Abstract: Calcium is an essential nutrient that induces a distinctive taste quality, but the sensing mechanism of calcium in the tongue is poorly understood. A recent study linked calcium to T1R3 receptor. Here, we propose another system for calcium taste involving the extracellular calcium-sensing receptor (CaSR). This G protein-coupled receptor that responds to calcium and magnesium cations is involved in calcium homeostasis regulating parathyroid and kidney functions. In this study, CaSR was found in isolated taste buds from rats and mice. It was expressed in a subset of cells in circumvallate and foliate papillae, with fewer cells in the fungiform papillae. This is the first evidence in mammals that locates CaSR in gustatory tissue and provides the basis for better understanding not only calcium taste but also the taste of multiple CaSR agonists.
Abstract: Gustatory and anticipatory cephalic stimuli during a meal yield nutritional information and aid efficient food digestion. Mammals, including humans, can detect the amount of dietary protein and its quality via cephalic relay to initiate proper digestion in the upper gastrointestinal (GI) tract. In addition to gustatory stimuli, visceral sensing by the abdominal vagus conveys primary afferent nutritional information from the digestive system to the brain. Electrophysiological studies indicated that abdominal vagal afferents, which were innervated into the stomach and intestine sending information to the brain, were activated by luminal glutamate. Histochemical analysis also revealed the existence of a glutamate signalling system (metabotrophic glutamate receptors) in the GI tract. Luminal glutamate in the stomach and intestine provides the efferent reflection of the abdominal vagus, supporting the modulation of exocrine and endocrine excretion during digestion. These results strongly indicate that glutamate has regulatory effects on the food digestive processes through the gut nutrient-sensing system. It plays physiological and nutritional roles and initiates digestion in the stomach as well as anticipates subsequent processes in the small intestine and the liver. We reviewed recent studies on glutamate physiology in the gut including our research, and discussed the physiological significance of dietary free glutamate in the regulation of gut function, focusing on the visceral sensation from the stomach.
Abstract: The appropriate recognition of nutrients in gastrointestinal tract has an important role in the maintenance of healthy body functions. Perception of nutrients in gastrointestinal tract influences not only the regulation of gastrointestinal functions such as digestion and absorption, but also the subsequent formation of ingestive behaviors and food preferences. Glutamate is widely distributed amino acids, approximately 15-45% in the dietary protein as the total of free- and bound-forms. To note is that, glutamate is implicated in numerous physiological and metabolic functions in the body as a nutrient, in addition, free glutamate in the food is also known as the source of umami, one of the five basic taste. Taste is basically percepted on the tongue in the oral cavity via the specific taste sensors (receptors and/or transporters), however, the recent studies suggest the functional roles of taste sensors in the regulation of gastrointestinal tract. Interestingly, we recently found the presence of specific glutamate-sensing systems in the lumen of the stomach. In addition, we have been vigorously studying the physiological roles of dietary free glutamate in the body through experimental animals and human. In this topic, we review the beneficial roles of dietary free glutamate in the regulation of gastrointestinal functions and propose some application of the benefits for the clinical gastrointestinal disorders.
Abstract: l-glutamate not only confers cognitive discrimination for umami taste in the oral cavity, but also conveys sensory information to vagal afferent fibers in the gastric mucosa. We used RT-PCR, western blotting, and immunohistochemistry to demonstrate that mGluR1 is located in glandular stomach. Double staining revealed that mGluR1 is found at the apical membrane of chief cells and possibly in parietal cells. Moreover, a diet with 1% l-glutamate induced changes in the expression of pepsinogen C mRNA in stomach mucosa. These data suggest that mGluR1 is involved in the gastric phase regulation of protein digestion.
Abstract: Recent advancements in molecular biology in the field of taste perception in the oral cavity have raised the possibility for ingested nutrients to be "tasted" in the upper gastrointestinal tract. The purpose of this study was to identify the existence of a nutrient-sensing system by the vagus in the rat stomach. Afferent fibers of the gastric branch increased their firing rate solely with the intragastric application of the amino acid glutamate. Other amino acids failed to have the same effect. This response to glutamate was blocked by the depletion of serotonin (5-HT) and inhibition of serotonin receptor(3) (5-HT(3)) or nitric oxide (NO) synthase enzyme. Luminal perfusion with the local anesthesia lidocaine abolished the glutamate-evoked afferent activation. The afferent response was also mimicked by luminal perfusion with a NO donor, sodium nitroprusside. In addition, the NO donor-induced afferent activation was abolished by 5-HT(3) blockade as well. Altogether, these results strongly suggest the existence of a sensing system for glutamate in the rat gastric mucosa. Thus luminal glutamate would enhance the electrophysiological firing rate of afferent fibers from the vagus nerve of the stomach through the production of mucosal bioactive substances such as NO and 5-HT. Assuming there is a universal coexistence of free glutamate with dietary protein, a glutamate-sensing system in the stomach could contribute to the gastric phase of protein digestion.
