Effects of mechanical ventilation versus apnea on bi-ventricular pressure-volume loop recording.

Respiration changes intrathoracic pressure and lung volumes in a cyclic manner, which affect cardiac function. Invasive ventricular pressure-volume (PV) loops can be recorded during ongoing mechanical ventilation or in transient apnea. No consensus exists considering ventilatory mode during PV loop recording. The objective of this study was to investigate the magnitude of any systematic difference of bi-ventricular PV loop variables recorded during mechanical ventilation versus apnea. PV loops were recorded simultaneously from the right ventricle and left ventricle in a closed chest porcine model during mechanical ventilation and in transient apnea (n=72). Variables were compared by regression analyses. Mechanical ventilation versus apnea affected regression coefficients for important PV variables including right ventricular stroke volume (1.22, 95% CI [1.08-1.36], p=0.003), right ventricular ejection fraction (0.90, 95% CI [0.81-1.00], p=0.043) and right ventricular arterial elastance (0.61, 95%CI [0.55-0.68], p<0.0001). Right ventricular pressures and volumes were parallelly shifted with Y-intercepts different from 0. Few left ventricular variables were affected, mainly first derivatives of pressure (dP/dt(max): 0.96, 95% CI [0.92-0.99], p=0.016, and dP/dt(min): 0.92, 95% CI [0.86-0.99], p=0.026), which might be due to decreased heart rate in apnea (Y-intercept -6.88, 95% CI [-12.22; -1.54], p=0.012). We conclude, that right ventricular stroke volume, ejection fraction and arterial elastance were mostly affected by apnea compared to mechanical ventilation. The results motivate future standardization of respiratory modality when measuring PV relationships.

Qualitative and quantitative DECT pulmonary angiography in COVID-19 pneumonia and pulmonary embolism.

AIM: To assess differences in qualitative and quantitative parameters of pulmonary perfusion from dual-energy computed tomography (CT) pulmonary angiography (DECT-PA) in patients with COVID-19 pneumonia with and without pulmonary embolism (PE). MATERIALS AND METHODS: This retrospective institutional review board-approved study included 74 patients (mean age 61±18 years, male:female 34:40) with COVID-19 pneumonia in two countries (one with 68 patients, and the other with six patients) who underwent DECT-PA on either dual-source (DS) or single-source (SS) multidetector CT machines. Images from DS-DECT-PA were processed to obtain virtual mono-energetic 40 keV (Mono40), material decomposition iodine (MDI) images and quantitative perfusion statistics (QPS). Two thoracic radiologists determined CT severity scores based on type and extent of pulmonary opacities, assessed presence of PE, and pulmonary parenchymal perfusion on MDI images. The QPS were calculated from the CT Lung Isolation prototype (Siemens). The correlated clinical outcomes included duration of hospital stay, intubation, SpO2 and death. The significance of association was determined by receiver operating characteristics and analysis of variance. RESULTS: One-fifth (20.2%, 15/74 patients) had pulmonary arterial filling defects; most filling defects were occlusive (28/44) located in the segmental and sub-segmental arteries. The parenchymal opacities were more extensive and denser (CT severity score 24±4) in patients with arterial filling defects than without filling defects (20±8; p=0.028). Ground-glass opacities demonstrated increased iodine distribution; mixed and consolidative opacities had reduced iodine on DS-DECT-PA but increased or heterogeneous iodine content on SS-DECT-PA. QPS were significantly lower in patients with low SpO2 (p=0.003), intubation (p=0.006), and pulmonary arterial filling defects (p=0.007). CONCLUSION: DECT-PA QPS correlated with clinical outcomes in COVID-19 patients.

Atrial fibrillation and stroke in adult patients with atrial septal defect and the long-term effect of closure.

