1%), 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 microM carbachol (control 63.5 +/- 7.5% versus 9.0 +/- 3.2%), 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 microM) significantly potentiated (P < 0.01), to a similar extent (approximately 2 fold), relaxation to 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 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 Rmax for anandamide (control, 94.3 +/- 4.0%; 10 mM TEA, 60.7 +/- 4.4%; n = 5) but had no effect on the Rmax to carbachol or A23187. 6. BaCl2 (100 microM), considered to be selective for blockade of inward rectifier K+ channels, had no significant effect on relaxations to carbachol or 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; A23187, 5.84 +/- 0.04; anandamide, 5.95 +/- 0.02; n = 4 for each). 7. Apamin (1mu M), a selective blocker of small conductance, Ca2+-activated, K+ channels (SKCa), 4-aminopyridine (1mM), a blocker of delayed rectifier, voltage-dependent, K+ channels (Kv), and ciclazindol (10mu M), an inhibitor of Kv and adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on A23187 or anandamide responses. 8. Glibenclamide (10mu M), a KATP inhibitor and charybdotoxin (100 or 300nM), a blocker of several K+ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50nM), an inhibitor of large conductance, Ca2+-activated, K+ channels (BKCa), had no significant effect on the relaxation responses, either alone or in combination with apamin (1muM). Also, a combination of apamin (1muM) with either glibenclamide (10muM) or 4-aminopyridine (1mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to A23187 or anandamide. 9. A combination of apamin (1muM) with charybdotoxin (100nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to A23187. Apamin (1muM) and charybdotoxin (300nM) together consistently inhibited the response to A23187, while apamin (1muM) and ciclazindol (10muM) together inhibited relaxations to both carbachol and 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 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.     

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival

The relationship between Ca2+ release ("Ca2+ sparks") through ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and KCa channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (-40 mV) and intracellular Ca2+ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x-y) scanning confocal microscope and the fluorescent Ca2+ indicator, fluo-3. Virtually all (96%) detectable Ca2+ sparks were associated with the activation of a spontaneous transient outward current (STOC) through KCa channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at -40 mV). Approximately 41% of STOCs occurred without a detectable Ca2+ spark. The amplitudes of the Ca2+ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca2+ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca2+ sparks, was 33 pA at -40 mV. The mean amplitude of the "sparkless" STOCs was smaller, 16 pA. The very significant increase in KCa channel open probability (>10(4)-fold) during a Ca2+ spark is consistent with local Ca2+ during a spark being in the order of 1-100 microM. Therefore, the increase in fractional fluorescence (F/Fo) measured during a Ca2+ spark (mean 2.04 F/Fo or approximately 310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates KCa channels. These results indicate that the majority of ryanodine receptors that cause Ca2+ sparks are functionally coupled to KCa channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs.     

Mechanisms of relaxation of coronary artery by hypoxia

This study was designed to clarify the dependency of hypoxic coronary vasodilation (HCD) on the endothelium and the role of the K+ channels on HCD in the rabbit coronary artery. HCD was investigated in an isolated left circumflex coronary artery precontracted with prostaglandin F2 alpha. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit (KH) solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and was maintained for 15 approximately 25 min. Hypoxia elicited a vasodilation in the precontracted coronary artery with and without endothelium. There was no difference between the amplitude of the HCD induced by two consecutive hypoxic challenges and the effects of 20% O2 + 5% CO2 + 75% N2 and 95% O2 + 5% CO2 control K-H solution of subsequent responses to hypoxia. Inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HCD. Blockades of the tetraethylammonium chloride-sensitive K+ channel abolished HCD. Apamin, a blocker of the small conductance Ca(2+)-activated K+ (KCa) channel, and iberiotoxin, a blocker of the large conductance KCa channel had no effect on HCD, respectively. Glibenclamide, a blocker of the ATP-sensitive K+ (K+ATP) channel, reduced HCD. Cromakalim, an opener of the K+ATP channel, relaxed the coronary artery precontracted with prostaglandin F2 alpha. The degree of relaxation by cromakalim was similar to that by hypoxia while glibenclamide reduced both hypoxia- and cromakalim-induced vasodilatations. In conclusion, these results suggest that HCD is independent on endothelium and HCD is considered to be induced by activation of K+ATP channel.     

Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells

1. We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of Ca2+ release from endothelial intracellular stores to nitric oxide (NO) production evoked by increases in shear stress and by acetylcholine in rabbit aorta. 2. Experiments were performed before and following incubation with either the endoplasmic reticulum Ca(2+)-ATPase inhibitors cyclopiazonic acid (CPA, 10 microM) and thapsigargin (TSG, 1 microM) or ryanodine (30, 100 microM) which binds to a specific endoplasmic reticulum Ca(2+)-release channel. 3. In cascade bioassay all three agents induced relaxations of the recipient ring (CPA, 24.4 +/- 3.8%; TSG, 51.5 +/- 10.6%; ryanodine, 17.4 +/- 1.6%) which were significantly attenuated by preincubation of the donor with 100 microM NG-nitro-L-arginine methyl ester (L-NAME). However, in isolated rings, only CPA and TSG induced L-NAME-sensitive relaxations (CPA 52.7 +/- 6.5%; TSG 61.3 +/- 7%). 4. Addition of superoxide dismutase (SOD) to the donor perfusate evoked relaxations of the recipient ring in cascade bioassay (13.3 +/- 1.4%, n = 22). Prior administration of SOD attenuated relaxations to TSG (23.2 +/- 3.8% n = 4) and ryanodine (1.7 +/- 0.8%, n = 4), and pre-incubation with TSG and ryanodine blunted SOD-induced responses (4 +/- 1.5%, n = 4 and 8.9 +/- 1.1%, n = 4, respectively). By contrast, no interaction was observed between the relaxations evoked by SOD and CPA. In isolated rings, SOD exerted no direct relaxant and did not modulate relaxations to CPA, TSG or ryanodine. 5. In cascade bioassay studies time-averaged shear stress was manipulated with dextran (1-4% w/v, 800000 MW) to increase perfusate viscosity. NO-dependent relaxation of the recipient ring induced by increased perfusate viscosity was significantly attenuated by CPA (P < 0.01; n = 6) and TSG (P < 0.05; n = 7), but not by ryanodine (n = 6). 6. Endothelium-dependent relaxations to acetylcholine (0.1-30 microM) in cascade bioassay and in isolated aortic ring preparations were markedly attenuated by pretreatment with CPA and TSG, but were unaffected by ryanodine. Ryanodine and CPA caused only a small attenuation of endothelium-independent relaxations to sodium nitroprusside (0.001-10 microM), whereas TSG had no effect. 7. We conclude that release of Ca2+ from CPA- and TSG-sensitive endothelial stores is necessary for NO release evoked by acute flow changes and agonists in rabbit abdominal aorta. Ca(2+)-induced Ca2+ release via the ryanodine-sensitive release channel plays no direct role in these responses. Free radical interactions may complicate the interpretation of findings in cascade bioassay compared with isolated ring preparations.     

Pituitary adenylate cyclase activating peptides relax human pulmonary arteries by opening of KATP and KCa channels

BACKGROUND: Pituitary adenylate cyclase activating peptides (PACAPs) are potent endothelium independent dilators of human coronary arteries; however, their effects on human pulmonary arteries are unknown. METHODS: The vasorelaxant effects of PACAP27 on human pulmonary segmental arteries were studied and the specific potassium (K+) channel regulatory mechanisms in the vasorelaxant effects were tested by means of isometric contraction experiments. RESULTS: PACAP27 produced dose dependent relaxations of 10 microM rings preconstricted with prostaglandin F2 alpha (PGF2 alpha) with half maximal relaxation (IC50) at 17 nM. Pretreatment of the vessels with the ATP sensitive K+ (KATP) channel blocker glibenclamide (1 microM) or with the Ca2+ activated K+ (KCa) channel blocker iberiotoxin (100 nM) inhibited the PACAP27 induced relaxation. CONCLUSIONS: These results provide evidence that PACAPs are potent vasodilators of human pulmonary arteries and that this relaxation might be mediated by opening of KATP and KCa channels.     

Effects of halothane and isoflurane on bradykinin-evoked Ca2+ influx inbovine aortic endothelial cells