Abstract: Cilnidipine is a 1,4-dihydropyridine derived L/N-type calcium channel dual blocker possessing neuroprotective and analgesic effects which are related to its N-type calcium channel inhibitory activity. In order to find specific N-type calcium channel blockers with the least effects on cardiovascular system, we performed structure-activity relationship study on APJ2708, which is a derivative of cilnidipine, and found a promising N-type calcium channel blocker 21b possessing analgesic effect in vivo with a 1600-fold lower activity against L-type calcium channels than that of cilnidipine.
Abstract: Melatonin, a pineal secretory product synthesized from tryptophan, has been found to be effective against neurotoxicity. The present study was aimed at demonstrating the effectiveness of melatonin in vivo in reducing ischemia-induced cerebral edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.) just prior to 1 hr of MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. In the saline-treated control rats, increases in T2-weighted signals (water content) were clearly observed in the striatum and in the cerebral cortex. In the melatonin-treated group, total volume of edema was reduced by 51.6% compared with control group (P < 0.01). The protective effect of melatonin against edema was more clearly observed in the cerebral cortex (reduced by 59.8%, P < 0.01) than in the striatum (reduced by 34.2%, P < 0.05). Edema volume in a coronal slice was the greatest at the level of the bregma. Suppression of cerebral edema by melatonin was more effective posterior than anterior to the bregma. Melatonin appeared to reduce the volume of the edematous sites rather than to shift the signal intensity distribution. The present MRI study clearly demonstrates the effectiveness of melatonin against cerebral edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments associated with ischemic stroke.
Abstract: Gustatory and anticipatory cephalic stimuli that are detected during a meal yield nutritional information and aid in the efficient digestion of food. It is possible that animals can detect the amount of dietary food and its quality via cephalic relay to initiate proper digestion in the alimentary tract. The abdominal vagus conveys primary afferent nutritional information from the digestive system to the brain and modulates gastrointestinal motor and secretory activity that ensures the food digestion through local and central reflexes. Almost all the 5-HT in the body exists in the enterochromaffin (EC) cells in the alimentary tract, but the physiological significance of the mucosal 5-HT is not well elucidated. In the present paper, we reviewed recent advances in studies on gut nutrient perception and proposed the hypothesis that 5-HT derived from EC cells acts as a general transmitter of gut nutrient sensing by the abdominal vagus.
Abstract: The Ca(2+) channel-blocking efficacy of the cyproheptadine derivative AH-1058 (4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-1-[(E)-3-(3-methoxy-2-nitro)phenyl-2-propenyl]piperidine hydrochloride) was quantitatively assessed using isolated guinea pig cardiomyocytes. AH-1058 (0.001 - 10 microM) and its mother compound cyproheptadine (1 - 100 microM) reduced the Ca(2+) currents elicited from the holding potential of -80 or -40 mV. The IC(50) values for cyproheptadine at holding potentials of -80 and -40 mV were 42.44 and 7.75 microM, respectively, whereas those for AH-1058 were 4.91 and 0.32 microM, respectively, whose potency was equivalent to those of the typical Ca(2+) channel blocker verapamil. These results suggest that the introduction of the cinnamil structure to cyproheptadine can generate a potent L-type Ca(2+) channel-blocking compound as potent as verapamil.
Abstract: Systemic administration (i.v.) of serotonin (5-HT) evoked a transient vagal afferent nerve discharge, bradycardia, and hypotension in the rat. The half-effective dose of 5-HT for nerve discharge was 13 micro g/kg. The time- and dose-dependent kinetics of the nerve discharge rate were similar to the change of heart rate. The afferent neuronal discharge was mimicked by a selective 5-HT3 receptor agonist, 1-phenylbiguanide hydrochloride (PBA), and inhibited by a selective 5-HT3 antagonist, granisetron. The 5-HT(3/4) agonist, cisapride partially activated the vagus nerve, but the 5-HT4 agonist, RS6733 had no effect on the vagal afferent activity. Intra-gastric perfusion of lidocaine, moreover, abolished the 5-HT-induced vagal activation. These results indicate that the 5-HT transmission signal in the gastric mucosa inputs to the brain stem via 5-HT3 receptor-mediated vagal nerve afferent.