OBJECTIVE: To estimate the risk of atrial fibrillation (AF) and stroke and the impact of closure in patients with atrial septal defect (ASD) compared with a general population cohort. METHODS: All adult Danish patients (>18 years) diagnosed with ASD from 1977 to 2009 (N=1168) were identified through population-based registries. Using Cox regression, we compared ASD patients' risk of AF and stroke with an age-matched and gender-matched comparison cohort. We computed prevalence proportions of anticoagulation and antiarrhythmic medicine use before and after closure and described stroke-related mortality. RESULTS: Median follow-up was 9.6 years (range 1-33 years). Patients with ASD had a higher risk of first-time AF (adjusted HR 8.2; 95% CI 6.6 to 10.2) after closure than the comparison cohort, but with no difference between transcatheter and surgical closure (HR 1.5, 95% CI 0.6 to 3.5). Patients without prevalent AF had a 10-year cumulative incidence of AF of 11% (95% CI 9% to 14%) after closure compared with 2% (95% CI 1.8% to 2.5%) in the comparison cohort. Patients with ASD with prevalent AF continued to use anticoagulation medicine after closure/diagnosis. Patients with ASD had increased risk of stroke without closure (adjusted HR 2.6; 95% CI 1.4 to 3.0) and with closure (adjusted HR 2.0; 95% CI 1.4 to 2.7). Risk of stroke after closure was related to AF (HR adjusted for AF 1.3; 95% CI 0.9 to 1.9). CONCLUSIONS: Patients with ASD had a higher risk of first-time AF after closure than the comparison cohort. There was no effect of closure on the use of AF-related medicine in patients with prevalent AF.

Ischaemic stroke prevention in patients with atrial fibrillation and high bleeding risk: opportunities and challenges for percutaneous left atrial appendage occlusion.

Patients with atrial fibrillation (AF) are at an increased risk of ischaemic stroke. The efficacy of stroke prevention with vitamin K antagonists in these patients has been well established. However, associated bleeding risks may offset the therapeutic benefits in patients with risk factors for bleeding. Despite improvements achieved by novel oral anticoagulants, bleeding remains a clinically relevant problem, especially gastrointestinal bleeding. Percutaneous occlusion of the left atrial appendage (LAA) may be considered as an alternative stroke prevention therapy in AF patients with a high bleeding risk. This paper explores patient groups in whom oral anticoagulation may be challenging and percutaneous LAA occlusion (LAAO) has a potentially better risk-benefit balance. The current status of LAAO and future directions are reviewed, and particular challenges for LAA occlusion requiring further clinical data are discussed. This article is a summary of the Third Global Summit on LAA occlusion, 15 March 2013, Barcelona, Spain.

Comparison of two sulfonylureas with high and low myocardial K(ATP) channel affinity on myocardial infarct size and metabolism in a rat model of type 2 diabetes.

AIMS/HYPOTHESIS: Sulfonylureas (SUs) may impair outcome in patients with acute coronary syndrome. Most experimental studies of the myocardial effects of SU treatment are performed in non-diabetic models. We compared the effect of two widely used SUs, glibenclamide (gb) and gliclazide (gc), with high and low myocardial K(ATP) channel affinity, respectively, at therapeutic concentrations on infarct size, left ventricular (LV) function and myocardial glycogen, lactate and alanine content before and after ischaemia/reperfusion (I/R). METHODS: Non-diabetic Wistar and diabetic Goto-Kakizaki rat hearts were investigated in a Langendorff preparation. Gb (0.1 µmol/l) and gc (1.0 µmol/l) were administrated throughout the study. Infarct size was evaluated after 120 min of reperfusion. Myocardial metabolite content was measured before and after ischaemia. RESULTS: Infarct size was smaller in diabetic hearts than in non-diabetic hearts (0.33 ± 0.03 vs 0.51 ± 0.05, p < 0.05). Gb increased infarct size (0.54 ± 0.04 vs 0.33 ± 0.03, p < 0.05) and reduced post-ischaemic LV developed pressure (60 ± 3 vs 76 ± 3 mmHg, p < 0.05) and coronary flow (4.9 ± 0.5 vs 7.1 ± 0.4 ml min(-1) g(-1), p < 0.05) in gb-treated diabetic rats compared with untreated diabetic rats. On comparing gb-treated diabetic rats with untreated diabetic rats, glycogen content was reduced before (9.1 ± 0.6 vs 13.6 ± 1.0 nmol/mg wet weight, p < 0.01) and after ischaemia (0.9 ± 0.2 vs 1.8 ± 0.2 nmol/mg wet weight, p < 0.05), and lactate (4.8 ± 0.4 vs 3.2 ± 0.3 nmol/mg wet weight, p < 0.01) and alanine (1.38 ± 0.12 vs 0.96 ± 0.09 nmol/mg wet weight, p < 0.05) contents were increased during reperfusion. Gc-treatment of diabetic and non-diabetic rats did not affect any of the measured variables. CONCLUSIONS/INTERPRETATIONS: Gb, but not gc, exacerbates I/R injury and deteriorates LV function in diabetic hearts. These effects of gb on diabetic hearts may be due to detrimental effects on myocardial carbohydrate metabolism.

