Hisayuki Uneyama

Selectivity of dihydropyridines for cardiac L-type and sympathetic N-type Ca2+ channels

The blocking effects of cilnidipine and other dihydropyridines on L-type cardiac Ca2+ channels (I(Ca,L)) and N-type sympathetic Ca2+ channel currents (I(Ca,N)) were studied using a whole-cell patch-clamp technique. At -80 mV, cilnidipine had little inhibitory effect below concentrations of 1 microM on I(Ca,L) (IC50 value; 17 microM). However, 1 microM cilnidipine strongly shifted the steady-state inactivation curve of I(Ca,L) toward negative potentials without changing the current-voltage relationship. Each action of cilnidipine was characterized by a high affinity for the inactivated channel in preference to the resting channel. The IC50 values of dihydropyridines for I(Ca,L) were in the range between 0.01 and 10 microM, and those for I(Ca,N) were between 3 and 30 microM. Cilnidipine had the strongest affinity for I(Ca,N) among the dihydropyridines tested. These results suggest that cilnidipine did not cause hypotension-evoked tachycardia deficiency by depression of cardiac L-type channels but by sympathetic N-type channels blockade.

Caffeine response in pyramidal neurons freshly dissociated from rat hippocampus

The effect of caffeine on the CA1 pyramidal neurons freshly dissociated from rat hippocampus was investigated with nystatin-perforated patch technique under voltage-clamp condition. Caffeine evoked a transient outward current (Icaffeine) in a concentration-dependent manner at a holding potential of -40 mV. The activation and inactivation of Icaffeine were accelerated with increasing caffeine concentration. The reversal potential for Icaffeine was close to K+ equilibrium potential. The Icaffeine was not blocked by apamin and 4-aminopyridine but suppressed by charybdotoxin, tetraethylammonium, quinine and Ba2+. Thus, the pharmacological characteristics of Icaffeine were similar to those of Ca(2+)-activated K+ current having a large conductance (IC), which generates a fast afterhyperpolarization (a.h.p.). Icaffeine was depressed by pretreatment with a membrane-permeant Ca2+ chelator (BAPTA-AM) and by depletion of the Ca(2+)-induced Ca2+ release (CICR) pool with ryanodine. A blocker of CICR sites, procaine, potently depressed the Icaffeine. In the absence of the extracellular Ca2+, an application of 10 mM caffeine depleted the caffeine-sensitive Ca2+ pools. Icaffeine recovered in an exponential fashion in the presence of the extracellular Ca2+. It was concluded that rat hippocampal pyramidal neurons have a caffeine-sensitive Ca2+ pool. Furthermore, the Ca2+ released from the pool evokes K+ current similar to IC current and hyperpolarizes the neurons.

Caffeine and related compounds block inhibitory amino acid-gated Cl- currents in freshly dissociated rat hippocampal neurones.

1 The effects of caffeine and related compounds on responses mediated by inhibitory amino acids were investigated in freshly dissociated rat hippocampal pyramidal neurones by conventional and nystatin perforated patch‐clamp techniques. 2 Glycine and γ‐aminobutyric acid (GABA) evoked Cl− currents in hippocampal neurones. The half‐maximum effective concentrations (EC50) of glycine and GABA were 8.5 × 10−5 and 5 × 10−6 m, respectively. 3 Caffeine reversibly inhibited both 10−4 m glycine‐ and 10−5 m GABA‐induced Cl− currents in a concentration‐dependent manner. The half‐maximum inhibitory concentrations (IC50) of caffeine were 4.5 × 10−4 m for the glycine response and 3.6 × 10−3 m for the GABA response. 4 Caffeine shifted the concentration‐response curve of IGly to the right without affecting the maximum response. 5 The inhibitory action of caffeine did not show voltage‐dependency. 6 The blocking action of caffeine was not affected by intracellular perfusion with 5 mm BAPTA or by pretreatment with the protein kinase A inhibitor, H‐8. This excludes the participation of Ca2+ or cyclic AMP in the inhibitory action of caffeine. 7 Caffeine failed to inhibit the augmentations of aspartate‐ and N‐methyl‐d‐aspartate (NMDA) ‐gated current by glycine, suggesting that caffeine has no effect on the allosteric glycine binding site on the NMDA receptor. 8 The inhibitory effects of some xanthine derivatives on IGly were compared. The inhibitory potency of those compounds on IGly was in the order of pentoxifylline > theophylline ≥ caffeine > paraxanthine > IBMX ≥ theobromine > dyphylline. Xanthine had no effect. 9 The results indicate that methylxanthines including caffeine may act directly on the glycine receptor Cl− channel complex in rat hippocampal pyramidal neurones. The blockade of the inhibitory amino acid response by methylxanthines may be involved in the excitatory side effects of methylxanthines in the mammalian central nervous system.