Abstract: Little is known about the psychobehavioral consequences of a dietary deficiency of the amino acid, L-lysine. This report demonstrates that a 4-d long L-lysine deficiency in rats interfered with the normal circadian release of the neurotransmitter serotonin, but not dopamine, measured by in vivo microdialysis in the central nucleus of the amygdala. L-Lysine deficiency was induced by feeding rats a L-lysine-deficient diet. Controls were pair-fed a L-lysine-sufficient diet. Footshock stress-induced anxiety, measured in an elevated plus-maze paradigm, and wrap-restraint stress-stimulated fecal excretion were significantly greater in the L-lysine-deficient rats than in the controls. We conclude that a severe deficiency of dietary L-lysine enhances serotonin release in the amygdala, with subsequent changes in psychobehavioral responses to stress.
Abstract: Reduction of cerebral edema, an early symptom of ischemia, is one of the most important remedies for reducing subsequent chronic neural damage in stroke. Melatonin, a metabolite of tryptophan released from the pineal gland, has been found to be effective against neurotoxicity in vitro. The present study was aimed to demonstrate the effectiveness of melatonin in vivo in reducing ischemia-induced edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.): just prior to 1 h MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. Increases in T2-weighted signals in ischemic sites of the brain were clearly observed after MCA occlusion. The signal increase was found mainly in the striatum and in the cerebral cortex in saline-treated control rats. In the melatonin-treated group, the total volume of cerebral edema was reduced by 45.3% compared to control group (P < 0.01). The protective effect of melatonin against cerebral edema was more clearly observed in the cerebral cortex (reduced by 56.1%, P < 0.01), while the reduction of edema volume in the striatum was weak (reduced by 23.0%). The present MRI study clearly demonstrated that melatonin is effective in reducing edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments.
Abstract: 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.
Abstract: Irregular functions in Ca2+ channels are intimately involved in many aspects of cardiovascular diseases. We can obtain a wide variety of L-type Ca2+ channel antagonists to treat hypertension and angina pectoris. Dihydropyridines (DHPs) have, first of all, been extensively developed due to their high selectivity for L-type Ca2+ channel and safety in pharmacological aspects. In contrast, many lines of evidence suggest that clinical efficacy of those DHPs are limited and undesirable effects are sometimes observed because of the specific distribution of L-type Ca2+ channels. As well as the L-type, peripherally distributed N-type Ca2+ channel plays a key role in cardiovascular regulation through autonomic nervous system. Recently, we developed a unique DHP derivative, cilnidipine (FRC8653) which has a dual antagonistic action on both L-type and N-type Ca2+ channels. Our recent studies with this DHP have made it clear that the N-type Ca2+ channel is also a new therapeutic target in cardiovascular diseases. We review the recent advances in pharmacology of the N-type Ca2+ channel and therapeutic implications of their antagonists.
Abstract: 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.
Abstract: 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.
Abstract: Megakaryocytes respond to externally applied agonists showing a periodic K+ current that reflects oscillation in cytoplasmic calcium concentration ([Ca2+]i). We have revealed several signal transducing factors that are involved in the K+ current oscillation of megakaryocytes. In this megakaryocyte system, it is relatively easy to determine what point of the signal transduction pathway a drug affects. In addition, as a progenitor cell, megakaryocytes resemble platelets which have important roles in many diseases. Therefore, this experimental system can be used for evaluation of new drugs.
Abstract: The effect of imipramine on the ATP-evoked release of dopamine was analyzed in parallel with its effects on the rise in the intracellular Ca2+ concentration ([Ca2+]i) and current induced by ATP in rat pheochromocytoma PC12 cells. Imipramine (10-300 microM) inhibited the ATP-evoked release of dopamine and rise in [Ca2+]i in a concentration-dependent fashion though the effect of imipramine on the release was slightly more obvious. Imipramine also inhibited the ATP-activated inward current at a similar concentration range. These results show a new pharmacological profile of imipramine, namely the inhibition of P2X2 receptors.