Effect of the oral hypoglycaemic sulphonylurea glibenclamide, a blocker of ATP-sensitive potassium channels, on walking distance in patients with intermittent claudication.

AIMS: The oral hypoglycaemic sulphonylurea glibenclamide stimulates endogenous insulin secretion through blockade of ATP-sensitive potassium (KATP) channels on pancreatic beta cells, but also blocks cardiovascular KATP channels, leading to increased peripheral vascular resistance and reduced peripheral blood flow in non-diabetic subjects. Therefore, this study examined whether a single oral dose of glibenclamide adversely affected the pain-free or maximal walking distance in patients with intermittent claudication. METHODS: In a double-blind, randomized crossover study, 12 non-diabetic patients with intermittent claudication were given a single oral dose of glibenclamide (5.25 mg) or placebo separated by a washout period of 1 week. A treadmill test was carried out 180 min after glibenclamide/placebo intake for determination of pain-free and maximal walking distance. Plasma glucose concentrations were kept constant by an euglycemic clamp. Changes in ankle/brachial blood pressure index (ABI), serum insulin, and serum glibenclamide were also assessed. RESULTS: The pain-free walking distance was 62.8 +/- 9.8 metres (mean +/- sem) after glibenclamide and 52.6 +/- 5.9 metres after placebo (P = 0.52). The maximal walking distance was 142.7 +/- 18.7 metres after glibenclamide and 132.6 +/- 16.6 metres after placebo (P = 0.23). The ABI was not significantly changed by glibenclamide compared with placebo. Serum glibenclamide was 0.51 +/- 0.08 microm 180 min after administration of the drug. Glibenclamide produced an 8-fold increase in circulating insulin compared with placebo (P < 0.001). CONCLUSIONS: Glibenclamide given as a single oral dose commonly used in glucose-lowering drug therapy does not reduce pain-free or maximal walking distance in non-diabetic patients with intermittent claudication.

Ischaemic preconditioning does not protect the heart in obese and lean animal models of type 2 diabetes.

