alpha 1-Adrenoceptor stimulation partially inhibits ATP-sensitive K+ current in guinea pig ventricular cells: attenuation of the action potential shortening induced by hypoxia and K+ channel openers

Effects of alpha 1-adrenoceptor stimulation on the action potential shortening produced by K+ channel openers (KCOs) or hypoxia and on the ATP-sensitive K+ current (IK.ATP) activated by KCOs were examined in guinea-pig ventricular cells by using conventional microelectrode and patch-clamp techniques. In papillary muscles, nicorandil (1 mM) or cromakalim (30 microM) markedly shortened the action potential duration (APD) (to 51 +/- 2% and 40 +/- 5% of each control value). Addition of 100 microM methoxamine, an alpha 1-adrenoceptor agonist, partially but significantly reversed the KCOs-induced APD shortening (to 69 +/- 3% and 50 +/- 4% of each control value). The APD-prolonging effect of methoxamine was antagonized by 1 microM prazosin (alpha 1-antagonist) and 100 nM WB4101 (alpha 1A-antagonist) but not by 10 microM chloroethylclonidine (alpha 1B-antagonist). In papillary muscles exposed to a hypoxic, glucose-free solution, APD declined gradually. In the presence of 100 microM methoxamine or 10 microM glibenclamide, the hypoxia-induced action potential shortening was significantly inhibited. In single ventricular myocytes, the KCOs increased a steady-state outward current that was abolished by glibenclamide (1 microM), thereby suggesting that these KCOs activate IK.ATP. Methoxamine (100 microM) significantly inhibited the nicorandil-induced IK.ATP by 18 +/- 5% and the cromakalim-induced IK.ATP by 16 +/- 2%. 4 beta-Phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, failed to mimic the alpha 1-adrenoceptor-mediated inhibition of the nicorandil-induced outward current. Staurosporine (30 nM), a protein kinase C inhibitor, also failed to affect the partial inhibition of IK.ATP by methoxamine. Neither intracellular loading of heparin (100 micrograms/ml), an inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release inhibitor, nor IP3 (20 microM) plus inositol 1,3,4,5-tetrakisphosphate (IP4 5 microM) could affect the inhibitory action of methoxamine. In conclusion, alpha 1A-adrenergic stimulation partially inhibits IK.ATP in cardiac cells. Neither protein kinase C activation nor IP3 formation appears to be involved in the partial inhibition of IK.ATP. The alpha 1A-adrenoceptor-mediated inhibition of IK.ATP may be deleterious for ischemic myocardium and partly offset the cardioprotective effect of KCOs because attenuation of action potential shortening may potentially increase Ca2+ influx in ischemic cells.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,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 alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM 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 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,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 ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-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 ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds

1. Ouabain, an inhibitor of Na+/K+ ATPase induces the release of acetylcholine from central and myenteric cholinergic neurones principally due to partial depolarization of the cell membrane. The effect of ouabain has been examined on neurogenic contractions in the guinea-pig ileum arising from either electrical field stimulation or from naloxone in morphine-exposed preparations. 2. Guinea-pig isolated ileum preparations were stimulated transmurally (0.1 Hz, 0.3 ms, 200 mA) to elicit contractions of the myenteric plexus-longitudinal smooth muscle. 3. Incubation with morphine (0.3 microM, 60 min) was followed by naloxone (1 microM) which produced withdrawal contractions in 16/26 preparations (median of 10.7 [2.2-40.0]% of a maximal contracture to KCl (60 mM)). 4. In parallel experiments, ouabain (1 microM) was added to the tissue before exposure to morphine (0.3 microM, 60 min). Naloxone (1 microM) subsequently displayed a withdrawal contraction in all 26/26 tissues (57.9 [30.5-151.7]% of a maximal contracture to KCl (60 mM). 5. Ouabain neither affected the concentration-dependent contractions of guinea-pig ileum produced by carbachol nor the inhibition of electrically-evoked contraction produced by morphine (0.3 microM). 6. The muscarinic antagonist atropine (0.1 microM) antagonized control naloxone withdrawal responses. The atropine resistant component, evident in ouabain-treated tissues, was blocked by SR140333((S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenyla cetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2. 2]-octane, chloride), a substance P antagonist. 7. Clonidine (alpha2-adrenoceptor agonist) inhibited electrically-evoked contractions. Exposure to the alpha2-adrenoceptor antagonist RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline), resulted in a contracture which was not significantly enhanced by ouabain (1 microM). 8. Ouabain selectively potentiates the naloxone-induced withdrawal contraction following acute exposure to morphine the major components of which are mediated by both acetylcholine and substance P.     

Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats

1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.     

Importance of diabetes mellitus and systemic hypertension rather than completeness of revascularization in determining long-term outcome after coronary balloon angioplasty (the LDCMC registry). Lady Davis Carmel Medical Center

To characterize KC 12291 (1-(5-phenyl-1,2, 4-thiadiazol-3-yl-oxypropyl)-3-[N-methyl-N-[2-(3,4-dimethoxy phenyl) ethyl] amino] propane hydrochloride), a newly synthezised inhibitor of voltage-gated Na+ channels, the effects of the agent on Na+ current and ischemia-induced Na+ overload were investigated in isolated cardiomyocytes, atria and saline-perfused hearts. As measured by the patch clamp technique, KC 12291 (1 microM) significantly reduced peak Na+ current after activation of voltage-gated Na+ channels in rat cardiomyocytes. Partial depolarization enhanced the inhibitory effects during steady state conditions of the channel. In isolated guinea pig atria, 1 microM KC 12291 had no effect on contractility under basal conditions but effectively delayed the onset and reduced the extent of anoxic contracture. The concentration-response curve was clearly shifted to the left when atria were partially depolarized by increased extracellular K+. As measured by 23Na NMR spectroscopy in isolated perfused guinea pig hearts, intracellular Na+ rose more than four-fold in a linear fashion during 60 min of low-flow ischemia. KC 12291 (1 microM) prevented Na+ overload within the initial 12 min of ischemia; thereafter the slope of Na+ accumulation was identical to controls. Electrical excitability of hearts, evaluated by intracardial ECG, completely ceased within 15 min after the onset of ischemia. KC 12291 (1 microM) accelerated this process by more than 6 min. The data provide first evidence that KC 12291 reduces Na+ influx through voltage-gated Na+ channels during ischemia and thus delays Na+ overload by enhancing the inexcitability of the heart.     

Angiotensin II AT1 receptors and Na+/K+ ATPase in human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells (HUVECs) at passage 4 specifically bound 70 +/- 12 fmol [3,5-3H]Tyr4-Ile5-angiotensin (Ang) II/mg protein, with a Kd of 0.9 +/- 0.36 nM. Binding was eliminated in cells preincubated with a monoclonal antibody (6313/G2) raised against the subtype AT1 of the Ang II receptor. Freshly seeded HUVECs were positive for 6313/G2 antibody by immunocytochemistry, and such immunoreactivity was still retained at passage 4. Incubation of HUVECs for 20 min with different concentrations of Ang II provoked a significant increment in Na+/K+ ATPase activity compared with controls, in a dose- and time-dependent manner. Maximal response was obtained with 1000 pM Ang II after 20 min stimulation and resulted in a 2.2-fold increment in Na+/K+ ATPase activity. This stimulation was abolished when cells were incubated with 1000 pM Ang II in the presence of 1 microM of the specific AT1 subtype inhibitor, DuP753. Moreover, preincubation of HUVECs with 6313/G2 or with 1 mM dithiothreitol also inhibited the stimulatory effect of Ang II. These results suggest that the AT1 receptor subtype mediates the Na+/K+ ATPase response to Ang II in these cells.     

Biomarkers for malignancy in the columnar-lined esophagus

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.     

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.     

Age-dependent decrease in the negative inotropic effect of carbachol on isolated human right atrium

On isolated, electrically driven human right atrial strips, carbachol (10(-8)-10(-3) M) concentration-dependently decreased force of contraction prestimulated with 1 microM forskolin; maximal negative inotropic effects of carbachol (10(-6)-3 x 10(-6) M), however, were in atria from patients aged < 25 years (mean age: 16.8 +/- 2.0 years, n = 9) significantly larger than in patients aged 50-69 years (mean age: 62.5 +/- 0.7 years, n = 33) and were further decreased in patients aged > 70 years (mean age: 73.8 +/- 0.6 years, n = 11). We conclude that, in human right atrium, the recently described age-dependent decrease in muscarinic M2 receptor density is accompanied by a decrease in negative inotropic effects.     

