Adenosine induces prolonged anti-beta-adrenergic effects in guinea-pig papillary muscle

A sustained anti-beta-adrenergic effect of adenosine has been reported. This study was initiated to investigate this topic and especially elucidate the role of protein kinase C (PKC). Contractile force amplitude and action potential duration at 90% repolarization (APD90) were measured in guinea-pig papillary muscles before and after 5 min challenge with 5 nm isoproterenol. Protocols contained 30 min exposure to the test agents adenosine 33 microm (ado), adenosine + PKC-inhibitor bisindolylmaleimide 20 nM (ado + BIM), PKC-activator 1,2-dioctanoyl-sn-glycerol 10 microm (DOG) and alpha-agonist phenylephrine 5 microm (phe). Isoproterenol was given at the end of test exposure and after 15 min washout. Results are mean +/- SEM of percentage-change, P < or = 0.05 considered significant and labelled *. The first isoproterenol challenge significantly increased contractile force (27 +/- 7%*) in the control group. Responses in the test groups were 2 +/- 4 (ado), 1 +/- 5 (ado + BIM), 14 +/- 4* (DOG), 0 +/- 2% (phe). After washout of adenosine, DOG and phenylephrine, isoproterenol induced 3 +/- 8 (ado), 23 +/- 5* (ado + BIM), 13 +/- 5* (DOG), 15 +/- 7% (phe) increase in test groups compared with 22 +/- 5%* increase in contractile force in the control group. After 45 min washout of adenosine the inotropic response was still significantly reduced compared with control (29 +/- 4 vs. 79 +/- 8%*). Isoproterenol stimulation shortened APD90 in controls at both time points (5 +/- 1%* and 4 +/- 1%*), with no significant shortening in test groups. Adenosine induces sustained anti-beta-adrenergic effects on contractile force as well as APD90. A role for PKC in signal transduction is supported with respect to contractile force.     

Positive inotropic and sustained anti‐β‐adrenergic effect of diadenosine pentaphosphate in human and guinea pig hearts. Role of dinucleotide receptors and adenosine receptors

Aim: Diadenosine polyphosphates are present intracellularly and in extracellular fluid due to release from secretory vesicles in platelets, chromaffin cells and other cells. This study investigates effects of diadenosine pentaphosphate (AP₅A) on heart muscle function. Methods: Contractile force amplitude and action potential duration at 90% repolarization (APD₉₀) were measured after challenge with AP₅A 50 μm or isoproterenol 50–70 nM in guinea pig papillary muscles. Isoproterenol was given immediately after AP₅A‐exposure or after 45 min washout. AP₅A was combined with antagonists to the purinergic P2 receptor (suramin 100 μm ), the dinucleotide receptor [diinosine pentaphosphate 30 μm (IP₅I)] or adenosine receptors [8‐(P‐sulfophenyl) theophylline 50 μm (8‐SPT)]. Results: Results are %‐change (mean ± SEM) from value before exposure. AP₅A increased contractile force by 22 ± 3%* (*P < 0.05), and IP₅I abolished this. AP₅A prolonged APD₉₀ by 7 ± 2%*. AP₅A significantly reduced response to isoproterenol acutely from 31 ± 4* (controls) to 9 ± 4% and after 45 min washout from 61 ± 14* (controls) to 16 ± 5%. 8‐SPT abolished the sustained effect. Increase in contractile force by AP₅A was confirmed in human atria trabecula preparations. Conclusion: AP₅A increased contractile force and prolonged APD₉₀. Contractile force increased by stimulation of the dinucleotide receptor in guinea pig myocardium. The sustained anti‐β‐adrenergic effect of AP₅A was due to adenosine receptor stimulation.     

Positive inotropic and sustained anti-beta-adrenergic effect of diadenosine pentaphosphate in human and guinea pig hearts. Role of dinucleotide receptors and adenosine receptors