AIMS/HYPOTHESIS: The prevalence of type 2 diabetes mellitus is increasing worldwide with obese diabetic patients constituting the majority of this population. Type 2 diabetes is associated with increased morbidity and mortality after acute myocardial infarction. Previous experimental studies of ischaemia-reperfusion tolerance in diabetes have only been performed in animal models of type 1 diabetes mellitus, yielding conflicting data. The aim of the present study was to characterise and compare the tolerance to ischaemia and effects of ischaemic preconditioning (IPC) in hearts from obese Zucker diabetic fatty (ZDF) and lean Goto-Kakizaki (GK) type 2 diabetic rats, using non-obese Zucker and Wistar rats as respective controls. METHODS: The two rat strains were divided into 8 groups. The ZDF study (n=47) consisted of: Control -IPC, Control +IPC, ZDF -IPC and ZDF +IPC. The GK study (n=38) consisted of: Control -IPC, Control +IPC, GK -IPC and GK +IPC. Hearts, which were studied in a Langendorff preparation perfused with Krebs-Henseleit buffer, were subjected or not to IPC (+IPC, -IPC) before 50 minutes of regional ischaemia and 120 minutes reperfusion. RESULTS: Ischaemic reperfusion injury was smaller in obese (p<0.05) and lean (p<0.05) type 2 diabetic animals than in their respective control animals. IPC reduced ischaemic reperfusion injury during reperfusion in non-diabetic control rats (p<0.01), but failed to protect hearts from both diabetic animal models. Post-ischaemic haemodynamic recovery was impaired in the ZDF rats compared to both control and GK rats (p<0.05). CONCLUSIONS/INTERPRETATION: Ischaemic preconditioning does not protect hearts from obese or lean type 2 diabetic animals. However, the susceptibility of the type 2 diabetic myocardium to ischaemic damage is lower than in non-diabetic hearts. The method described here could be used as a tool to study the pathogenesis of increased cardiovascular morbidity and mortality in type 2 diabetes.

[Spontaneous dissection of the left main coronary arteries in three women]. / Spontan dissektion af venstre koronararteries hovedstamme hos tre kvinder.

Spontaneous dissection of the coronary artery is a rare cause of sudden death and myocardial infarction. We report three cases in women aged 32, 38, and 55 years. One patient was one week post partum. In one case all three coronary arteries were involved. Two patients underwent coronary artery bypass grafting and one died of acute heart failure. The epidemiology, aetiology, and clinical manifestations are briefly described. We suggest that coronary angiography should be considered in young women with acute myocardial infarction and few risk factors of atherosclerosis.

Vascular smooth muscle contraction is an independent regulator of endothelial nitric oxide production.

This investigation was conducted to determine whether endothelial nitric oxide (NO) production is regulated by vascular smooth muscle contraction. Unperfused ring segments of rat aorta and mesenteric artery were studied using isometric tension recording (n = 6-8 in all experiments). Following a reference contraction to K+ 80 mM (100%), arteries were left either unstimulated or stimulated by different concentrations of K+ or prostaglandin F2alpha (PGF2alpha) to induce different levels of vascular precontraction. N(G)-nitro-L-arginine methyl ester (L-NAME 0.1-300 microM) or NS 2028 (0.03-3 microM), which is a new specific inhibitor of the NO-sensitive guanylate cyclase, was then added at increasing concentrations to evaluate endothelial NO production. L-NAME and NS 2028 produced a concentration-dependent vasoconstrictor response which was progressively enhanced with increasing levels of precontraction. For L-NAME, this amounted in aorta to (% of reference contraction): 35+/-1% and 105 +/- 4% (precontraction by K(+) 20 and 30 mM) and 22+/-1%, 89+/-1%, 138+/-1% and 146+/-2% (precontraction by PGF2alpha 0.5, 1, 2 and 3 microM). A similar coupling was found in the mesenteric artery. A precontraction as little as 2% was enough to trigger a vasoconstrictor response to L-NAME. In contrast, L-NAME and NS 2028 had no effect in non-contracted arteries, not even when passive mechanical stretch was increased by 100%. The results suggest (i) that endothelial NO formation is progressively increased with increasing vascular tone, and (ii) that vascular isometric contraction per se stimulates endothelial NO formation. It is concluded, that active vascular smooth muscle contraction is an independent regulator of endothelial NO production.

Nitroglycerin-mediated vasorelaxation is modulated by endothelial calcium-activated potassium channels.