Protective effect of l-cis-diltiazem on hypercontracture of rat myocytes induced by veratridine

The protective effect of l-cis-diltiazem, the stereoisomer of d-cis-diltiazem, was studied against the veratridine-induced hypercontracture of rat myocytes. Veratridine increased both [Na+]i and [Ca2+]i, but did not cause hypercontracture in the absence of extracellular Ca2+. Both l-cis-diltiazem (0.1-10 microM) and d-cis-diltiazem (10-30 microM) inhibited the hypercontracture and the increase in [Ca2+]i in a concentration-dependent manner. However, l-cis-diltiazem did not exert a negative inotropic effect in K+ (20 mM)-depolarized rat papillary muscles even at a dose of 10 microM. As seen in the case of tetrodotoxin, l-cis-diltiazem and d-cis-diltiazem also suppressed the increase in [Na+]i. The results show that l-cis-diltiazem prevents the veratridine-induced hypercontracture of myocytes by suppression of the [Ca2+]i increase. The attenuation of the [Ca2+]i increase by l-cis-diltiazem was not dependent on inhibition of Ca2+ channels, but was partly due to inhibition of excessive Na+ entry via veratridine-modified Na+ channels.     

Synthesis and calcium antagonistic activity of 8-[N-[2-(3,4-dimethoxy- phenyl)ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4]thiazepi ne fumarate

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl) ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine (1). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC50 = 23 microM, n = 7), and a negative inotropic effect in papillary muscles (IC50 = 2.7 microM, n = 7) and left atria (IC50 = 4 microM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC50: 70 +/- 5 microM) and low (IC50: 129 +/- 34 microM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC50: 80 +/- 13 microM) in chick dorsal root ganglion neurons. In addition nicotine-induced currents were potently inhibited (IC50: 6 +/- 0.7 microM) in bovine chromaffin cells.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.     

Purine and pyrimidine nucleotides inhibit a noninactivating K+ current and depolarize adrenal cortical cells through a G protein-coupled receptor

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that sets the resting membrane potential and may mediate depolarization-dependent cortisol secretion. External ATP stimulates cortisol secretion through activation of a nucleotide receptor. In whole-cell patch clamp recordings from bovine AZF cells, we found that ATP selectively inhibited IAC K+ current by a maximum of 75.7 +/- 3% (n = 13) with a 50% inhibitory concentration of 1.3 microM. A rapidly inactivating A-type K+ current was not inhibited by ATP. Other nucleotides, including ADP and the pyrimidines UTP and UDP, also inhibited IAC, whereas 2-methylthio-ATP (2-MeSATP) and CTP were completely ineffective. The rank order of potency for six nucleotides was UTP = ADP > ATP > UDP > 2-MeSATP = CTP. At maximally effective concentrations, UTP, ADP, and UDP inhibited IAC current by 81.4 +/- 5.2% (n = 7), 70.7 +/- 7.2% (n = 4), and 65.2 +/- 7.9% (n = 5), respectively. Inhibition of IAC by external ATP was reduced from 71. 3 +/- 3.2% to 22.8 +/- 4.5% (n = 18) by substituting guanosine 5'-O-2-(thio) diphosphate for GTP in the patch pipette. Inhibition of IAC by external ATP (10 microM) was markedly suppressed (to 17.3 +/- 5.5%, n = 9) by the nonspecific protein kinase antagonist staurosporine (1 microM) and eliminated by substituting the nonhydrolyzable ATP analog 5-adenylyl-imidodiphosphate or UTP for ATP in the pipette. ATP-mediated inhibition of IAC was not altered by the kinase C antagonist calphostin C, the calmodulin inhibitory peptide, or by buffering the intracellular (pipette) Ca++ with 20 mM 1,2-bis-(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid. In current clamp recordings, ATP and UTP (but not CTP) depolarized AZF cells at concentrations that inhibited IAC K+ current. These results demonstrate that bovine AZF cells express a nucleotide receptor with a P2Y3 agonist profile that is coupled to the inhibition of IAC K+ channels through a GTP-binding protein. The inhibition of IAC K+ current and associated membrane depolarization are the first cellular responses demonstrated to be mediated through this receptor. Nucleotide inhibition of IAC proceeds through a pathway that is independent of phospholipase C, but that requires ATP hydrolysis. The identification of a new signaling pathway in AZF cells, whereby activation of a nucleotide receptor is coupled to membrane depolarization through inhibition of a specific K+ channel, suggests a mechanism for ATP-stimulated corticosteroid secretion that depends on depolarization-dependent Ca++ entry. This may be a means of synchronizing the stress-induced secretion of corticosteroids and catecholamines from the adrenal gland.     

