Biomedical subjects

N Akaike

Publications and source records attributed to N Akaike.

At least 127 records · Page 7Linked to original sources

PubMed1995

Colocalization of ATP and nicotinic ACh receptors in the identified vagal preganglionic neurone of rat.

1. Effects of exogenous adenosine 5'-triphosphate (ATP) and acetylcholine (ACh) were investigated on acutely dissociated preganglionic neurones in the dorsal motor nucleus of vagus (DMV) of rats using whole-cell patch clamp recording methods. 2. The DMV neurones identified by retrograde transport of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) fixed onto the cervical vagal nerve bundle were large in size (25-35 microns diameter) and bipolar or tripolar in shape. 3. About 90% of DiI labelled DMV neurones responded to both ATP (10(-4) M) and ACh (10(-4) M) with inward currents at a holding potential (Vh) of -40 mV. 4. The ATP-induced current (IATP) and the ACh-induced current (IACh) reversed their polarities at membrane potentials between +5 and +15 mV, indicating that ATP and ACh increase the membrane permeability to cations. 5. The inhibitory potency of Reactive Blue on 5 x 10(-4) M IATP is more effective (concentration for half-inhibition (IC50), 4.4 x 10(-7) M) than suramin (IC50, 6.0 x 10(-6) M). In addition, alpha,beta-methylene ATP up to 10(-4) M could not induce any current. As intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) did not block the IATP, the IATP was mediated not by guanosine triphosphate (GTP) binding protein, but rather by ligand-gated ionic channels, presumably via P2X receptors. 6. Currents produced by ACh were due to activation of nicotinic receptors because they were mimicked by nicotine and carbachol, and blocked by hexamethonium. In addition, muscarine evoked no response. 7. Only 25% of nucleus tractus solitarii (NTS) neurones and no hypoglossal neurones responded to the exogenous application of ATP. 8. These results suggest that vagal preganglionic neurones colocalize functionally nicotinic and P2X purinergic receptors.

Acetylcholine↗

PubMed1995

Pharmacological studies on mechanisms involved in Ca2+ oscillations in rat megakaryocytes.

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.

Adenosine Triphosphate↗

PubMed1995

Muscarinic receptor agonist-induced increases in cytosolic Ca2+ concentrations in chick ciliary ganglion cells.

We used fura-2 microfluorometry to examine the mechanism underlying the muscarinic receptor agonist-induced increase in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick ciliary ganglion neurons. The order of potencies of muscarinic agonists in increasing [Ca]in was: oxotremorine M > muscarine > methacholine > oxotremorine > bethanechol. The muscarine-induced increase in [Ca]in persisted after treatment with thapsigargin, which blocked caffeine- and muscarinic agonist-induced Ca2+ release. The muscarine-sensitive [Ca]in increase was inhibited by both L- and N-type Ca2+ channel blockers but potentiated by an L-type Ca2+ channel agonist. Muscarine was effective in increasing [Ca]in in the presence of a desensitizing concentration of nicotine, and simultaneous addition of maximal doses of muscarine and nicotine caused an additive increase in [Ca]in. On the other hand, nicotine-, ATP-, and high K(+)-induced increase in [Ca]in was markedly potentiated during continuous stimulation with muscarine. These results suggest that muscarinic receptor stimulation increases Ca2+ influx passing through voltage-dependent Ca2+ channels. However, the muscarine-induced Mn2+ influx was observed in only some muscarine-sensitive cells, suggesting that muscarine-induced depolarization is too weak to overcome the inhibitory effect of Mn2+ on Ca2+ channels.

Animals↗

PubMed1995

Block of P-type Ca2+ channels in freshly dissociated rat cerebellar Purkinje neurons by diltiazem and verapamil.

We investigated the effects of organic Ca2+ channel blockers, diltiazem and verapamil, on the high voltage-activated P-type Ca2+ channels in freshly isolated rat Purkinje neurons. Both diltiazem and verapamil blocked P-type Ca2+ channel current without any change in the current-voltage relation. The block was concentration-dependent. In the presence of these agents, the inactivation curve was shifted to hyperpolarizing potentials. The characteristics of block of P-type Ca2+ channels by diltiazem and verapamil are similar to that of L-type Ca2+ channels. These results indicate that both benzothiazepine and phenylalkylamine react with P-type Ca2+ channels and suggest that some structural features common to which operate in both L-type and P-type Ca2+ channels may be involved in drug binding to these channels.

