Herbert M. Himmel

Sphingosylphosphorylcholine-biological functions and mechanisms of action.

Compared to the lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), little information is available regarding the molecular mechanisms of action, metabolism and physiological significance of the related sphingosylphosphorylcholine (SPC). S1P and LPA have recently been established as agonists at several G-protein-coupled receptors of the EDG family, S1P additionally serves an intracellular second messenger function. Several cellular effects of SPC can be explained by low-affinity binding to and activation of S1P-EDG receptors. However, certain cellular and subcellular actions of SPC are not shared by S1P, suggesting that SPC, which has been identified in normal blood plasma, ascites and various tissues, is a lipid mediator in its own right. This concept was corroborated by the recent discovery of specific high-affinity G-protein-coupled SPC receptors. In this article, our present knowledge on cellular actions and biological functions of SPC will be reviewed.

EFFECTS OF THE NEW CLASS III ANTIARRHYTHMIC DRUG E-4031 ON MYOCARDIAL CONTRACTILITY AND ELECTROPHYSIOLOGICAL PARAMETERS

The effects of the new class III antiarrhythmic agent E-4031 were investigated in different guinea pig cardiac preparations. In left atria, E-4031 (10 8–10-5 M) prolonged the functional refractory period up to 45% and reduced the frequency of spontaneously beating right atria by 32%. In papillary muscles, E-4031 (3 x 10 -8–3 x 10 -7 M) reversibly prolonged the action potential duration (APD70) of fast and slow APs by 65 and 51%, respectively. Vmax resting potential, and AP amplitude (APA) were not altered. In isolated ventricular myocytes, E-4031 reversibly prolonged the APD90 from 275 ms (control) to 1,496 ms (10-6 M). pD2 value 6.5. The current changes that underlie the AP-prolonging effect were also studied in ventricular myocytes: in concentrations up to 10-5 M), E-4031 did not affect the Na+ or Ca2+ inward current but reduced the delayed rectifier (1k) tail current by 76% (10-7 M). Contractility was enhanced by E-4031 in isolated atria by 20% (3 x 10-7 M) and increased cell shortening in ventricular myocytes. Thus, the class III antiarrhythmic action of E-4O3I is due to a selective reduction of outward currents.

Characterization of the effects of the new inotropic agent BDF 9148 in isolated papillary muscles and myocytes of the guinea‐pig heart

1 The cardiotonic agent BDF 9148 (4‐[3′‐(1″‐benzhydryl‐azetidine‐3″‐oxy)‐2′‐hydroxypropoxy]‐1H‐indole‐2‐carbonitrile) is structurally related to DPI 201‐106 (4‐[3′‐(4″‐benzhydryl‐1″‐piperazinyl)‐2′‐hydroxypropoxy]‐1H‐indole‐2‐carbonitrile) which is known to modify cardiac sodium channels. In guinea‐pig papillary muscles, both compounds increase force of contraction with similar concentration‐response curves. Like DPI 201‐106, BDF 9148 prolongs the action potential duration in a tetrodotoxin‐sensitive manner. With high concentrations (> 3 μm), however, the action potential duration shortens again. In order to elucidate the underlying changes in membrane currents, we have investigated the effects of BDF 9148 in isolated ventricular myocytes of the guinea‐pig heart. 2 In isolated cells, a concentration of 1 μm BDF 9148 prolonged the action potential duration and markedly enhanced unloaded cell shortening, indicating that the procedure of cell isolation does not abolish the effect of the drug. 3 Membrane currents were studied with the single electrode voltage clamp technique. With clamp steps from −80 mV to −40 mV, BDF 9148 (1 μm) induced a slowly decaying inward current which was suppressed by tetrodotoxin. Therefore, like DPI 201‐106, BDF 9148 slows the inactivation of the sodium channels. 4 In order to quantify the effects of BDF 9148 and DPI 201‐106 on sodium current inactivation, we have measured the inward current amplitude still present at 100 ms after a depolarizing clamp step from −80 mV to −30 mV. Both drugs increased this current component in a concentration‐dependent manner; however, BDF 9148 had a larger effect in the low concentration range. 5 The calcium current was inhibited by BDF 9148 and DPI 201‐106 in a concentration‐dependent manner; the pD2 values were 5.70 and 5.95, respectively. 6 The two compounds are thought to produce similar positive inotropic effects by imposing a sodium load on the muscle cells via modification of the sodium channels. The differences in action potential duration could be due to different contributions of ionic currents other than sodium or calcium currents and of pump and exchange currents. At present, there is not sufficient data to identify clearly distinct current components responsible for the differences in action potential prolongation.;