AIM: Diadenosine polyphosphates are present intracellularly and in extracellular fluid due to release from secretory vesicles in platelets, chromaffin cells and other cells. This study investigates effects of diadenosine pentaphosphate (AP5A) on heart muscle function. METHODS: Contractile force amplitude and action potential duration at 90% repolarization (APD90) were measured after challenge with AP5A 50 microm or isoproterenol 50-70 nM in guinea pig papillary muscles. Isoproterenol was given immediately after AP5A-exposure or after 45 min washout. AP5A was combined with antagonists to the purinergic P2 receptor (suramin 100 microm), the dinucleotide receptor [diinosine pentaphosphate 30 microm (IP5I)] or adenosine receptors [8-(P-sulfophenyl) theophylline 50 microm (8-SPT)]. RESULTS: Results are %-change (mean +/- SEM) from value before exposure. AP5A increased contractile force by 22 +/- 3%* (*P <0.05), and IP5I abolished this. AP5A prolonged APD90 by 7 +/- 2%*. AP5A significantly reduced response to isoproterenol acutely from 31 +/- 4* (controls) to 9 +/- 4% and after 45 min washout from 61 +/- 14* (controls) to 16 +/- 5%. 8-SPT abolished the sustained effect. Increase in contractile force by AP5A was confirmed in human atria trabecula preparations. CONCLUSION: AP5A increased contractile force and prolonged APD90. Contractile force increased by stimulation of the dinucleotide receptor in guinea pig myocardium. The sustained anti-beta-adrenergic effect of AP5A was due to adenosine receptor stimulation.     

β ‐Adrenoceptor agonist sensitizes the dopamine‐1 receptor in renal tubular cells

The renal effects of dopamine are mainly mediated via the dopamine‐1 receptor (D1 receptor). This receptor is recruited from intracellular compartments to the plasma membrane by dopamine and atrial natriuretic peptide (ANP), via adenylyl cyclase activation. We have studied whether isoproterenol, a β‐adrenoceptor (β‐AR) agonist that may interact with dopamine in the regulation of rat renal Na+, K⁺‐adenosine triphosphatase (ATPase) activity, can recruit D1 receptors to the plasma membrane. The spatial regulation of D1 receptors was examined using confocal microscopy techniques in LLCPK cells and the functional interaction between dopamine and isoproterenol was examined by studying their effects on Na+, K⁺‐ATPase activity in microdissected single proximal tubular segments from rat. Isoproterenol was found to translocate the D1 receptors from the interior of the cell towards the plasma membrane. The recruitment of dopamine 1 receptors was found to be cyclic adenosine phosphate (cAMP) dependent, while protein kinase C (PKC) activation was not involved. The functional studies on Na+, K⁺‐ATPase activity showed that the effect of isoproterenol was abolished by a D1‐like receptor antagonist (SCH 23390), and mediated via protein kinase A (PKA) and PKC dependent pathways. The results provide an explanation for the interaction between G protein‐coupled receptors. The effects of isoproterenol on Na+, K⁺‐ATPase activity can be explained by a heterologous recruitment of D1 receptors to the plasma membrane.     

Dopamine‐induced inhibition of Na+‐K+‐ATPase activity requires integrity of actin cytoskeleton in opossum kidney cells

The present study evaluated the importance of the association between Na⁺‐K⁺‐ATPase and the actin cytoskeleton on dopamine‐induced inhibition of Na⁺‐K⁺‐ATPase activity. The approach used measures the transepithelial transport of Na⁺ in monolayers of opossum kidney (OK) cells, when the Na⁺ delivered to Na⁺‐K⁺‐ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 μg mL^–1) induced increase in short‐circuit current (I_sc) was prevented by ouabain (100 μM ) or removal of apical Na⁺. Dopamine (1 μM ) applied from the apical side significantly decreased (29 ± 5% reduction) the amphotericin B‐induced increase in I_sc, this being prevented by the D₁‐like receptor antagonist SKF 83566 (1 μM ) and the protein kinase C (PKC) inhibitor chelerythrine (1 μM ). Exposure of OK cells to cytochalasin B (1 μM ) or cytochalasin D (1 μM ), inhibitors of actin polymerization, from both cell sides reduced by 31 ± 4% and 36 ± 3% the amphotericin B‐induced increase in I_sc and abolished the inhibitory effect of apical dopamine (1 μM ), but not that of the PKC activator phorbol‐12,13‐dibutyrate (PDBu; 100 nM ). Colchicine (1 μM ) failed to alter the inhibitory effects of dopamine. The relationship between Na⁺‐K⁺‐ATPase and the concentration of extracellular Na⁺ showed a Michaelis–Menten constant (K_m) of 44.1 ± 13.7 mM and a V_max of 49.6 ± 4.8 μA cm^–2 in control monolayers. In the presence of apical dopamine (1 μM ) or cytochalasin B (1 μM ) V_max values were significantly (P < 0.05) reduced without changes in K_m values. These results are the first, obtained in live cells, showing that the PKC‐dependent inhibition of Na⁺‐K⁺‐ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na⁺‐K⁺‐ATPase.     