Animals↗

PubMed1995

Regional difference of high voltage-activated Ca2+ channels in rat CNS neurones.

The diversity of high voltage-activated (HVA) Ca2+ channels in rat CNS neurones was investigated with the nystatin perforated patch recording configuration. The neurones were freshly dissociated from rat substantia nigra, ventromedial hypothalamus, tuberomammillary nucleus, nucleus tractus solitarius, hippocampal CA1 region and cerebellum. Five different types of HVA Ca2+ channels were distinguished pharmacologically; dihydropyridine sensitive L-type, omega-conotoxin-GVIA sensitive N-type, omega-agatoxin-IVA sensitive P-type, omega-conotoxin-MVIIC sensitive Q-type, and R-type which is insensitive to these organic Ca2+ antagonists. The results showed clearly that the five subtypes of HVA Ca2+ channels differ considerably in their distribution among various CNS regions.

Animals↗

PubMed1995

Attenuated inhibition by levofloxacin, l-isomer of ofloxacin, on GABA response in the dissociated rat hippocampal neurons.

The effects of ofloxacin (OFLX) and its isomers, levofloxacin (LVFX) and DR-3354, on the gamma-aminobutyric acid (GABA)-induced Cl- current in acutely dissociated rat hippocampal CA1 neurons were investigated using nystatin perforated patch recording configuration under voltage-clamp conditions. At 10(-5) M these 3 compounds themselves did not affect the GABA response. Biphenylacetic acid (BPA) at 10(-5) M also had no effect on the GABA response, but BPA greatly suppressed the GABA response in combination with these 3 compounds without affecting the reversal potential of GABA response. The inhibitory effects of OFLX and DR-3354 on the GABA response were stronger than that of LVFX. LVFX inhibited the response in a competitive and voltage-independent manner. The results suggest that LVFX has lower CNS adverse effects, such as convulsions, compared to OFLX.

Animals↗

PubMed1995

Not Ca2+ but CAMP is the second messenger for morphological changes in rat megakaryocyte.

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.

Adenosine Diphosphate↗

PubMed1995

Caffeine rapidly decreases potassium conductance of dissociated outer hair cells of guinea-pig cochlea.

The effects of caffeine on the outer hair cells (OHCs) freshly dissociated from guinea-pig cochlea were investigated with the whole-cell patch-clamp technique, in both the conventional and the nystatin perforated patch-clamp configurations under voltage-clamp condition. Application of caffeine (> 1 mM for 10-30 s) induced an inward current (Icaffeine) with decrease of conductance in a dose-dependent manner at a holding potential (VH) of -60 mV. The reversal potential of Icaffeine (Ecaffeine) was close to the K+ equilibrium potential. The Icaffeine was not affected by Ca(2+)-free external solution. The internal perfusion of the Ca2+ chelator BAPTA had no effect on Icaffeine. The Icaffeine was not modulated by the external application of H-8 or staurosporine and by the internal perfusion of GDP-beta S. The amplitude of Icaffeine was the largest at the basal region of OHCs when caffeine was locally applied by the 'puffer' method. These results suggest that caffeine induces a decrease in membrane potassium conductance of the OHCs mainly at the basal region without mediating the intracellular signaling pathway.

Animals↗

PubMed1995

Gramicidin-perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride.