The ABC maltose transporter

OBJECTIVE: To define the electrophysiologic mechanism(s) by which MCI-154, a putative Ca2+ sensitizer, produces a positive inotropic response without a positive chronotropic response, we examined effects of MCI-154 on the action potential of atrial preparations and the membrane currents of atrial myocytes. METHODS: The action potentias were recorded from left atrial and sinoatrial node preparations of guinea pigs by the use of standard microelectrode techniques. The whole-cell membrane currents were recorded from enzymatically-dissociated guinea pig atrial myocytes using conventional patch clamp techniques. RESULTS: In isolated left atria, MCI-154 increased the developed tension in a concentration-dependent manner. MCI-154 at concentrations of 10 and 100 microM increased the action potential duration (APD) in left atria stimulated at 0.5 Hz. In sinoatrial node preparations MCI-154 at a concentration of 100 microM produced a negative chronotropic response and prolonged APD. In single right atrial myocytes, MCI-154 at concentrations of 10 and 100 microM failed to increase the inward L-type Ca2+ current, but decreased the delayed rectifier K+ current (IK) in a concentration-dependent manner. MCI-154 decreased IK elicited by short depolarizing pulses more markedly than that induced by long depolarizing pulses. In addition, MCI-154 produced only a little inhibition of IK in the presence of E-4031, a specific blocker of rapidly activating component of IK (IKr). CONCLUSIONS: MCI-154 preferentially blocks IKr and the inhibitory action on IKr may be partly involved in the negative chronotropic and positive inotropic responses in atrial preparations.     

Erosion and controlled release properties of semisolid vesicular phospholipid dispersions

Single adult guinea-pig and rat ventricular cardiac myocytes were used to study the effects of two members of the omega3 class of polyunsaturated fatty acids, docosahexaenoic acid and eicosapentaenoic acid, on the electrical and mechanical activity of cardiac muscle. Docosahexaenoic acid and eicosapentaenoic acid reduced the electrical excitability of both guinea-pig and rat cells in a dose-dependent manner. Both agents produced a dose-dependent negative inotropic response in guinea-pig cells but in the rat cells there was first a dose-dependent positive inotropic effect at low concentrations (< 10 microM) followed by a negative inotropic effect at higher concentrations (> 10 microM). Possible mechanisms by which these agents affect contraction were studied using conventional electrophysiological techniques. The polyunsaturated fatty acids reduced the action potential duration and the plateau potential of the guinea-pig cells in a simple, dose-dependent manner. In contrast, the effect on the rat action potential mirrored the inotropic effect. At low concentrations (< 10 microM) there was a concentration-dependent increase in action potential duration followed by a concentration-dependent decrease at higher concentrations (> 10 microM). Both polyunsaturated fatty acids decreased the fast Na+ current and the L-type Ca2+ current in a concentration-dependent but not use-dependent manner in cells from both species. In the rat cells these agents inhibited the transient outward current resulting in an increase in the duration of the rat action potential. The effects of polyunsaturated fatty acids on the Ca2+, Na+ and K+ currents underlie these changes in the action potentials in guinea-pig and rat heart cells. The effects on the L-type Ca2+ current and action potential duration can also explain both the simple negative inotropic effects of the agents on the guinea-pig cells and the more complex effects on the rat cells. These effects of polyunsaturated fatty acids on membrane currents may account for their anti-arrhythmic properties.     

Effects of angiotensin II on inotropy and intracellular Ca2+ handling in normal and hypertrophied rat myocardium

The direct inotropic effect of angiotensin II on the myocardium is still controversial and little information exists as to its potential modification by heart disorders. Therefore, this study performed simultaneous measurements of isometric force and intracellular Ca2+ concentrations ([Ca2+]i) in left ventricular papillary muscles from sham-operated and aortic-banded rats at 10 weeks post-surgery. Angiotensin II (10(-6) M) induced a reduction of peak systolic [Ca2+]i (0.56 +/- 0.03 to 0.48 +/- 0.04 microM; P<0.05) and a parallel but insignificant diminution of developed tension (10.5 +/- 1.3 to 9.6 +/- 0.8 mN/mm2) in normal papillary muscles from sham-operated animals. Hypertrophied papillary muscles from aortic-banded rats demonstrated a significant decline in both peak systolic [Ca2+]i (0.51 +/- 0.02 to 0.44 +/- 0.01 microM; P<0.05) and developed tension (8.4 +/- 1.1 to 6.8 +/- 1.7 mN/mm2; P<0.05) after addition of angiotensin II. The time courses of the mechanical contraction and the intracellular Ca2+ signal were prolonged by angiotension II in both groups. Isoproterenol dose-dependently increased developed tension and peak systolic [Ca2+]i in papillary muscles from sham-operated rats. In contrast, the positive inotropic response to isoproterenol was markedly reduced in hypertrophied muscles despite a seemingly unimpaired increase in peak systolic [Ca2+]i. Pretreatment with angiotensin II (10(-6) M) resulted in a significant attenuation of the systolic [Ca2+]i response to isoproterenol stimulation in both normal and hypertrophied papillary muscles. Neither the bradykinin B2 antagonist icatibent (10(-6) M) nor the nitric oxide (NO) inhibitor L-NMMA (10(-6) M) abolished the depressant effects of angiotension II. Thus, ANG II induces a parallel decline of the mechanical performance and Ca2+ availability in rat myocardium. These effects are more distinct in hypertrophied than in normal muscle and become accentuated during beta-adrenergic stimulation. The underlying mechanism is not associated with the NO pathway but might involve a negative functional coupling between the angiotensin and beta-adrenergic-receptor complex.     