OBJECTIVE: Recent in vitro data suggest, large conductance calcium-activated K+ channels (BKCa) modulate the vascular response to nitric oxide (NO). The in vivo implications and the characteristics of this interaction are not clear. This study firstly investigates whether modulation of BKCa affects the vascular response to nitroglycerin (NTG)-derived NO in vivo and in the isolated heart and secondly examines the influence of endothelial BKCa on NTG-mediated vasodilation in vitro. METHODS: The hypotensive effect of NTG was measured in conscious, chronically catheterized rats during i.v. infusions of iberiotoxin (IbTX, a selective inhibitor of BKCa) or placebo. Similarly, NTG-induced flow-changes in the isolated perfused rat heart were examined before and after IbTX treatment (0.1 microM). Concentration-relaxation curves to NTG in the presence of various K+ channel modulating agents were performed in vitro on porcine coronary arteries with and without intact endothelium. RESULTS: I.v. infusion of IbTX reduced the in vivo hypotensive effect of NTG by 55% (before IbTX: 32.0 +/- 3.0 mmHg, vs. after IbTX: 14.5 +/- 3.2 mmHg, P < 0.05) and nearly abolished NTG-induced increase in coronary flow in the isolated perfused heart (P < 0.05). In vitro, this effect depended on an intact endothelium (endothelium intact segments; NTG: pD2 = 5.8 +/- 0.1, Emax = 97.6 +/- 3.2% vs. NTG + IbTX: pD2 = 4.9 +/- 0.2, Emax = 49.7 +/- 6.2%, P < 0.05; endothelium denuded segments; NTG: pD2 = 6.9 +/- 0.1, Emax = 104.0 +/- 1.4% vs. NTG + IbTX: pD2 = 6.7 +/- 0.1, Emax = 100 +/- 1.2%, P > 0.05). CONCLUSION: The results suggest, that modulation of endothelial BKCa significantly affects NTG-induced vasorelaxation in vitro, in the isolated perfused heart and in vivo.

Hydralazine-induced vasodilation involves opening of high conductance Ca2+-activated K+ channels.

The purpose of this study was to investigate whether high conductance Ca2+-activated K+ channels (BK(Ca)) are mediating the vasodilator action of hydralazine. In isolated porcine coronary arteries, hydralazine (1-300 microM), like the K+ channel opener levcromakalim, preferentially relaxed contractions induced by K+ (20 mM) compared with K+ (80 mM). In addition, concentration-relaxation curves for hydralazine (pD2 = 5.38 +/- 0.06; Emax = 85.9 +/- 3.6%) were shifted 10-fold to the right by the BK(Ca) blockers tetraethylammonium (1 mM) and iberiotoxin (0.1 microM). In contrast, nimodipine (a Ca2+-entry blocker), relaxed contractions induced by K+ (20 mM) and K+ (80 mM) equally and nimodipine-induced relaxations were neither antagonized by tetraethylammonium nor by iberiotoxin. In isolated perfused rat hearts, hydralazine (1 microM) increased coronary flow by 28.8 +/- 2.7%. Iberiotoxin (0.1 microM) suppressed this response by 82% (P < 0.05). In conscious, chronically catheterized rats the hypotensive response to hydralazine (0.6 mg kg(-1) min(-1)) was significantly reduced by 41% during infusion of iberiotoxin (0.1 mg kg(-1)). It is concluded, that opening of BK(Ca) takes part in the mechanism whereby hydralazine produces vasodilation.

Inducible nitric oxide synthase (iNOS) in the human heart: expression and localization in congestive heart failure.