BACKGROUND: Volatile anesthetics, such as halothane and isoflurane, have been reported to affect the endothelium mediated relaxation of vascular smooth muscle cells. Because the activity of the constitutive nitric oxide synthase in endothelial cells depends on the availability of intracellular Ca2+, there is a definite possibility that the observed inhibitory effect of volatile anesthetics involves an action on the agonist-evoked internal Ca2+ mobilization and/or Ca2+ influx in these cells. Therefore, a study was undertaken to determine how halothane and isoflurane affect the Ca2+ signalling process in vascular endothelial cells. METHODS: The effect of halothane and isoflurane on the Ca2+ response to bradykinin of bovine aortic endothelial (BAE) cells was investigated using the fluorescent Ca2+ indicator fura-2. Halothane or isoflurane was applied either to resting cells or after bradykinin stimulation. The agonist-evoked Ca2+ influx in BAE cells was estimated by measuring either the rate of fura-2 quenching induced by Mn2+ or the increase in cytosolic Ca2+ concentration initiated after readmission of external Ca2+ after a brief exposure of the cells to a Ca(2+)-free external medium. The effects of halothane on cell potential and intracellular Ca2+ concentration were measured in cell-attached patch-clamp experiments in which a calcium-activated K+ channel and an inward rectifying Ca(2+)-independent K+ channel were used as probes to simultaneously monitor the intracellular Ca2+ concentration and the cell transmembrane potential. In addition, combined fura-2 and patch-clamp cell-attached recordings were carried out, to correlate the variations in internal Ca2+ caused by halothane and the activity of the Ca(2+)-dependent K+ channels, which are known in BAE cells to regulate intracellular potential. Finally, a direct action of halothane and isoflurane on the gating properties of the Ca(2+)-activated K+ channel present in these cells was investigated in patch-excised inside-out experiments. RESULTS: The results of the current study indicate that the initial Ca2+ increase in response to bradykinin stimulation is not affected by halothane, but that pulse applications of halothane (0.4-2 mM) or isoflurane (0.5-1 mM) reversibly reduce the sustained cytosolic Ca2+ increase initiated either by bradykinin or by the Ca2+ pump inhibitor thapsigargin. In addition, halothane appeared to dose-dependently inhibit the Ca2+ influx evoked by bradykinin, and to cause, concomitant to a decrease in cytosolic Ca2+ concentration, a depolarization of the cell potential. Halothane failed, however, to affect internal Ca2+ concentration in thapsigargin-treated endothelial cells, which were depolarized using a high K+ external solution. Finally, halothane and isoflurane decreased the open probability of the Ca(2+)-dependent K+ channel present in these cells. CONCLUSIONS: These observations suggest that the effects of halothane and isoflurane on Ca2+ homeostasis in BAE cells reflect, for the most part, a reduction of the thapsigargin- or bradykinin-evoked Ca2+ influx, which would be consequent to a cellular depolarization caused by an inhibition of the Ca(2+)-dependent K+ channel activity initiated after cell stimulation.     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Endothelin-A receptors mediate vascular smooth-muscle response to moderate acidosis in the canine tibial nutrient artery

Perfusate pH may influence the tone of vascular smooth muscle by affecting the release of endothelium-derived vasoactive factors or by directly modulating function of the smooth muscle. This study was designed to investigate the role of endothelium-derived factors on acidosis-induced responses of isolated canine tibial nutrient artery suspended in an organ chamber for the measurement of isometric contractile force. To investigate the specific role of the endothelium in half the rings, the endothelium was removed mechanically. Concentration-response curves to KCl were obtained in the absence or presence of inhibition of two important endothelium-derived relaxing factors, nitric oxide and prostacyclin, and an inhibitor of receptors for the endothelium-derived contracting factor, endothelin-1. Acidification of the perfusate from pH 7.45 to 7.0 significantly attenuated the contractions to KCl in arterial rings with endothelium (the mean of the effective concentration causing 50% of the maximal response for KCl at pH 7.45 and 7.0 was 12.31 +/- 0.40 nM and 14.60 +/- 0.55 nM, respectively). This difference was abolished by mechanical removal of the endothelium. In rings with endothelium, inhibition of nitric oxide or prostacyclin did not abolish the attenuation of KCl-induced contractions occurring with acidosis (the mean of the effective concentration causing 50% of the maximal response for KCl at pH 7.45 and 7.0 was 11.18 +/- 0.60 nM and 13.60 +/- 0.60 nM, respectively). Inhibition of endothelin-A receptors did not alter contractions to KCl at pH 7.45. However, the acidosis-induced attenuation of contractions with KCl was abolished by the endothelin-A-receptor antagonist BQ-123 (the mean of the effective concentration causing 50% of the maximal response at pH 7.45 and 7.0 was 13.8 +/- 1.34 nM and 13.2 +/- 1.34 nM, respectively). These results suggest that acidosis-induced relaxation of canine tibial nutrient artery is endothelium dependent and that activation of endothelin-A receptors during acidosis is coupled to a release of an endothelium-derived relaxing factor.