Abstract: The regulatory effects of cyclic GMP on purinoceptor-operated cytoplasmic Ca2+ oscillation of rat megakaryocytes were investigated by using whole-cell patch-clamp technique. ATP-induced oscillatory K+ currents though Ca2+-activated K+ channels (I(KCa)S) were depressed by pretreatment with the guanylate cyclase activator, sodium nitroprusside, and a stable membrane-permeable cGMP analogue, 8-bromo-cGMP. The inhibition by sodium nitroprusside was blocked by treatment with a cyclic nucleotide-dependent protein kinase inhibitor, N-[2-(methylamino)]-5-isoquinolinesulfonamide x HCl (H-8) (10 microM), but not by a selective cAMP-dependent-protein kinase inhibitor, Rp-cAMPS (100 microM). The oscillatory I(KCa) directly evoked by intracellular D-myo-inositol-trisphosphate (IP3) perfusion was also inhibited by the application of sodium nitroprusside. The inhibitory effect of sodium nitroprusside disappeared when the ATP-induced oscillatory I(KCa) was changed to a monophasic sustained I(KCa) current by inhibition of Ca2+-ATPase. These results suggested that cGMP depressed Ca2+ mobilization by improving Ca2+-ATPase activity by phosphorylation.
Abstract: 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.
Abstract: Kinetic characteristics of thrombin receptor-mediated responses on rat megakaryocytes were examined by the use of the perforated patch clamp combined with the rapid drug exchange system termed the 'Y-tube method'. The application of thrombin evoked repetitive Ca(2+)-activated K+ current (IKCa) in a concentration-dependent manner. The characteristic features for thrombin-induced response compared with purinoceptor-induced response were the long latency, long washout time and fast desensitization. The similar IKCa as thrombin was induced by trypsin. Thrombin- and trypsin-induced IKCa were both inhibited by the protease inhibitor, SBTI, and the washout time for thrombin was markedly shortened (7.4 +/- 2.2 s) when thrombin was washed out by a solution containing soybean trypsin inhibitor. A synthetic thrombin receptor agonist peptide induced IKCa oscillation with shorter latency than thrombin.
Abstract: 1 The inhibitory effects of cilnidipine (FRC-8653) and various organic Ca2+ channel blockers on high voltage-activated Ba2+ currents (HVA IBa) in rat sympathetic neurones were examined by means of the conventional whole-cell patch-clamp recording mode under voltage-clamped conditions. 2 HVA IBa was classified into three different current components with subtype selective peptide Ca2+ channel blockers. No omega-Agatoxin IVA-sensitive (P-type) or omega-conotoxin MVIIC-sensitive (Q-type) current components were observed. Most (> 85%) IBa was found to consist of omega-conotoxin GVIA-sensitive N-type components. 3 The application of cilnidipine inhibited HVA 1Ba in a concentration-dependent manner. The Kd value for cilnidipine was 0.8 microM. Cilnidipine did not shift the current-voltage (I-V) relationship for HVA IBa, as regards the threshold potential and peak potential where the amplitude reached a maximum. 4 High concentration of three hypotensive Ca2+ channel blockers, nifedipine, diltiazem and verapamil, all inhibited HVA IBa in a concentration-dependent manner. The Kd values for nifedipine, diltiazem and verapamil were 131, 151 and 47 microM, respectively. A piperazine-type Ca2+ channel blocker, flunarizine, showed a relatively potent blocking action on IBa. The Kd value was about 3 microM. 5 These results thus show that cilnidipine potently inhibits the sympathetic Ca2+ channels which predominantly consist of an omega-Cg-GVIA-sensitive component. This blockade of the N-type Ca2+ channel, as well as the L-type Ca2+ channel by cilnidipine suggests that it could be used therapeutically for treatment of hypersensitive sympathetic disorders associated with hypertension.
