[Interventional electrophysiology in cardiac MRI : what is the current status?]. / Interventionelle Elektrophysiologie in der kardialen MRT : Wie ist der aktuelle Stand?

The ablation of simple and complex cardiac arrhythmias has become a first-line therapy in interventional cardiology and is mainly guided by conventional fluoroscopy. Cardiac magnetic resonance imaging (cMRI) allows exact three-dimensional (3D) visualization of complex anatomical structures and serves in the planning and implementation of ablation procedures. Post-procedural lesion visualization using cMRI can assess the success of ablation therapy and may distinguish potential complications. Performing ablation directly in the MRI scanner, with the option of anatomical substrate imagining, exact catheter navigation and real-time lesion visualization, holds the promise of improving success rates and safety in the interventional therapy of simple and complex arrhythmias.

In human hypercholesterolemia increased reactivity of vascular smooth muscle cells is due to altered subcellular Ca(2+) distribution.

There is evidence that, besides an attenuated endothelium-dependent relaxation, functional changes in smooth muscle contractility occur in experimental hypercholesterolemic animals. Unfortunately, little is known of the situation in human arteries, and the intracellular mechanisms involved in the modulation of vascular smooth muscle function in human hypercholesterolemia are still unclear. Thus, besides acetylcholine-induced endothelium-dependent relaxation, smooth muscle reactivity to KCl, norepinephrine (NE) and phenylephrine (PE) was evaluated in uterine arteries from 34 control individuals (CI) and 22 hypercholesterolemic patients (HC). Contractions to KCl, norepinephrine and phenylephrine were enhanced by 1.3-, 2.1- and 3.5-fold in vessels from HC. Furthermore, the Ca(2+) signaling in the perinuclear cytosol, which promotes cell contraction, and that of the subplasmalemmal region, which contributes to smooth muscle relaxation, were examined in freshly isolated smooth muscle cells. In cells from HC, increases in perinuclear Ca(2+) concentration ([Ca(2+)](peri)) in response to 30 mM KCl and 300 nM NE were increased by 67 and 93%, respectively. In contrast, the increase in the subplasmalemmal Ca(2+) concentration ([Ca(2+)](sub)) to 10 microM NE was reduced in cells from HC by 33%. No further differences in perinuclear and subplasmalemmal Ca(2+) signaling were found in cultured smooth muscle cells from CI and HC (primary culture 4-6 weeks after isolation). These data indicate a significant change in the subcellular Ca(2+) distribution in smooth muscle cells from HC. In addition, production of superoxide anions (O(2)(-)) was increased 3.8-fold in uterine arteries from HC. Treatment of smooth muscle cells with the O(2)(-)-generating mixture xanthine oxidase/hypoxanthine mimicked hypercholesterolemia on smooth muscle Ca(2+) signaling. From these findings, we conclude that during hypercholesterolemia, besides a reduced endothelium-dependent relaxation, changes in smooth muscle reactivity take place. Thereby, smooth muscle contractility is increased possibly due to the observed changes in subcellular Ca(2+) signaling. The observed increased O(2)(-) production in HC might play a crucial role in the alteration of smooth muscle function in hypercholesterolemia.

Human diabetes is associated with hyperreactivity of vascular smooth muscle cells due to altered subcellular Ca2+ distribution.