Suramin and reactive blue 2 are antagonists for a newly identified purinoceptor on rat megakaryocyte.

1 The effects of purinoceptor antagonists on ATP‐induced oscillatory K+‐currents in rat isolated megakaryocytes were investigated. 2 Both reactive blue‐2 (RB‐2), a selective antagonist of the P2Y purinoceptor, at concentrations of 0.3–10 μm and suramin, a non‐selective P2 purinoceptor antagonist, at 1–30 μm blocked the ATP‐induced oscillation in a concentration‐dependent manner. 3 RB‐2 and suramin also blocked the ADP‐induced K+‐current oscillation at the same concentration range as in the case of ATP. However, both suramin and RB‐2 had no effect on thrombin‐ and inositol 1,4,5‐trisphosphate (IP3)‐induced K+ current oscillation, indicating that they act as specific purinoceptor antagonists. 4 Thus, the purinoceptors on megakaryocytes show the properties of the P2 subtype according to their blockade by antagonists.

Blood oxygenation level-dependent response to intragastric load of corn oil emulsion in conscious rats.

The postingestive actions after intragastric or oronasal stimulation of fat have been well investigated. The blood oxygenation level-dependent (BOLD) signal changes, however, after intragastric load of corn oil emulsion have yet to be elucidated. Here, using functional magnetic resonance imaging, we investigated the BOLD signal response to gut corn oil emulsion in nonanesthetized rats. Intragastrically infused 7% corn oil emulsion induced a BOLD signal increase in several brain regions, including the bilateral amygdala, hippocampus and the ventral tegmental area. These results indicate that the limbic system responds to gut corn oil emulsion and that activation of this system could promote the reinforcing action for food with high fat content.

5-HT response of rat hippocampal pyramidal cell bodies

Serotonin (5-HT) responses of pyramidal neurones freshly dissociated from rat ventral hippocampal CA1 region were investigated by using nystatin-perforated whole-cell recording. These dissociated neurones lack most of the dendrites and axons. Application of nanomolar concentrations of 5-HT induced outward current with an increase of membrane conductance at a holding potential (VH) of -40 mV. The current was mimicked by alpha-methyl-5-HT (5-HT2 receptor family agonist), but not by 8-OH-DPAT (5-HT1 receptor family agonist). Ketanserin (5-HT2 receptor family antagonist) and spiperone (5-HT1A and 5-HT2 receptor family antagonist) blocked the current in a concentration dependent manner. These results suggests that 5-HT-induced outward current is mediated by the activation of 5-HT2 receptor family in the cell bodies of hippocampal pyramidal neurones.

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca2+ antagonist, cilnidipine, on voltage-dependent Ca2+ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca2+ channels at the low concentrations under 10(-6) M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca2+ current (I(Ca)) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I(Ca) at the low concentrations under 10(-7) and 10(-6) M cilnidipine blocked not only L- but also N-type HVA I(Ca). At the high concentration over 10(-6) M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca2+ channels. This is the first report that cilnidipine at lower concentration of 10(-6) M blocks both L-and N-type HVA I(Ca) in the hippocampal neurons.