Abstract: The effects of neuropeptide Y on isometric tension simultaneously measured with cytosolic Ca2+ concentration ([Ca2+]cyt) and Ca2+ sensitivity of contractile elements were studied in isolated canine basilar arteries. Neuropeptide Y (1-100 nM) increased [Ca2+]cyt and tension in a concentration-dependent and parallel manner, whereas 9,11-dideoxy-11 alpha,9 alpha-epoxymethano prostaglandin F2 alpha (U46619) (10-100 nM), a thromboxane A2 mimetic, produced a large contraction with a small increase in [Ca2+]cyt. Ca2+ channel antagonists such as d-cis-diltiazem (10 mM) abolished both [Ca2+]cyt and tension augmented by neuropeptide Y. In Ca(2+)-free solution containing 0.2 mM EGTA, neuropeptide Y did not change [Ca2+]cyt and tension, whereas U46619 transiently increased both of them. Furthermore, neuropeptide Y apparently did not affect the Ca2+ sensitivity when assessed in the artery permeabilized with Staphylococcus aureus alpha-toxin, whereas U46619 augmented it. These findings suggest that neuropeptide Y-induced contraction in the canine basilar artery is produced mainly by Ca2+ influx through L-type Ca2+ channels.
Abstract: In order to elucidate the role of neuropeptide Y (NPY) in cerebral circulation, we undertook the present study to examine the action of NPY itself, and the combined effects of NPY with other vasoconstrictor stimuli. NPY itself produced contractions of isolated canine basilar artery in a concentration-dependent manner, which was independent of the presence or absence of endothelium. C-terminal peptides of NPY (NPY12-36) and (NPY22-36) were weak agonists, while those without C-terminals were ineffective. The vasoconstriction produced by NPY was, however, strongly potentiated by increasing the K+ concentration in the medium up to 20 mM, or by pretreatment with tetraethylammonium, a K+ channel blocker and hemolysate containing oxyhemoglobin. NPY also augmented the contractile responses to prostaglandin F2 alpha, norepinephrine, and histamine, but not to serotonin. The contraction in response to NPY per se or in 20 mM K+ was effectively attenuated by Ca2+ antagonists such as d-cis-diltiazem, and in a Ca(2+)-free medium. These results suggest that in canine basilar artery, the activation of the Y1 receptor modulates the availability of the L-type Ca2+ channel, leading to enhance Ca2+ influx from the extracellular space and potentiate contractile effects of other cerebral vasoconstrictors. This might be involved in the cerebral vasospasm which occurs after subarachnoid hemorrhage.
Abstract: Extracellular application of ATP evoked the oscillatory K+ currents (IKCa) reflecting oscillation in cytoplasmic Ca2+ concentration ([Ca2-]i) of megakaryocyte isolated from rat bone marrow. We have reported that the [Ca2+], oscillation was regulated by intracellular Ca(2+)-pumping activity (Uneyama H.C. Uneyama and N. Akaike, 1993, J. Biol. Chem. 268, 168). Here we found that the Ca2+ pump of the megakaryocyte could be divided into at least two classes according to the sensitivity to phosphorylation-modulating drugs. The effects of protein kinase C and cyclic AMP-dependent protein kinase are complementary, and the effect of Ca2+/calmodulin is independent of the above two kinases. In addition, this is the first report concerning the physiological regulation of Ca(2+)-ATPase in living cells.
Abstract: ATP and thrombin both induced Ca2+ mobilization from intracellular Ca2+ store site of megakaryocyte, the progenitor cell of platelet (Uneyama C., Uneyama H. and Akaike N. (1993) J. Physiol. (Lond.), 470, 73-749). Since in platelet, thrombin is known as a strong agonist and ADP is known as a weak agonist, we further investigated the effect of these agonists on megakaryocyte. Thrombin induced Ca2+ mobilization, 5-hydroxy tryptamine (5-HT) release and aggregatory morphological changes in megakaryocyte, but ATP induced only Ca2+ mobilization. Thrombin-induced 5-HT release was inhibited by adenylate cyclase-activating drugs, and the morphological changes could be induced by H-8, an inhibitor of cAMP-dependent protein kinase. These results suggest that the Ca2+ mobilization is not sufficient to induce morphological changes, and the signal to cause morphological changes in megakaryocyte may be cAMP.
Abstract: We have found that adenine enhanced the purinoceptor-operated cytoplasmic Ca2+ oscillation in rat megakaryocytes at submillimolar concentrations. Guanine and other nucleic acid bases had no effect on this system. Adenine enhanced the reaction intensity but had no effect on the threshold concentration of ATP to evoke the oscillation.
Abstract: 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, purinoceptor, at concentrations of 0.3-10 microM and suramin, a non-selective P2 purinoceptor antagonist, at 1-30 microM 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.