Alterations of vascular smooth muscle function have been implicated in the development of vascular complications and circulatory dysfunction in diabetes. However, little is known about changes in smooth muscle contractility and the intracellular mechanisms contributing to altered responsiveness of blood vessels of diabetic patients. Therefore, smooth muscle and endothelial cell function were assessed in 20 patients with diabetes and compared with 41 age-matched control subjects. In rings from uterine arteries, smooth muscle sensitivity to K+, norepinephrine (NE), and phenylephrine (PE) was enhanced by 1.4-, 2.3-, and 9.7-fold, respectively, and endothelium-dependent relaxation was reduced by 64% in diabetic patients, as compared with control subjects. In addition, in freshly isolated smooth muscle cells from diabetic patients, an increased perinuclear Ca2+ signaling to K+ (30 mmol/l >73%; 60 mmol/l >68%) and NE (300 nmol/l >86%; 10 micromol/l >67%) was found. In contrast, subplasmalemmal Ca2+ response, which favors smooth muscle relaxation caused by activation of Ca2+-activated K+ channels, was reduced by 38% in diabetic patients as compared with control subjects, indicating a significant change in the subcellular Ca2+ distribution in vascular smooth muscle cells in diabetic patients. In contrast to the altered Ca2+ signaling found in freshly isolated cells from diabetic patients, in cultured smooth muscle cells isolated from control subjects and diabetic patients, no difference in the intracellular Ca2+ signaling to stimulation with either K+ or NE was found. Furthermore, production of superoxide anion (*O2-) in intact and endothelium-denuded arteries from diabetic patients was increased by 150 and 136%, respectively. Incubation of freshly isolated smooth muscle cells from control subjects with the *O2- -generating system xanthine oxidase/hypoxanthine mimicked the effect of diabetic patients on subcellular Ca2+ distribution in a superoxide dismutase-sensitive manner. We conclude that in diabetic subjects, smooth muscle reactivity is increased because of changes in subcellular Ca2+ distribution on cell activation. Increased *O2- production may play a crucial role in the alteration of smooth muscle function.

Potentiation of Ca2+ signaling in endothelial cells by 11,12-epoxyeicosatrienoic acid.

Incubation of endothelium with an increased epoxyeicosatrienoic acid (EET) concentration specifically augments the endothelium-dependent relaxation ascribed to endothelium-derived hyperpolarizing factor in porcine coronary arteries (Weintraub et al., Circ Res 1997;81:258-267). Experiments were designed to test whether such sustained increased levels of EETs in the environment of endothelial cells alters Ca2+ signaling. Changes in cytosolic Ca2+ were monitored in cultured porcine aortic endothelial cells (PAECs) and in the human endothelial EA.hy926 cell line after incubation (or not) with 5 microM 11,12-epoxyeicosatrienoic acid (EET). Although the mobilization of intracellular Ca2+ induced by 2 microM thapsigargin was not affected significantly, EET treatment augmented the capacitative Ca2+ entry evoked by the Ca(2+)-ATPase) inhibitor in both cell types. Similar observations were obtained by using histamine as a stimulant in EA.hy926 cells. As assessed in PAECs, 2 micrograms/ml triacsin C, a known inhibitor of the incorporation of EETs into phospholipids, did not significantly affect the potentiating action of EETs on Ca2+ signaling in response to thapsigargin. However, in solvent-control cells, triacsin C significantly reduced both the mobilization of Ca2+ from intracellular stores and the capacitative Ca2+ entry provoked by thapsigargin. Thus the EET-potentiating effect overcomes the inhibitory action of triacsin C on Ca2+ signaling in endothelial cells. Taken together, these results demonstrate that sustained increases in EETs may amplify Ca2+ signaling. However, contrary to the EET-induced augmentation of endothelium-dependent relaxation in the porcine coronary artery, resistance of this novel action of EETs to triacsin C suggests that the mechanism involved does not depend on incorporation into phospholipids.

Anandamide-induced mobilization of cytosolic Ca2+ in endothelial cells.

1. Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[3H]-arginine to L-[3H]-citruline. 5. The CB1 cannabinoid receptor antagonist SR141716A (1 microM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 microM anandamide. However, at 5 microM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 microM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/o proteins) had no significant effect on the mobilization of cytosolic Ca2+ evoked by either anandamide, or histamine. 7. Taken together the results demonstrate that anandamide mobilizes Ca2+ from a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca2+ in endothelial cells may account for the endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.

EDRF does not mediate coronary vasodilation secondary to simulated ischemia: a study on KATP channels and N omega-nitro-L-arginine on coronary perfusion pressure in isolated Langendorff-perfused guinea-pig hearts.

