Desuo Wang

Ionic basis of the electrophysiological actions of adenosine on cardiomyocytes.

The purpose of this review is to examine the role of the extracellular A1‐adenosine (Ado) receptor in modulating membrane potential and currents in cardiac cells. The cellular electrophysiological effects of adenosine are both cell type‐ and species‐dependent. In supraventricular tissues (SA, AV node, and atrium) of all species studied, the “direct” cAMP‐independent activation of the inwardly rectifying K+ current IKAdo seems to be the most important action of adenosine. This current is activated by both adenosine and acetylcholine and flows through K+ channels with unitary slope conductance of about 45 pS and an open time constant of 1.4 ms. The density of K+‐ACh,Ado channels is much less in ventricular than in atrial myocytes, and thus adenosine has little or no effect on the ventricular action potential. In atrial myocytes adenosine has a small inhibitory effect on basal L‐type calcium current (ICa,L), but no effect on T‐type calcium current (ICa,T). In ventricular myocytes, adenosine does not inhibit ICa,L (except ferret), ICa,T, or the sodium inward current INa. Adenosine has recently been shown to activate IKATP in ventricular membrane patches, but the relevance of this finding remains to be defined. Irrespective of cell type and species, adenosine inhibits membrane currents that are stimulated by β‐adrenergic agonists and other agents known to stimulate the activity of the enzyme adenylyl cyclase. This indirect cAMP‐dependent mechanism of action has been shown to be responsible for the inhibition by adenosine of isoproterenol‐stimulated ICa,L, delayed rectifier K+ current (IK), chloride current (ICl), the transient inward current ITi, and the pacemaker current IF. The importance of the actions of adenosine on membrane currents in modulation of atrial, ventricular, sinoatrial, and atrio‐ventricular nodal function are discussed. Likewise, the antiarrhythmic and proarrhythmic actions of adenosine are discussed and the clinical implications of these actions are noted.—Belardinelli, L., Shryock, J. C., Song, Y., Wang, D., Srinivas, M. Ionic basis of the electrophysiological actions of adenosine on cardiomyocites. FASEB J. 9, 359–365 (1995)

Cellular Basis for the Negative Dromotropic Effect of Adenosine on Rabbit Single Atrioventricular Nodal Cells

The effects of adenosine on action potentials, rate-dependent activation failure (the cellular basis for second-degree atrioventricular [AV] block), and the recovery of excitability in rabbit isolated single AV nodal cells were studied using the whole-cell patch-clamp technique. Adenosine (1 micromol/L) shortened the duration, depressed the amplitude, and reduced the rate of rise of the AV nodal cell action potential. Adenosine (10 micromol/L) caused a significant hyperpolarization (7 +/- 1 mV) of AV nodal cells. Adenosine increased the occurrence and the rate dependence of activation failure (Wenckebach periodicity) of AV nodal cells: this effect was concentration dependent and mediated by A1 adenosine receptors. The rate-dependent activation failure caused by adenosine was associated with a prolongation of the effective refractory period by 18 +/- 2 ms (P < .05), an increase in the duration of activation delay, and an elevation (from 0.22 +/- 0.04 to 0.30 +/- 0.03 nA, P < .05) of the threshold current amplitude required to activate AV nodal cells. The results suggest that the slowed recovery of excitability of AV nodal cells caused by adenosine forms the cellular basis for adenosine-induced second-degree AV block. Adenosine decreased ICa,L and activated IK,ADO of AV nodal cells. These actions of adenosine on ion currents may contribute to the effect of this nucleoside to depress excitability of AV nodal cells. The enhancement by adenosine of rate-dependent activation failure of AV nodal cells implies that the negative dromotropic effect of adenosine should be more pronounced during an episode of supraventricular tachycardia than during normal rhythm.