Ascorbic Acid–Induced Modulation of Venous Tone in Humans

Ascorbic acid appears to have vasodilatory properties, but the underlying mechanisms are not well understood. The aims of this study were to define the acute effects of locally infused ascorbic acid in human veins and to explore underlying mechanisms by using pharmacological tools in vivo. Ascorbic acid was infused in dorsal hand veins submaximally preconstricted with the &agr;1-adrenoceptor agonist phenylephrine or with prostaglandin F2&agr; in 23 healthy male nonsmokers, and the venodilator response was measured. Ascorbic acid produced dose-dependent dilation with maximum reversal of constriction of 38±4% in phenylephrine-preconstricted veins and of 51±13% in prostaglandin F2&agr;–preconstricted veins. Oral pretreatment with the cyclooxygenase inhibitor acetylsalicylic acid or local coinfusion of ascorbic acid and the nitric oxide synthase inhibitor NG-monomethyl-l-arginine had no effect, but coinfusion of ascorbic acid and methylene blue (to inhibit cGMP generation) abolished venodilation. Coinfusion of ascorbic acid and the nonselective potassium channel blocker quinidine abolished venodilation, whereas the inhibitor of ATP-dependent potassium channels glibenclamide had no effect. In cultured bovine endothelial cells, ascorbic acid did not affect intracellular calcium concentration but blunted the response to ATP or digitonin exposure. Ascorbic acid, in millimolar concentrations, dilates human hand veins, presumably by activation of vascular smooth muscle potassium channels through cGMP. This activation is independent of eNOS-mediated nitric oxide synthesis and cyclooxygenase products and does not involve ATP-dependent potassium channels.

Effects of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264 on cardiac ionic currents of guinea pig and rat ventricular myocytes.

The thiadiazinone enantiomers [+]-EMD 60263 and [-]-EMD 60264 ((+)-5-(1-(alpha-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4-tetrah ydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazine-2 -on) exhibit distinct stereoselectivity for Ca2+-sensitizing action ([+]-enantiomer) and phosphodiesterase inhibition ([-]-enantiomer). However, in isolated guinea pig papillary muscle, both compounds cause an action-potential prolongation that has been related to a nonselective depression of the delayed rectifier potassium current. Because [-]-EMD 60264 did not increase force of contraction despite phosphodiesterase inhibition, we postulated that one or several additional actions may oppose the anticipated positive inotropic effect. Therefore we investigated whether other membrane currents were also affected in voltage-clamped ventricular cardiomyocytes. Both [+]-EMD 60263 and [-]-EMD 60264 reduced sodium current as well as L-type calcium current in guinea pig ventricular myocytes, but steady-state inactivation or conductance curves of I(Na) and I(Ca) were not shifted along the voltage axis. Inward rectifier and transient outward current were studied in rat myocytes, but neither current was affected. We conclude that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca2+-sensitizing effect over its inhibitory actions on Na+ and Ca2+ current, whereas the negative inotropic effect of [-]-EMD 60264 may be caused by inhibition of I(Ca) predominating over PDE inhibition.

Local venous response to N-desethylamiodarone in humans

Amiodarone, a class III antiarrhythmic agent, is a potent vasodilator in vivo. Its main metabolite, N‐desethylamiodarone, contributes to the antiarrhythmic action of amiodarone after long‐term treatment. It is unknown whether N‐desethylamiodarone has acute vascular effects. The aim of this study was to explore the mechanism of action of N‐desethylamiodarone in human hand veins. Methods: The dorsal hand vein compliance technique was applied in 36 healthy male volunteers. In hand veins preconstricted with the α1–adrenergic receptor agonist phenylephrine or prostaglandin F2α, N‐desethylamiodarone and an inhibitor of nitric oxide formation (NG‐monomethyl‐L‐arginine, L‐NMMA) were infused in the presence or absence of a cyclooxygenase inhibitor (acetylsalicylic acid), and the venodilator effect was measured. Furthermore, N‐desethylamiodarone was infused after oral treatment with hydrocortisone or coinfused with α‐tocopherol. Additional experiments were carried out in bovine aortic endothelial cells to explore the effects of N‐desethylamiodarone on the intracellular Ca2+ concentration ([Ca2+]i).

Quantitation of hypoxanthine in plasma from patients with ischemic heart disease: adaption of a high-performance liquid chromatographic method

A high-performance liquid chromatographic method is described for the separation and quantitation of several purine compounds, including hypoxanthine. The isocratic separation of a standard mixture of nine compounds is achieved within 20 min on a reversed-phase Nucleosil 100-5C18 column, with a mobile phase of KH2PO4 (300 mM, pH 4.0)-methanol-acetonitrile-tetrahydrofuran (97.9:1:1:0.1, v/v). Uric acid, guanine, hypoxanthine, uridine, xanthine, allopurinol, inosine, guanosine and 7-methylxanthine were almost completely baseline-separated, with detection limits in the range 0.5-1.2 pmol per injection. The influence of the concentrations of buffer and tetrahydrofuran on the quality of separation are described. The within-day and the day-to-day precision were satisfactory (e.g. coefficients of variation of less than 1.5 and ca. 6.0%, respectively, for peak heights). The recovery of [3H]hypoxanthine added to samples was 86 +/- 1%. Hypoxanthine was quantified in human plasma samples obtained at various times during coronary artery bypass grafting. The hypoxanthine levels measured immediately after release of the aortic cross-clamp were significantly higher than those determined under control conditions (18.8 +/- 7.0 and 3.4 +/- 1.0 microM, respectively).

