Issei Seyama

Mechanism of nerve membrane depolarization caused by grayanotoxin I

1. The mechanism of depolarization of squid axon membranes caused by grayanotoxin I has been studied by means of internal perfusion and voltage clamp techniques.

The configuration of the P wave during mild exercise

Abstract A method of recording the average pattern of the P wave in the normal human electrocardiogram was described. The electrocardiogram was recorded with a tape recorder. The tape was then played back in reverse so that the SRQ spike could trigger the sweep circuit of both the cathoderay oscilloscope and the average transient computer. Fifty consecutive P waves were either superimposed photographically on the oscilloscope screen or computed by the average transient computer. The latter method was found to be suited for the P wave during exercise. The average pattern of the P wave invariably shows two tiny notches. During exertion the amplitude of the P wave increased, the P wave assumed a rather smooth contour, and notches disappeared in some instances. The P wave of the canine heart also increased in amplitude after the intravenous administration of epinephrine. The notches on the P wave disappeared and the peaked P wave was obtained. These observations suggest that the mechanism for the increase in the P-wave amplitude during exercise might be, in part, the synchronous excitation of the bilateral atrial chambers.

Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Nav1.2, Nav1.4, and Nav1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879–889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Nav1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257–262). Thus, we hypothesized that ciguatoxin-induced activation of the Nav1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 μm CTX3C preferentially affected the activation process of the Nav1.8 channel compared with those of the Nav1.2 and Nav1.4 channels. Importantly, without stimulation, 0.1 μm CTX3C induced a large leakage current (I L). The conductance of the I L calculated relative to the maximum conductance (G max) was 10 times larger than that of Nav1.2 or Nav1.4. To determine the molecular domain of Nav1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Nav1.4 and Nav1.8. Chimeras containing the N-terminal half of Nav1.8 exhibited a large response similar to wild-type Nav1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells

The effects of changing the intracellular concentrations of Ca2+ or Mg2+ ([Ca2+]i, [Mg2+]i) on Ca current (ICa) was studied in frog ventricular myocytes using the whole-cell and cell-attached patch clamp techniques. In the physiological range of [Mg2+]i an increase in [Ca2+]i enhancedICa whereas at lower [Mg2+]iICa was suppressed. The increase inICa caused by Ca2+ loading was not mediated by phosphorylation since the kinase inhibitors H-8 {N-[2-(methylamino)-ethyl]-5-isoquinolinesulphonamide dihydrochloride}, staurosporine and KN-62 {1-[N,O-bis(5-isoquinoline-sulphonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine} and a non-hydrolysable adenosine 5′-triphosphate analogue β,γ-methyleneadenosine 5′-triphosphate did not prevent the Ca2+-inducedICa increase.ICa was dramatically increased from 10 ± 6 (n = 4) to 71 ± 7 nA/nF (n = 4) when [Mg2+]i was lowered from 1.0 × 10−3 to 1.0 × 10−6 M at a [Ca2+]i of 10−8 M. The concentration response relation for inhibition of Ca channels by [Mg2+]i is modulated by [Ca2+]i. To account for the experimental results it is postulated that competitive binding of Ca2+ or Mg2+ to the Ca channel accelerates the transition of the channel from an active to a silent mode. Single-channel recordings support this hypothesis. The regulation may have clinical relevance in cytoprotection during cardiac ischaemia.

On site of action of grayanotoxin in domain 4 segment 6 of rat skeletal muscle sodium channel

Grayanotoxin I (GTX I) is a diterpenoid extracted from the family of Ericaceae that binds to Na+ channels and causes persistent activation. We investigated the interaction of GTX I with the amino acid residues I1575, F1579 and Y1586 in transmembrane segment D4S6 of μ1. In F1579A, GTX shifted the threshold potential about 50 mV in the hyperpolarizing direction and modified Na+ channels twice as efficiently as that in wild‐type. In contrast, these GTX‐effects were eliminated completely in the I1575A mutant and were reduced substantially in mutant Y1586A. Lysine substitution for F1579 significantly reduced and for Y1586 completely eradicated the GTX‐effect. Our data suggest that the GTX receptor site shares overlapping but non‐identical molecular determinants with BTX in D4S6 and has common molecular determinants in D1S6.