Pharmacological separation of early afterdepolarizations from arrhythmogenic substrate in DeltaKPQ Scn5a murine hearts modelling human long QT 3 syndrome

Aim: To perform an empirical, pharmacological, separation of early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in a genetically modified mouse heart modelling human long QT syndrome (LQT) 3. Methods: Left ventricular endocardial and epicardial monophasic action potentials and arrhythmogenic tendency were compared in isolated wild type (WT) and Scn5a+/Δ hearts perfused with 0.1 and 1 μm propranolol and paced from the right ventricular epicardium. Results: All spontaneously beating bradycardic Scn5a+/Δ hearts displayed EADs, triggered beats and ventricular tachycardia (VT; n = 7), events never seen in WT hearts (n = 5). Perfusion with 0.1 and 1 μm propranolol suppressed all EADs, triggered beats and episodes of VT. In contrast, triggering of VT persisted following programmed electrical stimulation in 6 of 12 (50%), one of eight (12.5%), but six of eight (75%) Scn5a+/Δ hearts perfused with 0, 0.1 and 1 μm propranolol respectively in parallel with corresponding alterations in repolarization gradients, reflected in action potential duration (ΔAPD₉₀) values. Thus 0.1 μm propranolol reduced epicardial but not endocardial APD₉₀ from 54.7 ± 1.6 to 44.0 ± 2.0 ms, restoring ΔAPD₉₀ from ?3.8 ± 1.6 to 3.5 ± 2.5 ms (all n = 5), close to WT values. However, 1 μm propranolol increased epicardial APD₉₀ to 72.5 ± 1.2 ms and decreased endocardial APD₉₀ from 50.9 ± 1.0 to 24.5 ± 0.3 ms, increasing ΔAPD₉₀ to ?48.0 ± 1.2 ms. Conclusion: These findings empirically implicate EADs in potentially initiating spontaneous arrhythmogenic phenomena and transmural repolarization gradients in the re‐entrant substrate that would sustain such activity when provoked by extrasystolic activity in murine hearts modelling human LQT3 syndrome.     

Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

Aims: Adenosine causes vasoconstriction of afferent arterioles of the mouse kidney through activation of adenosine A₁ receptors and Gi‐mediated stimulation of phospholipase C. In the present study, we further explored the signalling pathways by which adenosine causes arteriolar vasoconstriction. Methods and results: Adenosine (10^?7 m ) significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura‐2 fluorescence. Pre‐treatment with thapsigargin (2 μm ) blocked the vasoconstrictor action of adenosine (10^?7 m ) indicating that release of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP₃, is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 μm ) blocked the adenosine‐induced constriction whereas the protein kinase C inhibitor calphostin C had no effect. The calcium‐activated chloride channel inhibitor IAA‐94 (30 μm ) inhibited the adenosine‐mediated constriction. Patch clamp experiments showed that adenosine treatment induced a depolarizing current in preglomerular smooth muscle cells which was abolished by IAA‐94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 μm nifedipine (control 8.3 ± 0.2 μm, ado 3.6 ± 0.6 μm, ado + nifedipine 6.8 ± 0.2 μm) suggesting involvement of voltage‐dependent calcium channels. Conclusion: We conclude that adenosine mediates vasoconstriction of afferent arterioles through an increase in intracellular calcium concentration resulting from release of calcium from the SR followed by activation of Ca²⁺‐activated chloride channels leading to depolarization and influx of calcium through voltage‐dependent calcium channels.     

Characterization of trabecular bone density with ultra‐short echo‐time MRI at 1.5, 3.0 and 7.0 T – comparison with micro‐computed tomography

