Diclofenac, a Non-steroidal Anti-inflammatory Drug, Inhibits L-type Ca2+ Channels in Neonatal Rat Ventricular Cardiomyocytes

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitation-contraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than 3 micrometer, diclofenac inhibited reversibly the Na+ current and did irreversibly the L-type Ca2+ channels-mediated inward current (IC50=12.89+/-0.43 micrometer) in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type Ca2+ currents but not the Na+ current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type Ca2+ channel, leading to the impairment of E-C coupling in cardiac myocytes.

Lysophosphatidylcholine Increases Ca2+ Current via Activation of Protein Kinase C in Rabbit Portal Vein Smooth Muscle Cells

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase A(2), has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. Ca2+ influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying Ca2+ influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type Ca2+ current (ICa(L)) activity and to elucidate the mechanism of LPC-induced change of ICa(L) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased ICa(L) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of ICa(L) was not significantly changed by LPC. Staurosporine (100 nanometer) or chelerythrine (3 micrometer, which is a potent inhibitor of PKC, significantly decreased basal ICa(L), and LPC-induced increase of ICa(L) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal ICa(L) significantly, and LPC-induced enhancement of ICa(L) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased ICa(L) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of ICa(L) might be, at least in part, responsible for increased Ca2+ influx in atherosclerotic artery.

Voltage-dependent Ca2+ Current Identified in Freshly Isolated Interstitial Cells of Cajal (ICC) of Guinea-pig Stomach

The properties of voltage dependent Ca2+ current (VDCC) were investigated in interstitial cells of Cajal (ICC) distributed in the myenteric layer (ICC-MY) of guinea-pig antrum. In tissue, ICC-MY showed c-Kit positive reactions and produced driving potentials with the amplitude and frequency of about 62 mV and 2 times min(-1), respectively, in the presence of 1micrometer nifedipine. Single ICC-MY isolated by enzyme treatment also showed c-Kit immunohistochemical reactivity. These cells were also identified by generation of spontaneous inward current under K+-rich pipette solution. The voltage clamp experiments revealed the amplitude of - 329 pA inward current at irregular frequency. With Cs+-rich pipette solution at Vh=?80 mV, ICC-MY produced voltage-dependent inward currents (VDIC), and nifedipine (1micrometer) blocked VDIC. Therefore, we successfully isolated c-Kit positive single ICC from guinea-pig stomach, and found that ICC-MY potently produced dihydropiridine sensitive L-type voltage-dependent Ca2+ currents (VDCCL).

Characteristics of Potassium and Calcium Currents of Hepatic Stellate Cells (Ito) in Rat

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA) -sensitive current, 4-aminopyridine (4-AP) -sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15microM pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.

Depression of L-type Ca2+ and transient outward K+ currents in endotoxin-treated rat cardiac myocytes

Decreased cardiac contractility occurs in endotoxicosis, but little is known about the ionic mechanism responsible for myocardial dysfunction. In this study, we examined the changes in Ca2+ and K+ currents in cardiac myocytes from endotoxin-treated rat. Ventricular myocytes were isolated from normal and endotoxemic rats (ex vivo), that were treated for 10 hours with Salmonella enteritidis lipopolysaccharides (LPS; 1.5 mg/kg) intravenously. Normal cardiac myocytes were also incubated for 6 hours with 200 ng/ml LPS (in vitro). L-type Ca2+ current (ICa,L) and transient outward K+ current (Ito) were measured using whole cell patch clamp techniques. Peak ICa,L was reduced in endotoxemic myocytes (ex vivo; 6.00.4 pA/pF, P<0.01) compared to normal myocytes (control; 10.90.6 pA/pF). Exposure to endotoxin in vitro also attenuated ICa,L (8.40.4 pA/pF, P<0.01). The amplitude of Ito on depolarization to 60 mV was reduced in endotoxin treated myocytes (16.51.5 pA/pF, P<0.01, ex vivo; 20.00.9 pA/pF, P<0.01, in vitro) compared to normal myocytes (control; 24.71.0 pA/pF). There was no voltage shift in steady-state inactivation of ICa,L and Ito between groups. These results suggest that endotoxin reduces Ca2+ and K+ currents of rat cardiac myocytes, which may lead to cardiac dysfunction.

Effects of prostaglandin E2 on the spontaneous contractions and electrical activities of the antral circular muscle in guinea-pig stomach

The spontaneous contractions of gastric smooth muscles are regulated by slow waves, which are modulated by both nervous system and humoral agents. This study was designed to examine the effects of Prostaglandin E2 (PGE2) on the contractile and electrical activities of antral smooth muscles in guinea-pig stomach, using an intracellular recording technique. To elucidate the underlying mechanism for its effect on contractility, ionic currents were also measured using a whole-cell patch clamp method. The basal tone by PGE2 was variable, whereas the magnitude of phasic contractions was reduced (19.0 +/- 2.1%, n=19). The resting membrane potentials were hyperpolarized (-4.4+/-0.5 mV, n=10), and plateau potentials were lowered (-2.9+/-0.5 mV, n=10). In most cases, however, the initial peak potentials of slow waves were depolarized more by PGE2 than those of control. The frequency of the slows wave was increased from 5.7+/-0.2 cycles/min to 6.5+/-0.2 (n-22). Voltage-operated Ca2+ currents were decreased by PGE2 (n=5). Voltage-operated K+ currents, both Ca-dependent and Ca-independent, were increased (n-5). These results suggest that PGE2 plays an important role in the modulation of gastric smooth muscle activities, and its inhibitory effects on the contractility and activities of slow waves are resulted from both decrease of Ca2+ currents and increase of K+ currents.

Effect of Kanamycin on Calcium Current of Rabbit Ventricular Myocyte

It has been reported that kanamycin, a useful agent in the treatment of gram-negative and other infectious disease, has a negative inotropic action in isolated cardiac muscle preparation and also it decreases the amplitude of high K+ -induced action potential of guinea pig atrium. These findings imply that kanamycin has a property of Ca2+ antagonist. In this study, the effects of kanamycin on cardiac Ca2+ current were investigated in isolated rabbit ventricular cells by using a whole cell clamp nethod.The results are summarized as follows ; 1) Kanamycin caused a depression fo cardiac Ca2+ current in a dose dependent manner and its effect was observed in a whole memberane potential renge. 2) The decreasing effect of kanamycin on Ca2+ current was inhibited by high Ca(2+)-Tyrode solution and such inhibition was also observed in high Sr2+-Tyrode solution, but in the case of Cd2+, well known inorganic Ca2+ antagonist, the current through Ca2+ channel was greatly decreased when the perfuaste was changed from high Ca(2+)-Tyrode to high Sr(2+)-Tyrode solution. 3) In the presense of kanamycin, the decreasing pattern of Ca2+current by repetitve depolarization was not specific as in the case of verapamil. 4) Neomycin decreased Ca2+ current similar to kanamycin.From the above result, it may be concluded that kanamycin has competitive antagonistin effect on cardiac Ca2+ current.