1. By the development of a new perforated patch method using gramicidin, the effects of GABA on neurones dissociated from the rat substantia nigra pars reticulata (SNR) were examined without disturbing the intracellular chloride concentration. 2. Using the patch pipette solution containing gramicidin (100 micrograms ml-1), the access resistance dropped to less than 20 M omega within 40 min after making the gigaohm seal. 3. Under current-clamp conditions, GABA caused a hyperpolarization accompanied by a blockade of spontaneous firing. Under voltage clamp at a holding potential (Vh) of -50 mV, GABA evoked an outward current by way of bicuculline- and picrotoxin-sensitive GABAA receptors. 4. A 10-fold change of extracellular chloride concentration resulted in a 58 mV shift of the reversal potential of GABA-induced outward current (EGABA), indicating that the membrane behaves like a chloride electrode in the presence of GABA. 5. The intracellular chloride activities (aCli), calculated with the Nernst equation using both extracellular chloride activity and EGABA values, ranged from 2.8 to 19.7 mM with a mean value of 9.5 mM. The aCli was not affected either by different pipette solutions or by different holding potentials more hyperpolarized than -40 mV. 6. In the recording from SNR neurones in brain slice using the gramicidin-perforated patch-clamp technique, the inhibitory and excitatory postsynaptic currents were recorded in different current directions and the former was blocked by bicuculline. 7. In conclusion, the gramicidin-perforated patch method will disclose previously unknown aspects of biological responses involving Cl-.

Amphotericin B↗

PubMed1995

Time-dependent rundown of GABA response in mammalian cns neuron during experimental anoxia.

Gamma-Aminobirtyric acid (GABA) is one of the major neurotransmitters in the mammalian central nervous system (CNS). The activation of post-synaptic GABAA receptor-chloride channel complex is thought to underlie inhibitory postsynaptic potentials ubiquitously in various CNS regions. GABAA receptors are modulated by convulsant, hypnotic-anticonvulsant, anxiolytic and anxiogenic agents and endogenous agents such as nurosteroids and intracellular calcium, ATP, and cyclic AMP. The function of GABAA receptor in CNS neuron is also affected by some pathophysiological processes, e.g., anoxia. For example, it is currently believed that delayed neuronal death after brain ischemia results from excessive cell excitability and/or loss of inhibition. In the present study, we investigated how the GABA-gated chloride current is affected by anoxic conditions. All experiments were carried out on neurons freshly dissociated from rat CNS by the use of both conventional and nystatin perforated patch recording configurations. The GABA response showed a considerable rundown with time in anoxic condition. The rundown was prevented by adding either ouabain or SPAI-I (Na+-K+ ATPase inhibitor-I), suggesting that the experimental anoxia reduced GABA response by decreasing intracellular ATP synthesis. This result was also confirmed by finding that the direct decrease of intracellular ATP concentration using a conventional whole-cell patch recording mode inhibited the GABA-gated chloride response in mammalian CNS neurons.

Adenosine Triphosphate↗

PubMed1995

Photolysis of a newly synthesized caged glycine activates the glycine receptor of rat CNS neurons.

The functions of 12 different newly synthesized caged glycines were examined on strychnine-sensitive glycine receptors on rat hypothalamic neurons using photochemical and physiological methods. Quantitative analysis of Cl- current induced by photolyzed glycines revealed N-[1-(2-nitrophenyl)ethylcarbonoxyl] glycine (N4-caged glycine) to be the most effectively photolyzed. The current amplitude induced by photolyzed N4-caged glycine was almost the same as that induced by normal glycine. We propose N-[1-(2-nitrophenyl)ethylcarbonoxyl] to be a useful photolabile moiety for new caged neurotransmitters.

Animals↗

PubMed1995

Changes of intracellular electrolyte contents in rat skeletal muscle during body suspension.

The CNS-mediated inhibition of active Na(+)-K+ transport in both "type S" muscle, soleus (SOL), and "type FF" muscle, extensor digitorum longus (EDL) was investigated in rats suspended horizontally. Plasma Na+ and K+ contents did not change during the suspension period. The relative wet weight of SOL decreased more than that of EDL by suspension. There was significant intracellular Na+ accumulation and K+ loss in both SOL and diaphragm of suspended rats. However, cerebrum, cerebellum, medulla oblongate, ventricle, liver, pancreas, kidney, intestine, aorta and EDL were spared from the intracellular Na+ accumulation and K+ loss. Sciatic nerve sectioning or cervical spinal cord transection recovered the Na+ and K+ contents in the SOL of suspended rats. The results indicate the existence of neural inhibition of the active Na(+)-K+ transport in skeletal muscle of the suspended rats.

Animals↗

PubMed1995

ATP responses in the embryo chick ciliary ganglion cells.