The goal of this study was to test the potential of ultra‐short echo‐time (UTE) MRI at 1.5, 3.0 and 7.0 T for depiction of trabecular bone structure (of the wrist bones), to evaluate whether T₂* relaxation times of bone water and parametric maps of T₂* of trabecular bone could be obtained at all three field strengths, and to compare the T₂* relaxation times with structural parameters obtained from micro‐computed tomography (micro‐CT) as a reference standard. Ex vivo carpal bones of six wrists were excised en bloc and underwent MRI at 1.5, 3.0 and 7.0 T in a whole‐body MR imager using the head coil. A three‐dimensional radial fat‐suppressed UTE sequence was applied with subsequent acquisitions, with six different echo times T_E of 150, 300, 600, 1200, 3500 and 7000 µs. The T₂* relaxation time and pixel‐wise computed T₂* parametric maps were compared with a micro‐computed‐tomography reference standard providing trabecular bone structural parameters including porosity (defined as the bone‐free fraction within a region of interest), trabecular thickness, trabecular separation, trabecular number and fractal dimension (D_k). T₂* relaxation curves and parametric maps could be computed from datasets acquired at all field strengths. Mean T₂* relaxation times of trabecular bone were 4580 ± 1040 µs at 1.5 T, 2420 ± 560 µs at 3.0 T and 1220 ± 300 µs at 7.0 T, when averaged over all carpal bones. A positive correlation of T₂* with trabecular bone porosity and trabecular separation, and a negative correlation of T₂* relaxation time with trabecular thickness, trabecular number and fractal dimension, was detected (p < 0.01 for all field strengths and micro‐CT parameters). We conclude that UTE MRI may be useful to characterize the structure of trabecular bone, comparable to micro‐CT. Copyright © 2014 John Wiley & Sons, Ltd.     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998     

Role of adenosine in exercise‐induced human skeletal muscle vasodilatation

The role of adenosine in exercise‐induced human skeletal muscle vasodilatation remains unknown. We therefore evaluated the effect of theophylline‐induced adenosine receptor blockade in six subjects and the vasodilator potency of adenosine infused in the femoral artery of seven subjects. During one‐legged, knee‐extensor exercise at ～48% of peak power output, intravenous (i.v.) theophylline decreased (P < 0.003) femoral artery blood flow (F_aBF) by ～20%, i.e. from 3.6 ± 0.5 to 2.9 ± 0.5 L min^?1, and leg vascular conductance (VC) from 33.4 ± 9.1 to 27.7 ± 8.5 mL min^?1 mmHg^?1, whereas heart rate (HR), mean arterial pressure (MAP), leg oxygen uptake and lactate release remained unaltered (P = n.s.). Bolus injections of adenosine (2.5 mg) at rest rapidly increased (P < 0.05) F_aBF from 0.3 ± 0.03 L min^?1 to a 15‐fold peak elevation (P < 0.05) at 4.1 ± 0.5 L min^?1. Continuous infusion of adenosine at rest and during one‐legged exercise at ～62% of peak power output increased (P < 0.05) F_aBF dose‐dependently to level off (P = ns) at 8.3 ± 1.0 and 8.2 ± 1.4 L min^?1, respectively. One‐legged exercise alone increased (P < 0.05) F_aBF to 4.7 ± 1.7 L min^?1. Leg oxygen uptake was unaltered (P = n.s.) with adenosine infusion during both rest and exercise. The present findings demonstrate that endogenous adenosine controls at least ～20% of the hyperaemic response to submaximal exercise in skeletal muscle of humans. The results also clearly show that arterial infusion of exogenous adenosine has the potential to evoke a vasodilator response that mimics the increase in blood flow observed in response to exercise.     

Separation of early afterdepolarizations from arrhythmogenic substrate in the isolated perfused hypokalaemic murine heart through modifiers of calcium homeostasis

Aims: We resolved roles for early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in Langendorff‐perfused hypokalaemic murine hearts paced from the right ventricular epicardium. Methods: Left ventricular epicardial and endocardial monophasic action potentials (MAPs) and arrhythmogenic tendency were compared in the presence and absence of the L‐type Ca²⁺ channel blocker nifedipine (10 nm –1 μm ) and the calmodulin kinase type II inhibitor KN‐93 (2 μm ). Results: All the hypokalaemic hearts studied showed prolonged epicardial and endocardial MAPs, decreased epicardial‐endocardial APD₉₀ difference, EADs, triggered beats and ventricular tachycardia (VT) (n = 6). In all spontaneously beating hearts, 100 (but not 10) nm nifedipine reduced both the incidence of EADs and triggered beats from 66.9 ± 15.7% to 28.3 ± 8.7% and episodes of VT from 10.8 ± 6.3% to 1.2 ± 0.7% of MAPs (n = 6 hearts, P < 0.05); 1 μm nifedipine abolished all these phenomena (n = 6). In contrast programmed electrical stimulation (PES) still triggered VT in six of six hearts with 0, 10 and 100 nm but not 1 μm nifedipine. 1 μm nifedipine selectively reduced epicardial (from 66.1 ± 3.4 to 46.2 ± 2.5 ms) but not endocardial APD₉₀, thereby restoring ΔAPD₉₀ from ?5.9 ± 2.5 to 15.5 ± 3.2 ms, close to normokalaemic values. KN‐93 similarly reduced EADs, triggered beats and VT in spontaneously beating hearts to 29.6 ± 8.9% and 1.7 ± 1.1% respectively (n = 6) yet permitted PES‐induced VT (n = 6), in the presence of a persistently negative ΔAPD₉₀. Conclusions: These findings empirically implicate both EADs and triggered beats alongside arrhythmogenic substrate of ΔAPD₉₀ in VT pathogenesis at the whole heart level.     