Selectivity of class-III antiarrhythmic action of clofilium in guinea pig ventricular myocytes

Clofilium is an antiarrhythmic agent with a supposedly predominant class-III action which is related to impairment of K+ channel function. We investigated membrane currents in cardiac myocytes isolated from guinea pig ventricle to evaluate the selectivity of action of clofilium on K+ currents. For measurement of action potentials or membrane currents, the single electrode patch clamp technique was applied in current- or voltage-clamp mode, respectively. Clofilium (30 mu M) irreversibly prolonged the action potential duration in guinea pig myocytes. In contrast, the concomitant reduction in plateau phase was completely reversible. The delayed rectifier K+ current Ikappa, was reduced. The rapidly activating component of Ikappa, which has been defined by its sensitivity to the compound E-4031, was also reduced by clofilium. The inward rectifier was slightly inhibited by the drug. Clofilium reversibly reduced L-type Ca2+ current. Sodium current was inhibited in a use-dependent manner. This effect was not reversible but proceeded after washout of the compound. Therefore, clofilium affects both inward and outward currents in mammalian cardiac myocytes in a similar concentration range. The effects on multiple membrane currents may contribute to the antiarrhythmic action of the drug.

Stereoselectivity of actions of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264.

The thiadiazinone derivative [+]-EMD 60263 ((+)-5-(1-(α-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4- tetrahydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4 -thiadiazine-2-on) is a Ca2+-sensitizing agent with only minor phosphodiesterase inhibitory activity. Our aim was to characterize the inotropic and electrophysiological effects of [+]-EMD 60263 and its enantiomer [-]-EMD 60264 in several cardiac muscle preparations. The Ca2+-sensitizing activity resided in the [+]-enantiomer only. [+]-EMD 60263 (3 µM) shifted the EC50 of Ca2+ for contractile activation of skinned fibers of pig heart from 2.41 µM to 0.73 µM, whereas [-]-EMD 60264 (30 µM) was ineffective. In Langendorff-perfused guinea pig hearts, [+]-EMD 60263 and [-]-EMD 60264 induced concentration-dependent positive and negative inotropic effects, respectively; both enantiomers reduced spontaneous heart rate but did not influence perfusion pressure. The maximum increase in force of human atrial trabeculae was 35 % of pre-drug control with [+]-EMD 60263 in comparison to 113 % with forskolin. In guinea-pig papillary muscles, [+]-EMD 60263 and [-]-EMD 60264 had opposite inotropic responses, however, both agents similarly prolonged action potential duration. Both enantiomers concentration-dependently blocked the rapidly activating component IKr of the delayed rectifier in guinea-pig myocytes. The block saturated at potentials positive to +30 mV, closely resembling the effects of the antiarrhythmic agent E-4031 which had been originally used to define IKr. It is concluded, that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca2+-sensitizing effect. The accompanying prolongation in action potential duration is caused by block of the IKr component of the delayed rectifier. While the inotropic effects are stereoselective, most of the electrophysiological actions are clearly independent of sterical configuration. The combination of Ca2+-sensitizing with class-III antiarrhythmic action may provide an interesting pharmacological profile of potential therapeutic use.

Subsensitivity of P2X but not vanilloid 1 receptors in dorsal root ganglia of rats caused by cyclophosphamide cystitis

The application of cyclophosphamide to rats was used to induce interstitial cystitis. Behavioural studies indicated a strong pain reaction that developed within 2 h and levelled off thereafter causing a constant pain during the following 18 h. Neurons prepared from L6/S1 dorsal root ganglia innervating the urinary bladder responded to the application of capsaicin or alpha,beta-methylene ATP (alpha,beta-meATP) with an increase of intracellular Ca2+ ([Ca2+]i). The [Ca2+]i responses to capsaicin were identical in the dorsal root ganglion cells of cyclophosphamide- and saline-treated rats, whereas alpha,beta-meATP induced less increase in [Ca2+]i in the cyclophosphamide-treated animals than in their saline-treated counterparts. Hence, alpha,beta-meATP-sensitive P2X3 and/or P2X2/3 receptors of L6/S1 dorsal root ganglion neurons were functionally downregulated during subacute pain caused by experimental cystitis. In contrast, capsaicin-sensitive vanilloid 1 receptors did not react to the same procedure. Thoracal dorsal root ganglia, not innervating the urinary bladder, were also unaltered in their responsiveness to alpha,beta-meATP by cyclophosphamide treatment.