The inducible nitric oxide (NO) synthase (iNOS or NOS2) generates a prolonged release of large amounts of NO which may be cytotoxic and/or inhibit myocyte contractility. It has been suggested that this mechanism specifically contributes to heart failure caused by dilated cardiomyopathy (DCM). To test this hypothesis we compared the myocardial amount and localization of iNOS in myocardial biopsies from patients with heart failure caused by either DCM or ischemic heart disease (IHD). During heart transplantation, myocardial biopsies collected from the diseased heart after explantation were frozen in liquid nitrogen. Twenty-two patients in NYHA class III-IV were included (DCM: n = 8; IHD: n = 14). In each biopsy, iNOS expression was assessed using reverse transcription polymerase chain reaction (RT-PCR), and visualized by immunohistochemistry. iNOS was detected in all biopsies. Intriguingly, the amount of iNOS mRNA (shown as iNOS cDNA normalized to GADPH cDNA) did not differ significantly between the two groups (DCM 30 +/- 7; IHD 20 +/- 6, mean +/- S.E.M., P > 0.05). Similarly, no inter-group differences in the amount of iNOS protein (Western) were observed. iNOS was invariably located to vascular endothelial and smooth muscle cells. In addition, an iNOS reaction in relation to the myocyte membrane was found in 4 of the 22 patients. These four patients (two from each group) had significantly (P < 0.05) higher iNOS/GADPH ratios (54 +/- 20) than patients without myocyte membrane iNOS reaction (17 +/- 15). In conclusion, iNOS is expressed in the myocardium of all patients with heart failure caused by either DCM or IHD. iNOS is located primarily and invariably in the endothelium and vascular smooth muscle cells of the myocardial vasculature and its expression appears to be associated with the condition of heart failure per se rather than related to the heart failure etiology.

Coronary vasorelaxant effect of levosimendan, a new inodilator with calcium-sensitizing properties.

We examined the action of levosimendan, a new Ca2+-sensitizing inodilator, on isolated porcine coronary arteries. Vessel rings were studied in isometric myographs. Arterial cyclic adenosine monophosphate (cAMP) levels were determined by radioimmunoassay. Levosimendan (10(-7)-10(-3) M) completely relaxed arteries preconstricted by prostaglandin F2alpha (PGF2alpha) with a pD2 (-logEC50) value of 3.99 +/- 0.05 (n = 6-9 in all experiments). Pretreatment with levosimendan also prevented contraction induced by PGF2alpha. The vasorelaxation produced by levosimendan (10(-7)-10(-3) M) was not attenuated by removal of the endothelium. Levosimendan (10(-7)-10(-3) M) relaxed contractions induced by 30 mM K+ as well as 80 mM K+, whereas the K+ channel opener levcromakalim selectively relaxed contraction induced by 30 mM K+. Neither the cyclooxygenase inhibitor indomethacin nor the beta-adrenoceptor blocker propranolol influenced levosimendan-induced vasorelaxation. The Ca2+-entry blocker isradipine failed to relax arteries precontracted by endothelin-1 in Ca2+-free/EGTA medium. However, levosimendan (10(-7)-3 x 10(-3) M) completely relaxed endothelin-1-induced contractions in this medium. Levosimendan potentiated the relaxant effect of a cAMP-stimulating drug, isoprenaline, but also that of nitroglycerin and isradipine. At a maximal effective concentration, it increased arterial tissue contents of cAMP twofold. In conclusion, levosimendan produces coronary vasorelaxation by a mechanism that seems to be endothelium independent and not mediated by K+ channel opening, Ca2+-entry blockade, release of cyclooxygenase products, or beta-adrenoceptor stimulation. Accumulation of cAMP may possibly participate in vasorelaxation at high concentrations of levosimendan, but a cAMP-independent mechanism seems to be involved at lower concentrations.

Characterization of NS 2028 as a specific inhibitor of soluble guanylyl cyclase.

1 The haeme-containing soluble guanylyl cyclase (alpha1beta1-heterodimer) is a major intracellular receptor and effector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle. 2 Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC50 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for Ki, the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min(-1) for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme's haem cofactor from 430 to 390 nm. 3 S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC50 17 nM) and by 17 structural analogues in a similar manner, albeit with different potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,- NH- or -CH2- decreased the inhibition. 4 In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC50 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC50 30 nM). 5 In prostaglandin F2alpha-constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC50 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 microM) suppressed the relaxant response to nitroglycerin from 88.3+/-2.1 to 26.8+/-6.4% and induced a 9 fold rightward shift (EC50 15 microM) of the concentration-relaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 microM), but did not affect the vasorelaxation to the KATP channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028. 6 In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 microM) did not affect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase. 7 NO-dependent relaxant responses in non-vascular smooth muscle were also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 microM), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The relaxant responses to electrical field stimulation of non-adrenergic, non-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 microM). 8 NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a change in the coordination of the haeme-iron of guanylyl cyclase is a likely inhibitory mechanism.