Angiotensin II Type 2 Receptor–Mediated Regulation of Rat Neuronal K+ Channels

We have previously shown that angiotensin II (Ang II), via AT2 receptors, increases whole-cell K+ current in cultured rat hypothalamus and brain stern neurons. We have now investigated the AT2 receptor-mediated effects of Ang II on the activity of single delayed rectifier K+ channels in cell-attached membrane patches. In control recordings (bath, 5.4 mmol/L K+; pipette, 140 mmol/L K+), two voltage-dependent channels were recorded with conductances of 34 +/- 4 and 56 +/- 6 pS, respectively (n = 6). When patches were excised, the channels reversed near a membrane potential expected for a K+ channel. In cell-attached patches (-40 mV), Ang II (100 nmol/L) increased open probability of the 56-pS K+ channel from 0.03 +/- 0.01 to 0.21 +/- 0.05 (n = 3). The selective AT2 receptor antagonist PD 123319 (1 mumol/L) but not the AT1 receptor antagonist losartan (1 mumol/L) blocked the actions of Ang II (n = 3). The selective AT2 receptor agonist CGP 42112 (100 nmol/L) produced similar effects to Ang II. Kinetic analysis of the Ang II effect showed that open-time histograms were best fit by two exponential functions. Ang II increased both open-time constants relative to control (control, tau 1 = 0.9 +/- 0.1 milliseconds, tau 2 = 2.3 +/- 0.3 milliseconds; Ang II, tau 1 = 3.1 +/- 0.4 milliseconds, tau 2 = 12.1 +/- 2.4 milliseconds), and PD 123319 blocked this effect (n = 3). The closed-time histogram was not affected by Ang II PD 123319, or losartan. These results suggest that activation of AT2 receptors modulates rat hypothalamus and brain stern neuronal whole-cell K+ current by increasing the open probability of a 56-pS K+ channel.

Angiotensin II regulation of intracellular calcium in astroglia cultured from rat hypothalamus and brainstem.

Abstract: This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1–100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura‐2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrodes solution (containing 2 mM CaCl2), the Ang II‐stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady‐state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 µM) inhibited the Ang II‐stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 µM) did not inhibit the Ang II‐stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II‐stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca2+‐free Tyrodes solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 µM), cyclopiazonic acid (10 µM), and ryanodine (10 µM), but not caffeine (1–10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 µM) and nifedipine (10 µM); in contrast, gadolinium (100 µM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.

Reducing the Late Sodium Current Improves Cardiac Function during Sodium Pump Inhibition by Ouabain

Inhibition by cardiac glycosides of Na+, K+-ATPase reduces sodium efflux from myocytes and may lead to Na+ and Ca2+ overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late INa) would reduce ouabains effect to cause cellular Na+ loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na+i) and high-energy phosphates using 23Na and 31P NMR, the amplitude of late INa using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late INa. Ouabain (1–1.3 μM) increased Na+i and late INa of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late INa inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na+i and late INa. Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late INa was also attenuated by the Ca2+-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4′-methoxybenzenesulfonyl)]amino-N-(4′-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na+ and Ca2+ overload is ameliorated by the inhibition of late INa.

Tetraethylammonium potentiates the activity of muscarinic potassium channels in guinea‐pig atrial myocytes

1 The modulation of native muscarinic potassium channels (KACh) by tetraethylammonium (TEA) was studied at 35oC in cell‐free patches from acutely dissociated guinea‐pig atrial myocytes. The channels were identified unambiguously by their conductance, inward rectification, rapid gating kinetics and pharmacological responses to muscarinic agonists and GTPγS. 2 Addition of 5 mm TEA to the cytoplasmic side of the patches almost doubled the open probability of KACh channels that had been activated maximally by GTPγS. In contrast even 30 mm TEA did not significantly potentiate the response to carbachol in whole‐cell recordings. 3 Unlike GTPγS, TEA alone did not activate KACh channels de novo, but in patches that showed spontaneous KACh activity, 5 mm TEA increased channel open probability fourfold in the absence of added sodium, ATP or guanine nucleotides. Furthermore, the effect of TEA was not blocked by 10 μm atropine or by 1 mm GDPβS, and subsequent addition of 0.1 mm GTPγS did not stimulate channel activity further in the presence of TEA. 4 Phosphatidylinositol 4,5‐bisphosphate (PIP2) also stimulates KACh channels under these conditions, but the kinetics of gating differ from channels stimulated by either TEA or GTP, which are very similar to one another. 5 The effects of TEA were not mimicked by tetramethyl‐ or tetrapentylammonium or by sodium or spermine, and TEA did not potentiate the activity of other inwardly rectifying potassium (KATP) channels in patches from cardiac myocytes. 6 We consider the possibility that TEA is mimicking the effect of an unidentified cellular factor, not sodium or PIP2, which normally occupies the TEA site on KACh channel proteins but which diffuses away when the patch is excised.