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells

1. The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea-pig with the whole-cell voltage-clamp technique. 2. No. 7943 at 0.1-10 microM suppressed the outward Na+/Ca2+ exchange current in a concentration-dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 microM. 3. No. 7943 at 5-50 microM suppressed also the inward Na+/Ca2+ exchange current in a concentration-dependent manner but with a higher IC50 value of approximately 17 microM. 4. In a concentration-response curve, No. 7943 raised the K(m)Ca2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/ Ca2+ exchange current. 5. The voltage-gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50S of approximately 14, 8 and 7 microM, respectively. 6. In contrast to No. 7943, 3', 4'-dichlorobenzamil (DCB) at 3-30 microM suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 microM, but did not affect the outward exchange current at these concentrations. 7. We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.     

Antagonist effects of seglitide (MK 678) at somatostatin receptors in guinea-pig isolated right atria

Somatostatin (SS) exerts a negative inotropic effect in isolated atria. Here we report that in guinea-pig isolated right atria, seglitide, a potent cyclic hexapeptide somatostatin agonist, behaves as a competitive somatostatin receptor antagonist with pA2 values against SS14, SS25 and SS28, of 6.50 +/- 0.40, 6.24 +/- 0.08 and 6.09 +/- 0.06, respectively. Seglitide had little or no effect on the negative inotropic action of carbachol or N6-cyclohexyladenosine. Our findings indicate that the receptor-response coupling characteristics of guinea-pig atria are such that in this preparation seglitide has low intrinsic activity and behaves specifically as a somatostatin receptor antagonist.     

Differential effects of etomidate, propofol, and midazolam on calcium and potassium channel currents in canine myocardial cells

BACKGROUND: Intravenous anesthetics etomidate, propofol, and midazolam produce negative inotropic effects of various degrees. The mechanism underlying these differences is largely unknown. METHODS: The effects of intravenous anesthetics on L-type Ca2+, transient outward and inward-rectifier K+ channel currents (ICa, IKto, and IK1) were compared in canine ventricular cells using the whole-cell voltage-clamp technique. ICa and IK were elicited by progressively depolarizing cells from -40 to +40 mV, and from -90 to +60 mV, respectively. The peak amplitude and time-dependent inactivation rate of ICa and IK were measured before, during, and after the administration of equimolar concentrations (5, 30, or 60 microM) of etomidate, propofol, or midazolam. RESULTS: Exposure to etomidate, propofol, and midazolam produced a concentration-dependent inhibition of ICa. Midazolam was the most potent intravenous anesthetic; at 60 microM, etomidate, propofol, and midazolam decreased peak ICa by 16 +/- 4% (mean +/- SEM), 33 +/- 5%, and 47 +/- 5%, respectively. Etomidate, propofol, and midazolam given in a 60-microM concentration decreased IKto by 8 +/- 3%, 9 +/- 2%, and 23 +/- 3%, respectively. IK1 was decreased by 60 microM etomidate and midazolam by 20 +/- 6% and 14% +/- 5%, respectively. Propofol had no effect on IK1. CONCLUSIONS: At equimolar concentrations, intravenous anesthetics decreased the peak ICa, IKto, and IK1 with various degrees of potency. Effects of anesthetics on ICa were significantly greater compared with their effects on K+ currents. These findings suggest that the negative inotropic actions of etomidate, propofol, and midazolam are related, at least in part, to decreased ICa. Some effects, such as IK inhibition, may partially antagonize effects of decreased ICa. Indeed, the final effect of these intravenous anesthetics on myocardium will be the sum of these and other sarcolemmal and intracellular effects.