Epidermal growth factor and insulin short‐term increase hPepT1‐mediated glycylsarcosine uptake in Caco‐2 cells

Aims: Little is known about the physiological regulation of the human intestinal di/tri‐peptide transporter, hPepT1. In the present study we evaluated the effects of epidermal growth factor (EGF) and insulin on hPepT1‐mediated dipeptide uptake in the intestinal cell line Caco‐2. Methods: Caco‐2 cells were grown on filters for 23–27 days. Apical dipeptide uptake was measured using [¹⁴C]glycylsarcosine([¹⁴C]Gly‐Sar). HPepT1 mRNA levels were investigated using RT‐PCR, cytosolic pH was determined using the pH‐sensitive fluorescent probe BCECF. Results: Basolateral application of EGF increased [¹⁴C]Gly‐Sar uptake with an ED₅₀ value of 0.77 ± 0.25 ng mL^?1 (n = 3?6) and a maximal stimulation of 33 ± 2% (n = 3?6). Insulin stimulated [¹⁴C]Gly‐Sar uptake with an ED₅₀ value of 3.5 ± 2.0 ng mL^?1 (n = 3?6) and a maximal stimulation of approximately 18% (n = 3?6). Gly‐Sar uptake followed simple Michaelis‐Menten kinetics. K_m in control cells was 0.98 ± 0.11 mM (n = 8) and V_max was 1.86 ± 0.07 nmol cm^?2 min^?1 (n = 8). In monolayers treated with 200 ng mL^?1 of EGF, K_m was 1.11 ± 0.05 mM (n = 5) and V_max was 2.79 ± 0.05 nmol cm^?2 min^?1 (n = 5). In monolayers treated with 50 ng mL^?1 insulin, K_m was 1.03 ± 0.08 mM and V_max was 2.19 ± 0.06 nmol cm^?2 min^?1 (n = 5). Kinetic data thus indicates an increase in the number of active transporters, following stimulation. The incrased Gly‐Sar uptake was not accompanied by changes in hPepT1 mRNA, nor by measurable changes in cytosolic pH. Conclusions: Short‐term stimulation with EGF and insulin caused an increase in hPepT1‐mediated uptake of Gly‐Sar in Caco‐2 cell monolayers, which could not be accounted for by changes in hPepT1 mRNA or proton‐motive driving force.     

Syndiospecific Living Block Copolymerization of Styrenic Monomers Containing Functional Groups,and Preparation of Syndiotactic Poly{(4‐hydroxystyrene)‐block‐[(4‐methylstyrene)‐co‐(4‐hydroxystyrene)]}

At –25°C, the sequential block copolymerizations of 4‐(tert‐butyldimethylsilyloxy)styrene (TBDMSS) and 4‐methylstyrene (4MS) were investigated by using a syndiospecific living polymerization catalyst system composed of (trimethyl)pentamethylcyclopentadienyltitanium (Cp*TiMe₃), trioctylaluminum (AlOct₃) and tris(pentafluorophenyl)borane (B(C₆F₅)₃). The number‐average molecular weight (M̄_n) of the poly(TBDMSS)s increased linearly with increasing the polymer yield up to almost 100 wt.‐% consumption of TBDMSS used as 1^st monomer. The M̄_n value of the polymer after the second monomer (4MS) addition continued to increase proportionally to the polymer yield. The molecular weight distributions (MWDs) of the polymers remained constant at around 1.05–1.18 over the entire course of block copolymerization. It was concluded that the block copolymerizations of TBDMSS and 4MS with the Cp*TiMe₃ /B(C₆F₅)₃ /AlOct₃ catalytic system proceeded with a high block efficiency. The ¹³C NMR analysis clarified that the block copolymers obtained in this work had highly syndiotactic structure. By the deprotection reaction of silyl group with conc. hydrochloric acid (HCl), syndiotactic poly{(4‐hydroxystyrene)‐block‐[(4‐methylstyrene)‐co‐(4‐hydroxystyrene)]} (poly[HOST‐b‐(4MS‐co‐HOST)]) was successfully prepared.     