Preclinical pharmacokinetics and metabolism of MNP001, a piperidine analog of 3-carbamyl compounds.

MNP001 is a newly synthesized 3‐carbamyl‐4‐methylpyrrole analog with dual pharmacophores simultaneously to inhibit phosphodiesterase type 4 (PDE4) and to antagonize L‐type calcium channels. The physicochemical properties of MNP001, including solubility, pKa, Log P, plasma protein binding and plasma/blood partitioning, were determined to support the pharmacokinetic characterization. The preclinical pharmacokinetic parameters were determined in an in vivo rat model and the metabolic pathways of MNP001 were characterized by incubating the compound in vitro in rat or human microsomes/supersomes cocktails. MNP001 was found to have a low solubility in simulated intestinal fluid but a high solubility in simulated gastric fluid. MNP001 is a highly lipophilic compound with a Log P value greater than 4. MNP001 was highly bound to the plasma protein and had an uneven partition between red blood cells and plasma. MNP001 exhibited a rapid absorption, broad distribution, slow systemic clearance and a low but pharmacologically relevant oral bioavailability in rats. The low oral bioavailability was possibly caused by the low aqueous solubility of MNP001 in the gastrointestinal tract. However, 8 h after oral dosing, the mean plasma level of MNP001 was able to remain about 2‐fold greater than the minimum effective concentration. The major metabolite of MNP001 was defined as a tetrahydropyridine product (MNP001‐M4) of CYP3A4‐mediated phase I oxidation. The possibility that the major metabolite MNP001‐M4 may have a comparable antihypertensive efficacy to MNP001 needs to be studied. Copyright

Alcohol abrogates intracellular Ca2+ elevation by angiotensin II and ATP in cultured rat astrocytes

Astrocytes have a robust and dynamic intracellular Ca activity in response to stimulation by various neurotransmitters and neuromodulators such as angiotensin II, glutamate, and ATP [1-4]. ATP evokes Ca bursts in astrocytes by activation of purinergic receptors [4] and angiotensin II (AngII) by stimulation of AT1 receptors [2]. Intracellular Ca mobilization by the peptide and high energy purine signaling molecules enhances astrocyte cycling excitatory neurotransmitter glutamate, which governs the social behavior and cognitive ability [5-7]. Acute alcohol overdose alters users’ social behavior and affects abusers’ cognition [8,9]. We hypothesize that alcohol impairs the intracellular Ca handling in astrocytes particularly in response to neurotransmitter/neuromodulator stimulation, which may play an important role in understanding alcohol intoxication. The impairment may relate to the development of alcohol addiction, dependence and tolerance. In this work, we studied the alteration of intracellular Ca signaling by alcohol treatment in cultured rat hippocampal astrocytes. The findings of this study enrich our understanding of ethanol intoxication and may lead to a new treatment target for alcoholism.

HPLC/MS/MS analysis of 3-carbamyl-4-methylpyrrole analog MNP001, a highly potent antihypertensive agent, in rat plasma

Chemically synthesized 3-carbamyl-4-methylpyrroles were characterized as a group of antihypertensive agents with dual-targeting mechanism to simultaneously inhibit type 4 phosphodiesterase (PDE4) and L-type calcium channels. A 5-butyl analog of the pyrrole family, MNP001, was found to have high potency in reducing animal blood pressure and heart rate. A method for measuring MNP001 using high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS/MS) was developed. The calibration curve for MNP001 showed good linearity with the value of correlation coefficient greater than 0.987 over the range of 0.25-500 ng/mL. The results for inter-day and intra-day precision as well as accuracy were acceptable according to the criteria established by FDA. The lower limit of quantification was 0.25 ng/mL. This method was quick, sensitive and sufficient for in vivo pharmacokinetic and pharmacodynamic studies on this novel antihypertensive pyrrole compound.