Several authors have alluded to the possible involvement of EDRF (NO) in ischemia-induced coronary artery dilation. Alternatively, it has been suggested that opening of ATP-dependent K channels could play a key role in this context. We studied the effects of sulfonylureas and NG-nitro-L-arginine (LNNA), a specific inhibitor of endothelial NO (EDRF) synthesis, on ischemia-induced coronary vasodilation in isolated Langendorff-perfused guinea pig hearts arrested with 15 mM KCl in normal Tyrode, and isolated pig coronary arteries precontracted with 43 mM KCl. In Isolated Langerdorff-perfused guinea pig heart, when hypoxia was simulated by switching 100% O2 in the perfusate to 100% N2, coronary perfusion pressure (CPP) fell from 90 cm H2O by 45 +/- 5 cm H2O. In the presence of LNNA, a specific inhibitor of NO synthetase in endothelial cells, CPP dropped by 44 +/- 6 cm H2O (n = 6; +/- SEM, no statistically significant). On biochemical simulation of ischemia (addition of iodoacetate [IAA]), CPP dropped 40 +/- 6 cm H2O, and in experiments performed under the same conditions but in the presence of LNNA, CPP dropped by 38 +/- 5 cm H2O (n = 6; +/- SEM; not statistically significant). When ischemia was simulated metabolically by equimolar replacement of 10 mM glucose with 2-deoxyglucose (DOG), an inhibitor of glycolysis CPP decreased by 24 +/- 1 cm H2O (n = 6; +/- SEM) after 15 minutes. This fall in CPP was almost prevented by 20 microM glibenclamide, whereas in the presence of 20 microM LNNA the DOG-induced decrease in CPP was not significantly inhibited, and CPP decreased by 22 +/- 2.6 cm H2O (n = 6; +/- SEM). In isolated pig coronary artery rings, maximal tension, achieved by depolarizing the smooth muscle cells by 43 mM KCl, decreased by 37 +/- 7% upon simulated hypoxia by replacing 100% O2 with 100% N2 in the perfusate (n = 6; +/- SEM) in arteries with intact endothelium. In arteries without endothelium, maximal tension also dropped by 35 +/- 6% (not statistically significant). In the same experiments the decrease in tension could be largely inhibited in the presence of 50 microM glibenclamide. Our results clearly show that in isolated perfused guinea pig hearts, as well as in isolated pig coronary arteries, EDRF does not play a decisive role in the coronary dilatory response to hypoxia and ischemia.

Mechanisms of L-NG nitroarginine/indomethacin-resistant relaxation in bovine and porcine coronary arteries.

1. Coronary arteries from bovines (BCA) and pigs (PCA) were used for measuring endothelium-dependent relaxation in the presence of L-NG nitroarginine and indomethacin. As some compounds tested have been found to have an inhibitory effect on autacoid-activated endothelial Ca2+ signalling, endothelium-dependent relaxation was initiated with the Ca2+ ionophore A23187. 2. The common compounds for modulating arachidonic acid release/pathway, mepacrine and econazole only inhibited L-NG nitroarginine-resistant relaxation in BCA not in PCA. In contrast, proadifen (SKF 525A) diminished relaxation in BCA and PCA. Mepacrine and proadifen inhibited Hoe-234-initiated relaxation in BCA and PCA, while econazole only inhibited Hoe 234-induced relaxation in PCA. Due to the multiple effects of these compounds, caution is necessary in the interpretation of results obtained with these compounds. 3. The inhibitor of Ca(2+)-activated K+ channels, apamin, strongly attenuated A23187-induced L-NG nitroarginine-resistant relaxation in BCA while apamin did not affect L-NG nitroarginine-resistant relaxation in PCA. 4. Pertussis toxin blunted L-NG nitroarginine-resistant relaxation in BCA, while relaxation of PCA was not affected by pertussis toxin. 5. Thiopentone sodium inhibited endothelial cytochrome P450 epoxygenase (EPO) in PCA but not in BCA, while L-NG nitroarginine-resistant relaxation of BCA and PCA were unchanged. Protoporphyrine IX inhibited EPO in BCA and PCA and abolished L-NG nitroarginine-resistant relaxation of BCA not PCA. 6. An EPO-derived compound, 11,12-epoxy-eicosatrienoic acid (11,12-EET) yielded significant relaxation in BCA and PCA in three out of six experiments. 7. These findings suggest that L-NG nitroarginine-resistant relaxation in BCA and PCA constitutes two distinct pathways. In BCA, activation of Ca(2+)-activated K+ channels via a pertussis-toxin-sensitive G protein and EPO-derived compounds might be involved. In PCA, no selective inhibition of L-NG nitroarginine-resistant relaxation was found.

[ACE inhibitors with SH groups have no effect of ATP-dependent K channels--a dissertation]. / SH-Gruppen-hältige ACE-Hemmer haben keinen Effekt auf ATP-abhängige K-Kanle--eine Dissertation.