Effects of a Novel Antihypertensive Drug, Cilnidipine, on Catecholamine Secretion From Differentiated PC12 Cells

Effects of a novel dihydropyridine type of antihypertensive drug, cilnidipine, on the regulation of the catecholamine secretion closely linked to the intracellular Ca2+ were examined using nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells. By measuring catecholamine secretion with high-performance liquid chromatography coupled with an electrochemical detector, we showed that high K+ stimulation evoked dopamine release from PC12 cells both before and after NGF treatments. Cilnidipine depressed dopamine release both from NGF-treated and untreated PC12 cells in a concentration-dependent manner. In contrast, inhibition by nifedipine was markedly decreased in the differentiated PC12 cells. With intracellular Ca2+ concentration ([Ca2+]i) measurements using fura 2, the elevation of high K+-evoked [Ca2+]i was separated into nifedipine-sensitive and -resistant components. The nifedipine-resistant [Ca2+]i increase was also blocked by cilnidipine, as well as omega-conotoxin-GVIA. By the use of the conventional whole-cell patch-clamp technique, the compositions of the high-voltage-activated Ca2+ channel currents in the NGF-treated PC12 cells were divided into types: L-type, N-type, and residual current components. It was also estimated that cilnidipine at 1 and 3 micromol/L strongly blocked the N-type current without affecting the residual current. These results suggest that cilnidipine inhibits catecholamine secretion from differentiated PC12 cells by blocking Ca2+ influx through the N-type Ca2+ channel, in addition to its well-known action on the L-type Ca2+ channel.

Effects of AH-1058, a new antiarrhythmic drug, on experimental arrhythmias and cardiac membrane currents.

AH-1058 is a newly synthesized antiarrhythmic agent. We investigated the antiarrhythmic and electrophysiological effects of AH-1058 in experimental arrhythmia models and isolated cardiomyocytes. In the ouabain-induced arrhythmia model of the guinea pig, pretreatment with AH-1058 (0.1-0.3 mg/kg, i.v.) delayed the appearance of premature ventricular complex (PVC) and ventricular fibrillation (VF) induced by intravenous infusion of ouabain. However, disopyramide (10 mg/kg, i.v.) delayed only that of PVC, and verapamil (1 mg/kg, i.v.) failed to affect the ouabain-induced ventricular arrhythmias. In the reperfusion-induced arrhythmia model of the rat, in which 5-min coronary occlusion and 10-min reperfusion were produced, AH-1058 (0.1-0.3 mg/kg, i.v.) inhibited the incidence of both ventricular tachycardia (VT) and VF, whereas disopyramide (5 mg/kg, i.v.) inhibited only reperfusion-induced VF. On the other hand, a higher dose of AH-1058 (1 mg/kg, i.v.) did not affect the aconitine-induced arrhythmias in rats, which were inhibited by disopyramide (5 mg/kg, i.v.). We also confirmed oral activity of AH-1058 in the reperfusion-induced arrhythmia model of the rat. AH-1058, at doses of 2-4 mg/kg, dose-dependently inhibited VT and VF. Electrophysiological experiments with patch-clamp techniques revealed that AH-1058 potently suppressed the L-type Ca2+ currents in isolated cardiomyocytes of the guinea pig. These results suggest that AH-1058 is a potent antiarrhythmic drug having a Ca2+ channel-blocking action. The antiarrhythmic profile of AH-1058 is different from that of disopyramide and verapamil.

Production of free glutamate in milk requires the leucine transporter LAT1.

The concentration of free glutamate (Glu) in rats milk is ∼10 times higher than that in plasma. Previous work has shown that mammary tissue actively transports circulatory leucine (Leu), which is transaminated to synthesize other amino acids such as Glu and aspartate (Asp). To investigate the molecular basis of Leu transport and its conversion into Glu in the mammary gland, we characterized the expression of Leu transporters and [(3)H]Leu uptake in rat mammary cells. Gene expression analysis indicated that mammary cells express two Leu transporters, LAT1 and LAT2, with LAT1 being more abundant than LAT2. This transport system is sodium independent and transports large neutral amino acids. The Leu transport system in isolated rat mammary cells could be specifically blocked by the LAT1 inhibitors 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH) and triiodothyronine (T3). In organ cultures, Glu secretion was markedly inhibited by these LAT1 inhibitors. Furthermore, the profiles of Leu uptake inhibition by amino acids in mammary cells were similar to those reported for LAT1. In vivo, concentrations of free Glu and Asp increased in milk by oral gavage with Leu at 6, 12, and 18 days of lactation. These results indicate that the main Leu transporter in mammary tissue is LAT1 and the transport of Leu is a limiting factor for the synthesis and release of Glu and Asp into milk. Our studies provide the bases for the molecular mechanism of Leu transport in mammary tissue by LAT1 and its active role on free Glu secretion in milk, which confer umami taste in suckling pups.