Effects of potassium channel openers in the isolated perfused hypokalaemic murine heart

Aim: We explored the anti‐arrhythmic efficacy of K⁺ channel activation in the hypokalaemic murine heart using NS1643 and nicorandil, compounds which augment I_Kr and I_KATP respectively. Methods: Left ventricular epicardial and endocardial monophasic action potentials were compared in normokalaemic and hypokalaemic preparations in the absence and presence of NS1643 (30 μm ) and nicorandil (20 μm ). Results: Spontaneously beating hypokalaemic hearts (3 mm K⁺) all elicited early afterdepolarizations (EADs) and episodes of ventricular tachycardia (VT). Perfusion with NS1643 and nicorandil suppressed EADs and VT in 7 of 13 and five of six hypokalaemic hearts. Provoked arrhythmia studies using programmed electrical stimulation induced VT in all hypokalaemic hearts, but failed to do so in 7 of 13 and five of six hearts perfused with NS1643 and nicorandil respectively. These anti‐arrhythmic effects were accompanied by reductions in action potential duration at 90% repolarization (APD₉₀) and changes in the transmural gradient of repolarization, reflected in ΔAPD₉₀. NS1643 and nicorandil reduced epicardial APD₉₀ from 68.3 ± 1.1 to 56.5 ± 4.1 and 51.5 ± 1.5 ms, respectively, but preserved endocardial APD₉₀ in hypokalaemic hearts. NS1643 and nicorandil thus restored ΔAPD₉₀ from ?9.6 ± 4.3 ms under baseline hypokalaemic conditions to 3.9 ± 4.1 and 9.9 ± 2.1 ms, respectively, close to normokalaemic values. Conclusion: These findings demonstrate, for the first time, the anti‐arrhythmic efficacy of K⁺ channel activation in the setting of hypokalaemia. NS1643 and nicorandil are anti‐arrhythmic through the suppression of EADs, reductions in APD₉₀ and restorations of ΔAPD₉₀.     

Nitric oxide modulates acetylcholine‐induced vasodilatation in the hepatic arterial vasculature of the dual‐perfused rat liver

The role of nitric oxide in the modulation of hepatic arterial vascular reactivity was investigated in an isolated dual‐perfused rat liver preparation. Twelve male Wistar rats (200–250 g) were anaesthetized with sodium pentobarbitone (60 mg kg^–1 i.p.). The livers were then excised and perfused in vitro through hepatic arterial and portal venous cannulae at constant flow rates. Concentration‐dependent dose–response curves to acetylcholine (10^–8–10^–5 M ), sodium nitroprusside (10^–6–5 × 10^–4 M ), and adenosine triphosphate (ATP) (10^–8–10^–5 M ) in the hepatic artery were constructed after the tone was raised by addition of methoxamine (3 μM L^–1). Acetylcholine‐induced vasodilatation in the hepatic artery was significantly attenuated with inhibition of nitric oxide synthase by using N^G‐nitro‐L ‐arginine methyl ester (30 μM ), E_max=51.7 ± 2.8 vs. 32.5 ± 3.1 mmHg, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. ATP‐induced hepatic arterial vasoconstriction which was significantly enhanced with L ‐NAME, E_max=94.0 ± 9.3 vs. 127.0 ± 8.0 mmHg, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. Sodium nitroprusside‐induced hepatic arterial vasodilatation remained unchanged with N^G‐nitro‐L ‐arginine methyl ester, E_max=57.0 ± 3.4 vs. 57.0 ± 4.1, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. The data from the present study suggest that acetylcholine‐induced vasodilatation in the intrahepatic arterial vasculature of the rat liver is at least, in part, mediated by the release of nitric oxide. In addition, ATP‐induced hepatic arterial vasoconstriction is also modulated by the release of nitric oxide (*P < 0.05, Student’s paired t‐test).     