ACE-inhibitors are used in the treatment of hypertension, and ischemic heart disease, chronic heart failure, cardiomyopathy and diabetic nephropathy. The effect of the ACE inhibitors is mainly due to the inhibition of the angiotensin converting-enzyme, but they also potentiate the effect of bradykinine. Sargent et al. have indicated, that SH-containing ACE-inhibitors show an effect on KATP-channel open probability in vascular smooth muscle. In our experiments, we used isolated bovine coronary arteries and guinea pig aortas, which were cut transversally and brought into Normal-Tyrode-solution. The vessels were precontracted with phenylephrine or U 46619, and after that a cumulative dose of the SH-containing ACE-inhibitors Captopril or Zofenopril was added to obtain a relaxation curve. In a second series we blocked the KATP-channels with glibenclamide to see if the relaxation could be attenuated. In bovine coronary arteries the relaxing effect of Captopril could not be attenuated by glibenclamide, a specific blocker of KATP-channels of vascular smooth muscle. In the guinea pig aorta, the relaxing effect of Zofenopril was also not effected by glibenclamide. The concentrations of Zofenopril were between 10(-7) and 10(-4) mol/l; the maximal effect could be seen at a concentration of 10(-5) mol/l. Experiments with the non SH-containing ACE-inhibitor Enalapril did also not show any statistically significant difference between the 2 groups of series. We conclude, that, in contrast to Sargent's conclusions, there is little or no effect on KATP-channels due to the presence of SH-groups.

KATP channel opening does not contribute significantly to the vasodilatory effect of SH-group-containing ACE inhibitors.

Angiotensin-converting enzyme (ACE) inhibitors are widely used in the management of hypertension, heart failure, and nephropathy. It has been suggested that ACE inhibitors containing the sulfhydryl group (SH) have an additional effect on KATP channels. To prove this hypothesis, we studied the effects of the SH-containing ACE inhibitors, captopril and zofenopril, on KATP channel opening of bovine coronary arteries and guinea pig thoracic aortas. Bovine coronary arteries were precontracted with the thromboxane A2 analogue, U46619, and guinea pig thoracic aortas were precontracted with phenylephrine and then relaxed with either captopril or zofenopril (n = 8). Inhibition of KATP channel opening with glibenclamide moderately attenuated the zofenopril-induced relaxation of guinea pig thoracic aorta. However, in the bovine coronary arteries, the relaxing effect of both captopril and zofenopril remained uneffected by glibenclamide. In experiments with enalapril (a non SH-containing ACE inhibitor; n = 6) on guinea pig thoracic aortas, no effect on KATP channels could be seen. From our experiments, we conclude that the postulated opening of KATP channels by SH-group-containing ACE inhibitors contributes little to the vasodilation of guinea pig thoracic aortas caused by ACE inhibitors, and that SH groups have no influence upon KATP channels of bovine coronary arteries.

Pharmacologic differentiation between endothelium-dependent relaxations sensitive and resistant to nitro-L-arginine in coronary arteries.

We investigated whether formation of endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF) in porcine and bovine endothelial cells (PAECs) was stimulated by different kinin receptors and studied pharmacologic differences and similarities between the two types of bradykinin-induced relaxation of bovine or porcine coronary arteries. Cultured PAECs were used for [3H]bradykinin binding assay and for measurement of the endothelial free [Ca2+]i by the fura-2/AM method. In organ bath studies with strips of bovine and porcine coronary arteries (endothelium intact), changes in length were recorded and cyclic GMP was measured by radioimmunoassay (RIA). Two bradykinin binding sites were detected, suggesting the presence of two subtypes of B2 kinin receptors. Bradykinin increased [Ca2+]i, and this action was antagonized by the B2 kinin receptor antagonist Hoe 140 and the K channel inhibitor tetrabutylammonium (TBA). Hoe 140 competitively antagonized the relaxing effects of bradykinin, whereas a B1 antagonist was inactive. L-omega N-nitro-arginine (L-NNA) diminished one part of bradykinin-induced relaxation and abolished the increases in cyclic GMP; TBA inhibited another part of the relaxing effect and attenuated (but not significantly) increases in cyclic GMP, and Hoe 140 completely inhibited relaxation and increases in cyclic GMP. The results indicate that the bradykinin response is mediated by biosynthesis of EDRF, which is sensitive to L-NNA, and of EDHF, which is sensitive to TBA.