The ATP responses in ciliary neurons acutely dissociated from chick ciliary ganglia were investigated using the nystatin perforated patch recording mode. ATP induced a transient inward current at a holding potential of -50 mV in a concentration-dependent manner. The half-maximum effective concentration of ATP was 8.2 microM and the Hill coefficient was 0.9. The current-voltage relationship of the ATP response revealed a strong inward rectification at potentials more positive than -30 mV and the reversal potential was near 0 mV. The relative potencies of purinoceptor agonists were in the order of ATP > 2-methylthio-ATP > ADP. Neither adenosine, AMP nor alpha, beta-methylene-ATP induced any response. The ATP-induced inward current was blocked by suramin, a selective P2 purinoceptor antagonist, in a concentration-dependent manner. The half-maximum inhibitory concentration was 4.5 microM. The cytosolic Ca2+ concentration was increased by ATP and suramin inhibited the increase in a concentration-dependent manner. These results suggest that ATP operates non-selective cation channels by acting on P2y purinoceptors and has a role in the excitation of ciliary neurons.

Adenosine Diphosphate↗

PubMed1995

Norepinephrine modulates high voltage-activated calcium channels in freshly dissociated rat nucleus tractus solitarii neurons.

The effects of norepinephrine on the low- and high-voltage-activated calcium channels in the neurons acutely dissociated from the nucleus tractus solitarius of 2- to 3-week-old rats were investigated in the nystatin perforated patch recording configuration under voltage-clamp conditions. The norepinephrine had no effect on the low voltage-activated calcium channel but inhibited the high voltage-activated calcium channel in a concentration-, time- and voltage-dependent manner. The norepinephrine slowed the activation phase of the high voltage-activated calcium channel current and the maximum inhibition was 30% of the total current amplitude measured 10 ms after the current activation. The inhibitory effect was eliminated by applying larger depolarizing prepulses. The pretreatment with pertussis toxin completely blocked the norepinephrine effect on high-voltage activated calcium channels, suggesting the contribution of pertussis toxin-sensitive Gi/Go-proteins to the norepinephrine-induced inhibition. Yohimbine but not prazosin nor propranolol antagonized the norepinephrine-induced inhibition, suggesting the involvement of alpha 2-adrenoceptor in norepinephrine-induced inhibition of the high voltage-activated calcium channels. omega-Conotoxin-GVIA, omega-agatoxin-IVA, nicardipine and omega-conotoxin-MVIIC blocked the high voltage-activated calcium channel current by 26, 9, 36 and 11% of the total current respectively, suggesting the existence of N-, P-, L- and Q-type calcium channels in the nucleus tractus solitarii neurons. The current being insensitive to these calcium channel antagonists, termed R-type calcium channel current, also existed. This residual R-type calcium channel was completely blocked by adding 200 microM CD2+. The norepinephrine significantly inhibited N- and P-type calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Mechanism underlying the ATP-induced increase in the cytosolic Ca2+ concentration in chick ciliary ganglion neurons.

We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]in was dependent on external Ca2+, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca2+ channel blockers. ATP was effective in increasing [Ca]in in the presence of a desensitizing concentration of nicotine (100 microM), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na+ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na+ influx through P2 purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca2+ influx through voltage-dependent Ca2+ channels. However, ATP still caused a small increase in [Ca]in under Na+-free conditions, and this [Ca]in increase was little affected by Ca2+ channel blockers. ATP also increased Mn2+ influx under Na+-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca2+ but also to Mn2+, in addition to monovalent cations.

Adenosine Triphosphate↗

PubMed1995

Dual mechanisms of GABAA response inhibition by beta-lactam antibiotics in the pyramidal neurones of the rat cerebral cortex.