Involvement of K+ channels in the relaxant effect of vasoactive intestinal peptide and atrial natriuretic peptide in isolated guinea-pig trachea.

The possible contribution of K+ channel activation to airway smooth muscle relaxation induced by vasoactive intestinal peptide (VIP) and atrial natriuretic peptide (ANP) was investigated in isolated guinea-pig trachea. Concentration-relaxation (CR) curves were assessed in preparations precontracted by 30 mM K+, 124 mM K+ or histamine either alone or in the presence of a K+ channel blocker: iberiotoxin (IbTX), glipizide, tetraethylammonium (TEA) or Ba2+. VIP completely relaxed contractions induced by histamine but had a lower effectiveness against those induced by 30 mM K+ and 124 mM K+. IbTX and TEA shifted the CR curve for VIP 5 and 14 times to the right, respectively. Glipizide and Ba2+ did not significantly antagonize the action of VIP. ANP relaxed contractions induced by histamine and 30 mM K+ but failed to relax those elicited by 124 mM K+. IbTX and TEA shifted the CR curve for ANP 8 and 46 times to the right, respectively. Glipizide and Ba2+ suppressed the maximal effect produced by ANP, and glipizide also shifted the CR curve to the left. The K+ channel opener levcromakalim relaxed tracheal contractions induced by histamine and 30 mM K+ but not those induced by 124 mM K+. Glipizide caused a 5-fold rightward shift of the CR curve for levcromakalim whereas IbTX shifted the curve to the left and increased the maximal relaxant effect. The Ca2+ channel blocker isradipine completely relaxed contractions induced by 30 mM K+ and 124 mM K+ but only partially relaxed those contracted by histamine. All four K+ channel blockers increased the maximal relaxant effect and shifted the CR curve for isradipine to the left. The results suggest that airway smooth muscle relaxation produced by VIP and ANP involves activation of large-conductance Ca(2+)-activated K+ channels (BKCa) and further that ANP may possibly activate other types of K+ channels additional to BKCa.

Potassium channel modulation: a new drug principle for regulation of smooth muscle contractility. Studies on isolated airways and arteries.