Pharmacological interaction experiments differentiate between glibenclamide-sensitive K+ channels and cyclic GMP as components of vasodilation by nicorandil.

The relaxant effect of the vasodilator drug, nicorandil, was studied in circular strips of bovine coronary arteries. To differentiate between relaxation caused by cyclic GMP (cGMP) and by hyperpolarization, the influence of cGMP was blocked with methylene blue and that of hyperpolarization with the inhibitor of ATP-dependent K+ channels, glibenclamide. Methylene blue and glibenclamide inhibited nicorandil-induced relaxation to similar extents. Cromakalim-induced relaxation but not that due to sodium nitroprusside (nitroprusside-Na) was inhibited by glibenclamide. Methylene blue inhibited the relaxation caused by nitroprusside-Na but not that due to cromakalim. The different modes of action of the two components of relaxation caused by nicorandil were studied in agonist-agonist interaction experiments. The interaction between nicorandil and nitroprusside-Na or 3-morpholino-sydnonimine (SIN-1) was overadditive in the absence of glibenclamide but additive, i.e. competitive, in the presence of glibenclamide. The interaction of nicorandil with cromakalim or pinacidil was overadditive in the absence of methylene blue but additive, i.e. competitive, in the presence of methylene blue. The results show that nicorandil relaxes smooth muscle through two independent mechanisms: ATP-dependent activation of K+ channels and stimulation of guanylyl cyclase resulting in increases in cGMP.

Molecular mechanism of action of nicorandil.

Nicorandil relaxes coronary vascular smooth muscle by stimulating guanylyl cyclase and increasing cyclic GMP (cGMP) levels (as shown first in our laboratory) as well as by a second mechanism resulting in activation of K+ channels and hyperpolarization. Therefore, we studied the relative contributions of either mechanism to the overall response in bovine circular strips of coronary arteries by simultaneously measuring changes in length and in cGMP levels through radioimmunoassay. Blockade by 10 microM methylene blue of the cGMP increases in strips precontracted by 1 microM of the thromboxane A2 analogue U46619 reduced nicorandil-induced relaxation to 30-50%, and there were no significant changes in cGMP levels. Suppression of the hyperpolarizing component of nicorandil by 80.4 mM K+ or 1 microM glibenclamide in precontracted strips reduced nicorandil relaxation to 50% (K+) or shifted the dose response to the right by a factor of two (glibenclamide) without alteration of increases in cGMP. A quantitative separation of both mechanisms of action was obtained by comparing the correlation between increases in cGMP and relaxation under conditions of inhibited versus noninhibited hyperpolarization. The results indicate that cGMP contributes to the total relaxing effect of nicorandil by 30-40% at low concentrations and 80-90% at high concentrations of nicorandil. From the experiments with glibenclamide, it can be concluded that the probable mechanism by which nicorandil hyperpolarizes is opening glibenclamide-sensitive K+ channels in coronary vascular smooth muscle and that this latter effect mimics those of other K+ channel openers such as cromakalim or pinacidil.

Cellular mechanisms of action of therapeutic nitric oxide donors.

A survey of the available literature leads to the conclusion that the most probable mechanism by which nitrovasodilators act, is by nitric oxide (NO) formation. This by itself or by formation of a nitrosothiol (e.g. nitroscocysteine) activates guanylyl cyclase which increases the production of cyclic guanosine monophosphate (cGMP). Endothelium-derived relaxing factor (EDRF), which later turned out to be or to form NO, relaxes smooth muscle by stimulating cGMP formation. The effect of cGMP is mediated by a cGMP-dependent protein kinase and causes a reduction in the intracellular concentration of free Ca2+ ions in the smooth muscle cell. The precise mechanism of this effect is not completely clear but sequestration into sarcoplasmatic reticulum seems to play a major role. In order to identify the nature of the endogenous stimulator of guanylyl cyclase, i.e. to decide whether it is a nitrosothiol or the free radical NO, we compared the effects of NO, nitrosocysteine and nitrosoglutathione on vascular relaxation and increases in cGMP levels in isolated bovine circular strips and on guanylyl cyclase activity in vitro. Induction of tolerance and of cross-tolerance between various NO donors was also investigated. Nitrosodium and nitrosoglutathione augmented cGMP and relaxed vascular smooth muscle slightly more powerfully than NO. The three agents induced slight tolerance after repeated administration without affecting cGMP rises or desensitizing guanylyl cyclase. Pretreatment of coronary strips with nitrosoglutathione caused largely similar cross-tolerance as did NO against nitroglycerin, SIN-1 and sodium nitroprusside. The similarities to NO characterize nitrosocysteine as its most likely precursor, e.g. as EDRF.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual mechanism of the relaxing effect of nicorandil by stimulation of cyclic GMP formation and by hyperpolarization.