Abstract: The effects of a mitochondrial uncoupler, FCCP, (carbonyl cyanide-p-trifluromethoxyphenyl-hydrazone) on the regulation of cytoplasmic Ca2+ were investigated in ATP-induced Ca2+ oscillation system of rat megakaryocyte. Application of FCCP did not induce any detectable Ca(2+)-activated K+ current (IKCa) but pretreatment with FCCP modulated the ATP-induced repetitive IKCa. FCCP abolished the IKCa induced by low concentrations of ATP. However, when the concentration of ATP was high, the uncoupler also changed the periodic current to a sustained one. Similar biphasic regulation by the uncoupler was observed in the case of IP3-evoked repetitive IKCa. These results indicate that FCCP inhibits both Ca2+ mobilization and elimination processes after IP3 liberation induced by agonist stimulation.
Abstract: 1. The intracellular mechanisms of serotonin (5-HT) response were investigated in dissociated rat hippocampal pyramidal neurons using the nystatin-perforated patch technique. 2. Under voltage-clamp conditions, 5-HT evoked outward currents (I5-HT) with an increase in membrane conductance at a holding potential of -40 mV. The outward current reversed at the K+ equilibrium potential, which shifted 59.4 mV with a 10-fold change in extracellular K+ concentration. 3. The first application of 5-HT on neurons perfused with Ca(2+)-free external solution induced outward currents of I5-HT but the amplitude was diminished dramatically with successive applications. Pretreatment with the membrane-permeant Ca2+ chelator 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) also diminished the I5-HT amplitude. 4. Pretreatment with pertussis toxin (PTX) had no effect on I5-HT. 5. The I5-HT was not cross-desensitized with the caffeine-induced outward current but with outward current mediated by the muscarinic acetylcholine receptor. Pretreatment with Li+ significantly enhanced the I5-HT, indicating that I5-HT is involved in the elevation of intracellular free Ca2+ released from inositol triphosphate (IP3)-sensitive Ca2+ store sites but not from the caffeine-sensitive ones. 6. The calmodulin (CaM) antagonists, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited I5-HT in a concentration-dependent manner. 7. The Ca2+/CaM-dependent protein kinase II inhibitor 1-[N,O-Bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosil]-4-phenylpiperazine depressed the I5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: 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.
Abstract: Extracellular application of ATP and ADP evoked the oscillatory K+ currents (IKCa) resulting from the periodic rise in cytoplasmic Ca2+ concentration ([Ca2+]i) of megakaryocyte isolated from rat bone marrow (Uneyama, H., Uneyama, C., and Akaike, N. (1992) Jpn. J. Pharmacol. 58, 231). The intracellular mechanism of ATP-induced cytoplasmic Ca2+ oscillation was investigated by the use of nystatin-perforated patch-clamp technique. Caffeine and ryanodine, which release Ca2+ from the Ca(2+)-induced Ca2+ release pool (CICR), and procaine, a blocker of Ca2+ release from CICR, had no effect on the IKCa oscillation in megakaryocyte. Thapsigargin and A23187 activated IKCa irreversibly by mobilizing Ca2+, but under Ca(2+)-free conditions they activated IKCa transiently. Intracellular application of inositol 1,4,5-triphosphate (IP3) also induced IKCa oscillation. Phorbol myristate acetate (PMA), a strong activator of protein kinase C (PKC), inhibited the oscillation completely. The inhibitory action of PMA was reversed by an inhibitor of PKC, staurosporin. The oscillation of ATP-induced IKCa was disrupted by staurosporin or calmodulin (CaM) antagonists such as W-7 and trifluoperazine, resulting in a transient and successive plateau-like IKCa. These results suggest that Ca2+ oscillation in megakaryocyte is caused by the interaction of both the Ca2+ release from IP3-sensitive Ca2+ pool and the Ca2+ uptake stimulated by PKC and Ca2+/CaM complex. Furthermore, forskolin, an activator of adenylate cyclase, and isobutylmethylxanthin, an inhibitor of phosphodiesterase, inhibited the frequency, latency, and current amplitude of the oscillation in a concentration-dependent manner. These reagents inhibited IP3-induced oscillation as well as an ATP-induced oscillation. Thus, the ATP-induced [Ca2+]i oscillation of rat megakaryocyte is also modulated by cAMP.