HLA‐A, ‐B and ‐DRB1 polymorphism in Koreans defined by sequence‐based typing of 4128 cord blood units

Human leucocyte antigen (HLA) alleles and haplotypes differ significantly among different ethnic groups, and high‐resolution typing methods allow for the detection of a wider spectrum of HLA variations. In this study, HLA‐A, ‐B and ‐DRB1 genotypes were analysed in 4128 cord blood units obtained from Korean women using the sequence‐based typing method. A total of 44 HLA‐A, 67 HLA‐B and 48 HLA‐DRB1 most probable alleles were identified. Of these, high‐frequency alleles found at a frequency of ≥5% were 6 HLA‐A (A*02:01, A*02:06, A*11:01, A*24:02, A*31:01, A*33:03), 5 HLA‐B (B*15:01, B*44:03, B*51:01, B*54:01, B*58:01) and 7 HLA‐DRB1 (DRB1*01:01, DRB1*04:05, DRB1*07:01, DRB1*08:03, DRB1*09:01, DRB1*13:02, DRB1*15:01) alleles. At each locus, A*02, B*15 and DRB1*04 generic groups were most diverse at allelic level, consisting of 8, 11 and 10 different alleles, respectively. Two‐ and three‐locus haplotypes estimated by the maximum likelihood method revealed 73 A‐B, 74 B‐DRB1 and 42 A‐B‐DRB1 haplotypes with frequencies of ≥0.3%. A total of 193 A‐B‐DRB1 haplotypes found at a frequency of ≥0.1% were presented, and the six most common haplotypes were A*33:03‐B*44:03‐DRB1*13:02 (4.6%), A*33:03‐B*58:01‐DRB1*13:02 (3.0%), A*24:02‐B*07:02‐DRB1*01:01 (2.7%), A*33:03‐B*44:03‐DRB1*07:01 (2.5%), A*30:01‐B*13:02‐DRB1*07:01 (2.2%) and A*24:02‐B*52:01‐DRB1*15:02 (2.1%). Compared with previous smaller scale studies, this study further delineated the allelic and haplotypic diversity in Koreans including low‐frequency alleles and haplotypes. Information obtained in this study will be useful for the search for unrelated bone marrow donors and for anthropologic and disease association studies.     

Muscle performance following fatigue induced by isotonic and quasi‐isometric contractions of rat extensor digitorum longus and soleus muscles in vitro

Aim: To study the effect of contraction mode on fatigue development. Methods: Muscle fatigue was induced by isotonic and quasi‐isometric contractions in rat soleus (SOL) and extensor digitorum longus (EDL) muscles, using identical stimulation protocol (60 Hz, 400 ms s^?1) for 100 s in SOL and 60 s in EDL. Fatigue was quantified as the decline in peak values of shortening, shortening velocity, relaxation and work during the isotonic contractions, and, correspondingly, of force, rate of force development, relaxation and work during the quasi‐isometric contractions. Maximal test contractions (60 Hz, 1.5 s) performed before and after fatigue were analysed for decline in force development (F_max), rate of force development (dF/dt_max) and relaxation (?dF/dt_max). Results: F _max declined to significantly lower values after isotonic than after quasi‐isometric fatiguing contractions (fatigued in percentage of unfatigued): 58.5 ± 6.4% vs. 64.4 ± 7.0% in SOL, and 30.4 ± 4.1% vs. 33.3 ± 3.6% in EDL, respectively. The same pattern was seen for dF/dt_max which decreased to: 46.3 ± 9.9% vs. 52.3 ± 8.5% in SOL, and 19.1 ± 4.3% vs. 22.3 ± 3.2% in EDL after isotonic and quasi‐isometric contractions, respectively. Similarly, when comparing fatigue development during the two contraction modes, the respective fatigue variables decreased more rapidly and to lower levels during isotonic vs. quasi‐isometric contractions. During maximal test contractions, the dynamic fatigue variables (±dF/dt_max) declined to significantly lower levels than F_max. Conclusions: Fatigue development was significantly larger during isotonic vs. quasi‐isometric contractions. The use of force as the only experimental fatigue variable may underestimate the functional impairment of fatigued muscle, neglecting the fatigue effect on time and length dimensions.     