In addition to previous results from our laboratory showing that nicorandil relaxed vascular smooth muscle by increasing cyclic GMP levels, it was shown to activate K-channels as well, an effect that also leads to relaxation. In the present study, we attempted to differentiate quantitatively between these two effects in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclic GMP. When the strips were contracted by the thromboxane A2 analogue U 46619 (1 microM) with 10 microM methylene blue added, nicorandil produced 30-50% relaxation without significant changes in cyclic GMP. When in U 46619-contracted strips the hyperpolarizing effect of nicorandil was suppressed by increasing extracellular K+ to 80.4 mM (30-fold), nicorandil caused only 52% relaxation, whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechanisms of relaxation by nicorandil was further achieved through calculation of the cyclic GMP-mediated component from a correlation between increases in cyclic GMP and percentage of relaxation as produced by nicorandil under conditions of inhibited hyperpolarization, i.e., in strips contracted with 1 microM U 46619 or 26.8 mM K+ (10-fold) and exposed to either 30-fold K+ or 10 mM Ba2+. Under both conditions, similar correlations between cyclic GMP and relaxation were obtained. Because U 46619, in addition to its contractile effect, partially antagonized the relaxation by nicorandil without changing cyclic GMP, the correlation was corrected for this effect and indicated a participation of cyclic GMP in the overall relaxant response of approximately 30-40% at low and less than or equal to 80-90% at high concentrations of nicorandil.

Evaluation of combined effects in dose-response studies by statistical comparison with additive and independent interactions.

An improved method for the evaluation of combined drug effects by means of dose-response curves (DRCs) is described. A drug, A, was tested in the absence and presence of a fixed concentration of another drug, B, mainly in organ-bath experiments with smooth muscle strips from bovine coronary arteries and tracheal muscle. The results of such experiments are expressed in terms of percent of maximum response. Median values, rather than mean values, for each concentration of drug A were determined in order to allow a comparison of observed with expected frequencies above or below median DRCs of additive and independent interactions. This comparison was done with the chi-square goodness-of-fit statistic. Advantage was taken of the curve-fitting program ALLFIT to construct DRCs. However, the method presented does not require a computer program. The results indicate that additive interactions point to actions of drugs at the same site inasmuch as they differ significantly from independent interactions. Overadditive interactions reflect differences between the sites of action. This may either be due to independent actions or to some kind of synergistic "cooperativity", for example, sequential interaction. The effects of the latter significantly exceed the effects expected for independently interacting compounds. This method appears applicable to compounds exerting all kinds of responses that can be described by DRCs.

Mechanisms of nitrate-induced vasodilatation and tolerance.

Nitrovasodilators have been found to relax vascular smooth muscle by stimulating soluble guanylate cyclase and thus by increasing the formation of cyclic GMP (cGMP). This nucleotide is responsible for relaxation, most likely by decreasing cytosolic free Ca2+ by one or several mechanisms. Repeated administration of organic nitrates causes tolerance development characterized by a diminished relaxing effect and an attenuated rise in cGMP. Experiments in isolated circular strips from bovine coronary arteries were performed in order to study the mechanism of tolerance development. It was found that after nitroglycerin (NG) pretreatment the response of the coronary strips to NG was less sensitive with respect to relaxation and increases in cGMP. These strips were also cross-tolerant against isosorbide-5-mononitrate, which by itself caused only little tolerance. With NG, the degree of tolerance development depended on the time and the concentration of NG pre-exposure. NG was found to stimulate guanylate cyclase (GC) in coronary supernatant provided that cysteine was added to the incubation medium. As in the intact strips, activation of GC by NG was attenuated when supernatants were preincubated with NG. It was found that addition of cysteine during incubation lessened the degree of desensitization but did not prevent it completely. Similarly, in coronary strips, tolerance development was lower when N-acetylcysteine was present during pre-exposure of the strips with NG. Considerably more effective in preventing tolerance development by about 50% was L-2-oxothiazolidine-4-carboxylate (OTC), a substance that easily penetrates into the cell and is transformed into cysteine by 5-oxo-prolinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Tolerance and cross-tolerance between SIN-1 and nitric oxide in bovine coronary arteries.