K+ channels play a key role in regulation of membrane potential and cell excitability. Several different types of K+ channels have been identified and the presence, characteristics and functions of these channels vary among different tissues. The 3 most important K+ channels in smooth muscle are the KATP (activated by a fall in intracellular ATP and a rise in nucleotide diphosphates and blocked by glibenclamide), BKCa (activated by a rise in intracellular Ca2+) and Kv (activated by depolarization). Cromakalim, pinacidil and nicorandil are members of a rapidly increasing group of novel drugs which open K+ channels. Opening of such channels leads to K+ efflux, membrane hyperpolarization, reduced excitability and smooth muscle relaxation. The purpose of the studies included in this thesis was to investigate this novel drug principle of K+ channel modulation on smooth muscle contractility of isolated airways and arteries and on neuroeffector transmission in airways. Smooth muscle contractility was measured in airway and vascular ring preparations suspended in isometric myographs. Neurotransmitter release was elicited by transmural electrical field stimulation. The major findings were: 1) Membrane depolarization by high extracellular K+ concentrations induced contraction of airway smooth muscle that was easily relaxed by Ca2+ antagonists and abolished in a Ca2+ free medium indicating that K+ contraction is triggered by Ca2+ influx through voltage-operated Ca2+ channels. Indomethacin was required to obtain reproducible responses upon repeated exposure to K+ suggesting that endogenous prostaglandins are released by K+ and interferes with its contractile effect. K+ depolarization was shown to be a valuable pharmacological tool for detection of drugs acting by K+ channel opening. The prototype K+ channel opener cromakalim relaxed contractions induced by 20-30 mM K+ but had no effect against contraction induced by 124 mM K+. This was a unique profile of action not shared by other types of airway and vascular smooth muscle relaxants. As the extracellular K+ concentration is raised the outward directed electrochemical gradient for K+ is reduced and at high K+ concentrations the effect of K+ channel opening is negligible. Although the K+ channel opener pinacidil had a higher relaxant potency against contraction induced by 30 mM K+ than by 124 mM K+, it still relaxed the latter contraction indicating an additional K+ channel independent mechanism of action of the drug. When K+ depolarization is used as a pharmacological tool, it is essential to maintain osmolarity. Addition of KCI directly to the tissue bath solution, which previously was a commonly applied technique, produced confounding and unwanted effects due to hyperosmolarity per se. 2) Pinacidil and cromakalim relaxed guinea-pig trachea either tone was spontaneous or induced by a range of airway spasmogens (histamine, PGF2 alpha, LTC4/LTD4 or carbachol) of relevance as asthma mediators. The relaxant effectiveness of the drugs was reduced when tone was elicited by carbachol. The airway smooth muscle relaxation produced by pinacidil and cromakalim was selectively blocked by the antidiabetic sulfonylureas glibenclamide, glipizide and glibornuride and also by phentolamine. These drugs are blockers of KATP which therefore indicates that this channel is the target for cromakalim and pinacidil in airway smooth muscle. Additional to the antagonistic action against K+ channel openers the sulfonylurea KATP blockers and phentolamine at higher concentrations relaxed airway smooth muscle by yet unknown mechanisms that seemed unrelated to KATP. 3) Cromakalim and pinacidil inhibited nerve-mediated e-NANC contractile responses in guinea-pig bronchi. Such responses are due to release of SP and related tachykinins from sensory nerve endings. These neuropeptides cause bronchoconstriction and airway inflammation and may possibly play an important role in the pathophysiology of asthma.

In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide.

BACKGROUND: In vitro data suggest that reduced bioconversion of nitroglycerin (NTG) to nitric oxide (NO) contributes to the development of vascular and hemodynamic tolerance to NTG. We examined the in vivo validity of this hypothesis by measuring NTG-derived NO formation by in vivo spin-trapping of NO in vascular tissues from nitrate-tolerant and -nontolerant rats. METHODS AND RESULTS: Five groups (n = 6 to 8 each) of conscious chronically catheterized rats received NTG (0.2 or 1 mg/h IV) for 72 hours (nitrate-tolerant groups). Four other groups received either NTG vehicle (placebo, for 72 hours) or were left untreated (control). Nitrate tolerance was substantiated by a reduced (55% to 85%) hypotensive response to NTG in vivo and a reduced relaxation to NTG in isolated aortic rings. NTG-derived NO formation in aorta, vena cava, heart, and liver was measured as NOFe(DETC)2 and NO-heme complexes formed in vivo during 35 minutes combined with ex vivo cryogenic electron spin resonance spectroscopy. NO formation was significantly (P < .05) increased in all tissues in nitrate-tolerant rats in an NTG dose-dependent manner. Furthermore, the amount of NO formed from a bolus dose of NTG (6.5 mg/kg over 20 minutes) was similar in nitrate-tolerant and -nontolerant rats. CONCLUSIONS: The results suggest that vascular and hemodynamic NTG tolerance occurs despite high and similar rates of NO formation by NTG in tolerant and nontolerant target tissues. This finding is compatible with the assumption that reduced biological activity of NO, rather than reduced bioconversion of NTG to NO, contributes to in vivo development of nitrate tolerance.