Influence of β‐Cyclodextrin on the Free‐Radical Copolymerization of N‐(4‐Methylphenyl)maleimide with N‐Vinylpyrrolidone in Water

Randomly methylated β‐cyclodextrin (me‐β‐CD) is used to include the hydrophobic monomer N‐(4‐methylphenyl)maleimide (MPM) ( 1 ) yielding the corresponding water‐soluble host‐guest structure 1a . Free‐radical copolymerization of 1a with N‐vinylpyrrolidone (NVP) ( 2 ) is performed and the reactivity ratios r₁ and r₂ are determined: 0.24 ± 0.03 (r₂) and 1.10 ± 0.05 (r_1a). This indicates a preferred incorporation of complexed maleimide into the copolymer chain. In contrast to that, the copolymerization of the uncomplexed monomers 1 and 2 is carried out using organic solvent (DMF/H₂O) showing reactivity ratios corresponding to nearly alternating copolymerization (r₂ = 0.09 ± 0.02; r₁ = 0.34 ± 0.03).

     

Endotoxin‐induced shock in the pig – limited effects of low and high concentrations of inhaled nitric oxide

Aim: This study was carried out to study the prophylactic effects of inhalation of nitric oxide (NO) before and during the induction of endotoxic shock. Methods: Eighteen anaesthetized pigs received an infusion of 10–20 μg kg^?1 endotoxin during 2 h after pre‐treatment with the cortisol‐synthesis inhibitor metyrapone. Three groups were tested (n = 6 each) and received 0, 0.2 or 20 ppm inhaled NO from 30 min before start of endotoxin infusion until 4 h after start of endotoxin. Both 0.2 and 20 ppm NO were able to improve blood gas values. Results: Area above curve values of arterial P₂/FiO₂ from 0 to 4 h were 0.83 ± 0.09 kPa h (control), 0.78 ± 0.22 (0.2 ppm NO, non‐significant) and 0.31 ± 0.06 (20 ppm NO, P < 0.01, Mann–Whitney U‐test, compared to control). Area under curve values of PCO₂ from 0 to 4 h were 3.96 ± 0.66 kPa h (control), 1.20 ± 0.46 (0.2 ppm NO, P < 0.05, Mann–Whitney U‐test, compared to control) and 2.78 ± 1.06 (20 ppm NO group, non‐significant). The increase in pulmonary arterial pressure (PAP) was partly prevented by 20 ppm NO, but not by 0.2 ppm NO at 4 h. Inhaled NO did not affect the levels of BAL fluid total protein, tumour necrosis factor‐α, interleukin‐8 and neutrophil counts. Conclusions: The addition of a high (20 ppm), but not a low (0.2 ppm), concentration of NO to the inhaled air during endotoxin shock improves arterial oxygen tension and reduces pulmonary artery pressure. Neither dose affects lung mechanics or inflammatory indices, in spite of being given prophylactically.     

Modulation of vascular contractile responses to α1-and α2-adrenergic and neuropeptide Y receptor stimulation in rats with ischaemic heart failure

In order to evaluate adaptational changes in vascular function in congestive heart failure (CHF), we studied the contractile responses of isolated arterial and venous blood vessels from rats suffering from CHF induced by coronary artery ligature, resulting in a myocardial infarction. The contractile responses of the basilar, femoral and renal arteries and of the iliac vein were examined in relation to adrenergic and neuropeptide Y (NPY) receptor function by the action of the α₁ agonist phenylephrine, the α₂ agonist clonidine and NPY. The contractile force was measured (in mN) and in% of K⁺-induced contraction as well as pD₂ to each agonist. When stimulated by a 60 mM K⁺-buffer solution, the femoral and renal arteries from CHF rats responded with a stronger contraction (E_max; 9.4 ± 0.6 and 9.8 ± 0.6mN) than the corresponding Sham vessels (E_max; 6.2 ± 0.7 and 5.6 ± 0.4 mN respectively, P < 0.001). On the contrary, the iliac vein of CHF responded less to K⁺ than the Sham iliac vein (E_mas 2.5 ± 0.2 and 3.7 ± 0.5 mN, P < 0.01). The CHF iliac vein responded with a weaker contraction when stimulated with phenylephrine (E_max 1.9 ± 0.4 mN) and showed a lower sensitivity (pD₂ 5.6 ± 0.1) than the corresponding sham vessel (E_max 5.7 ± 2.3mN and pD₂ 6.3 ± 0.5, P < 0.05). The CHF renal artery was less sensitive to clonidine (pD₂ 6.4 ± 0.6) than the Sham renal artery (pD₂ 7.2 ± 0.1, P < 0.05). The results indicate differences between CHF and Sham vessel segments according to both contractile capacity induced by K⁺-depolarization and to agonist induced contractile capacity and sensitivity. The differences are not of general nature but vary according to the vascular bed examined.