The molecular mechanism of tolerance development to nitrovasodilators, most prominent with nitroglycerin, associated with desensitization of guanylate cyclase is still unclear. Nitric oxide (NO) appears to be the common denominator of this group of drugs that leads to guanylate cyclase activation, followed by increases in levels of cyclic GMP and relaxation. It was therefore decided to study whether NO itself, which causes some tolerance, interferes with the actions of (a) SIN-1 and sodium nitroprusside, both of which are thought to act directly by NO formation, which explains why they cause little tolerance; and (b) with the actions of nitroglycerin, which stimulates cyclic GMP formation only in the presence of cysteine and causes pronounced (large) tolerance. Experiments were performed in circular strips of isolated de-endothelialized bovine coronary artery by measuring isotonic changes in length and cyclic GMP determined by radioimmunoassay. When the strips were treated with submaximal effective concentrations of NO, some tolerance was observed, as shown by moderate attenuation of the rises in cyclic GMP, and a rightward shift of the dose-response curve of the relaxing effects by a dose factor of 10 (DF = 10). Exposure to nitroglycerin, SIN-1, or sodium nitroprusside rendered the strips cross-tolerant to NO to a comparable extent as NO itself, suggesting that under these conditions the NO component of all of these drugs that caused similar tolerance is displayed. When the strips were treated with NO and subsequently challenged with nitroglycerin, SIN-1, or sodium nitroprusside, the NO cross-tolerance was uniformly lower than the tolerance to the challenging agent.(ABSTRACT TRUNCATED AT 250 WORDS)

[Mechanism of the vasodilating effect and blood platelet- antiaggregating activity of molsidomine and SIN-1]. / Mécanisme de l'effet vasodilatateur et de l'action anti-agrégante plaquettaire de la molsidomine et du SIN-1.

Present evidence indicates that the active metabolite SIN-1 of the prodrug molsidomine dilates vascular smooth muscle and inhibits platelet aggregation by a direct stimulatory effect on soluble guanylate-cyclase in the cytosol of vascular smooth muscle cells and platelets, respectively. Evidence from studies in bovine coronary arteries is presented to proof the causal relation between SIN-1 induced rises in cGMP and relaxation under a variety of pharmacological conditions. In contrast to organic nitrates, SIN-1, which activates guanylate-cyclase in vitro independently of the presence of added cysteine, does not cause tolerance. Tolerance most pronounced with nitroglycerin appears to be due to a direct inactivating effect of this drug on guanylate-cyclase.

Cyclic GMP in nicorandil-induced vasodilatation and tolerance development.

Nicorandil (SG-75) is a new organic nitrate with pronounced vasodilator properties. We studied whether nicorandil, in analogy to other nitrovasodilatators, exerted its relaxing effects on vascular smooth muscle by stimulating guanylate cyclase, and whether this effect was susceptible to tolerance development. Dose-response curves for the relaxing and cyclic guanosine monophosphate (cGMP) increasing effects of nicorandil were obtained in isolated strips of bovine coronary arteries and compared with those of other nitrovasodilatators. It was found that nicorandil dose-dependently relaxed the strips precontracted with 26.7 mM K+ and that this effect was closely associated with increases in cGMP levels (measured by RIA under various conditions). The correlation between relaxation and rises in cGMP was steeper than with other nitrovasodilatators, suggesting that nicorandil, in addition to its cGMP-mediated effect, also relaxed vascular smooth muscle by a cGMP independent mechanism. In contrast to nitroglycerin (NG), nicorandil caused little development of tolerance or cross-tolerance toward ISDN or IS-5-MN when tested after preincubation of the strips toward the respective substance. Pretreatment with N-acetylcysteine during the preincubation period prevented tolerance towards nicorandil. The results indicate that the relaxant effects of nicorandil consist of a larger cGMP-mediated component and a smaller one which is independent of this nucleotide.