1. The effects of beta-lactam antibiotics on the gamma-aminobutyric acid (GABA)-induced Cl- current were investigated in pyramidal neurones freshly dissociated from the rat frontal cortex by the use of a nystatin-perforated patch recording mode under voltage-clamp conditions. 2. The GABA-induced inward current increased in a concentration-dependent manner with an EC50 of 6.7 x 10(-6) M at a holding potential of -40 mV. The GABA response was accompanied by an increase in the membrane conductance and reversed at near the Cl- equilibrium potential. 3. All beta-lactams (penicillin, imipenem, aztreonam and cefotiam) inhibited the 10(-5) M GABA-induced response in a concentration-dependent manner with an IC50 and Hill coefficient of 1.3 x 10(-3) M and 0.64 for penicillin, 9.6 x 10(-4) M and 0.83 for imipenem, 2.5 x 10(-3) M and 9.99 for aztreonam, and 2.9 x 10(-4) M and 1.03 for cefotiam. 4. Imipenem inhibited the GABA-response competitively while penicillin inhibited the same response in a noncompetitive fashion. 5. The inhibitory action of imipenem showed no voltage-dependency, whereas the effect of penicillin was voltage-dependent. 6. It is thus proposed that some classes of beta-lactams, including imipenem may have a mechanism that is different from penicillin and competitively affects the GABAA receptor.

Animals↗

PubMed1995

Suppression of a nonselective cation conductance by substance P in cochlear outer hair cells.

Substance P (SP) hyperpolarizes outer hair cells (OHCs) of guinea pig cochlea. The cellular mechanisms of the SP response were investigated with the whole cell patch-clamp technique. SP induced outward currents in a dose-dependent manner at a holding potential of -60 mV in a concentration range between 3 x 10(-6) and 10(-4) M. SP decreased slope conductance between -60 and +20 mV. Ion substitutions in the external medium revealed that SP suppresses nonselective cation conductance with high permeability for Ca2+. The relative ion permeability of the channel modulated by SP was as follows: Ca2+ > Li+ approximately Cs+ approximately Na+ > Tris+. The potency of the agonist action was as follows: SP >> neurokinin A > neurokinin B. Peptide antagonists induced currents similar to those of SP. CP-96345, a selective nonpeptide antagonist for the neurokinin type 1 receptor, did not inhibit the SP-induced current. Intracellular dialysis of guanosine 5'-O-(2-thiodiphosphate) and pertussis toxin (PTX) suggests that a PTX-insensitive G protein is involved in the SP response. Neither the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid nor staurosporine (10(-6) M) affects the SP response. Local application of SP by a puffer pipette indicates that the SP receptors are distributed along the side of the OHC. These results suggest the possibility that the action of SP on the OHCs may not be mediated by the tachykinin receptors but rather by a tachykinin receptor-independent pathway. It is proposed that SP suppresses the nonselective cation conductance in the lateral wall of OHCs via a PTX-insensitive G protein.

Animals↗

PubMed1995

Somatostatin modulates high-voltage-activated Ca2+ channels in freshly dissociated rat hippocampal neurons.

1. The effects of somatostatin (SS) on the low-voltage-activated and high-voltage-activated (HVA) Ca2+ channels in pyramidal neurons acutely dissociated from the hippocampal CA1 region of 2- to 3-wk-old rats were investigated in a nystatin perforated-patch recording configuration under voltage-clamp conditions. 2. SS had no effect on the low-voltage-activated Ca2+ channel but did inhibit the HVA Ca2+ channel in a concentration-, time-, and voltage-dependent manner. 3. SS showed the activation phase of Ba2+ current (IBa) passing through HVA Ca2+ channels, and the maximum inhibition was 28% of the total current amplitude measured 10 ms after the current activation. The inhibitory effect was eliminated by applying larger depolarizing prepulses. Pretreatment with pertussis toxin (PTX) completely blocked the effect of SS on HVA IBa, suggesting the contribution of PTX-sensitive Gi/Go proteins to the SS-induced inhibition. 4. The applications of forskolin, 8-Br-cAMP, dibutyryl-guanosine 3'5'-cyclic monophosphate, staurosporine, and 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine did not affect either the control HVA IBa or the SS-induced inhibition of HVA IBa. 5. Pretreatment with protein kinase C (PKC) activators had no significant effect on HVA IBa but did remove the inhibition of HVA IBa by SS. 6. Omega-Conotoxin-GVIA, omega-agatoxin-IVA, nicardipine, and omega-conotoxin-MVIIC blocked HVA IBa by 27, 13, 38, and 9% of the total HVA current, respectively, which suggested the existence of N-, P-, L-, and Q-type HVA Ca2+ channels in the hippocampal CA1 pyramidal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