Abstract: 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 gamma-aminobutyric acid (GABA) evoked Cl- currents in hippocampal neurones. The half-maximum effective concentrations (EC50) of glycine and GABA were 8.5 x 10(-5) and 5 x 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 x 10(-4) M for the glycine response and 3.6 x 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 > or = caffeine > paraxanthine > IBMX > or = theobromine > dyphylline. Xanthine had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: 1. The effect of rolipram (ME3176) on ADP- and IP3-induced repetitive IK(Ca) in rat megakaryocyte was investigated by use of the nystatin perforated patch and conventional whole-cell patch-clamp techniques. 2. The ADP-induced IK(Ca) was depressed by treatment with rolipram in a concentration-dependent manner. The inhibition by rolipram disappeared after treatment with a cyclic nucleotide-dependent protein kinase inhibitor, H-8. The inhibition of IK(Ca) was also observed in the presence of cyclic AMP accumulating agents such as forskolin and isobutylmethylxanthine (IBMX). 3. Rolipram enhanced the inhibitory action of forskolin, suggesting that rolipram facilitates the accumulation of cyclic AMP by blocking its breakdown. Similar results was obtained with adenosine, an endogenous adenylate cyclase activator. 4. Intracellular application of inositol trisphosphate (IP3) induced repetitive IK(Ca) in megakaryocytes. The induced IK(Ca) was also inhibited by rolipram and by other cyclic AMP accumulating agents. 5. It was concluded that megakaryocytes possess rolipram-sensitive phosphodiesterase (PDE), which was not detected in platelets, but plays a distinct modulatory role in megakaryocytes for generating ADP-induced IK(Ca).
Abstract: 1. The responses of megakaryocytes isolated from rat bone marrow to externally applied adenosine triphosphate (ATP) were investigated in the whole-cell mode by the use of nystatin perforated patch-clamp technique. 2. ATP at 1-100 microM evoked periodic outward currents at a holding potential of -40 mV. The reversal potential of the currents was close to K+ equilibrium potential (EK) and the K+ channel blockers such as quinine and quinidine suppressed the currents, indicating that the outward currents are predominantly carried by K+. 3. Since it has been reported that adenosine diphosphate (ADP) evoked monophasic K+ current using a conventional whole-cell recording, we compared the results obtained by perforated and conventional patch-clamp techniques. The crucial difference between our results and previous results was due to the intracellular perfusion with internal solution containing a high concentration of EGTA by which both current shape and concentration response were modified. 4. The membrane permeable Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxy methyl ester; BAPTA AM), inhibited the K+ current concentration dependently, suggesting that ATP-induced oscillatory K+ currents are caused by changes in cytoplasmic free Ca2+ concentration ([Ca2+]i). 5. With increasing ATP concentration, the frequency and the maximum amplitude of K+ current oscillation increased and the latency of current, which is the period required to activate the first K+ current after ATP application, decreased. 6. ADP, 2-methylthio-ATP and ATP-gamma-S could also evoke the periodic K+ currents, but adenosine, uridine triphosphate (UTP) and alpha-beta-methylene adenosine 5'-triphosphate (AMP-CPP) failed. 2-Methylthio-ATP was the most potent agonist; next was ADP which showed a 10-30 times stronger effect than ATP. Cross-desensitization was observed between ATP and ADP, but not between ATP or ADP and thrombin. 7. Extracellular Ca2+ was not required for the ATP-induced K+ current activation, indicating that Ca2+ released from intracellular pools induced the oscillatory response. In addition, the agonist potency increased when extracellular Ca2+ concentration ([Ca2+]o) decreased, suggesting that the principal agonists might be ATP4- and ADP3-. 8. The results suggest the presence of a novel subtype of purinoceptor in the megakaryocyte plasma membrane which induces cytoplasmic Ca2+ oscillation and evokes periodic K+ current flux.
Abstract: The effect of cytoplasmic pH (pH(i)) on ATP-induced Ca2+ oscillation of rat megakaryocytes was investigated by the whole-cell patch-clamp technique. Megakaryocytes responded to extracellular ATP and showed the periodic activation of K+ channels, which reflects the oscillation in intracellular free Ca2+ concentration. Intracellular alkalinization by 20 mM NH4Cl resulted in inhibition of the oscillatory response, and intracellular acidification by 20 mM sodium acetate enhanced the response. NH4Cl also inhibited the Ins(1,4,5)P3-induced oscillation. Sodium acetate had no effect on the InsP3-induced oscillation, but enhanced the guanosine 5'-[gamma-thio]triphosphate-induced response. These results indicate that pH(i) modulates the ATP-induced cellular response at least at two points.
Abstract: 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.