A study of the electrical characteristics of sodium currents in single ventricular cells of the frog.

1. The generation of action potentials elicited from enzymatically dispersed ventricular cells from the frog, Rana catesbeiana, has been shown to be due to the influx of both Na+ and Ca2+. The maximum rate of rise, the amplitude and the duration at 50% repolarization of the action potential were estimated to be 26.4 +/‐ 5.1 V/s (n = 8), 110 +/‐ 2.7 mV (n = 8) and 601 +/‐ 180 ms (n = 8) at 15 degrees C, respectively. 2. Inward Na+ current (INa) was studied in these ventricular cells by the whole‐cell patch clamp technique in a medium where Ca2+ current was eliminated by substituting extracellular Mg2+ for Ca2+ and K+ current was suppressed by applying Cs+ intracellularly. All the voltage clamp experiments were carried out at 4 degrees C. 3. INa elicited by single depolarizing steps from a holding potential (VH) of ‐80 mV had a threshold of ‐50 mV and was maximal at ‐20 mV. Peak currents in normal Ringer solution containing 113.5 mM‐Na+ were of the order of 0.01‐0.02 mA/cm2. Maximum Na+ conductance (gNa) was calculated to be 5.9 mS/cm2. 4. Under normal conditions the reversal potential for INa was determined to be 50 mV, which is close to the value predicted from the Nernst equation. The reversal potential changed by 59 mV per tenfold change in the activity of extracellular Na+ (aNa). 5. The instantaneous relation between INa tail currents and membrane potential is linear, crossing the abscissa at the reversal potential for INa. 6. Reconstructions of INa were made in terms of the parameters of the Hodgkin‐Huxley model for the squid axon, using constants obtained from the frog ventricular cells. 7. The falling phase of INa and the development of inactivation measured by the double‐pulse method could be well fitted by a single‐exponential function. 8. The time course for recovery of INa from inactivation exhibited a single time constant.

Effect of urine pH changed by dietary intervention on uric acid clearance mechanism of pH-dependent excretion of urinary uric acid

BackgroundThe finding reported in a previous paper - alkalization of urine facilitates uric acid excretion - is contradictory to what one might expect to occur: because food materials for the alkalization of urine contain fewer purine bodies than those for acidification, less uric acid in alkaline urine should have been excreted than in acid urine. To make clear what component of uric acid excretion mechanisms is responsible for this unexpected finding, we simultaneously collected data for the concentration of both creatinine and uric acid in serum as well as in urine, in order to calculate both uric acid and creatinine clearances.MethodsWithin the framework of the Japanese government’s health promotion program, we made recipes which consisted of protein-rich and less vegetable-fruit food materials for H + -load (acidic diet) and others composed of less protein and more vegetable-fruit rich food materials (alkaline diet). This is a crossover study within some limitations. Healthy female students, who had no medical problems at the regular physical examination provided by the university, were enrolled in this consecutive 5-day study for each test. From whole-day collected urine, total volume, pH, organic acid, creatinine, uric acid, titratable acid and all cations (Na+,K+,Ca2+,Mg2+,NH4+) and anions (Cl−,SO42−,PO4−) necessary for the estimation of acid–base balance were measured. In the early morning before breakfast of the 1st, 3rd and 5th experimental day, we sampled 5 mL of blood to estimate the creatinine and uric acid concentration in serum.Results and discussionUrine pH reached a steady state 3 days after switching from ordinary daily diets to specified regimens. The amount of acid generated ([SO42−] + organic acid − gut alkali)was linearly related with the excretion of acid (titratable acid + [NH4+] − [HCO3−]), indicating that H + in urine is generated by the metabolic degradation of food materials. Uric acid and excreted urine pH retained a linear relationship, as reported previously. Among the five factors which are associated with calculating clearances for both uric acid and creatinine, we identified a conspicuous difference between acidic and alkaline diets in the uric acid concentration in serum as well as in urine; uric acid in the serum was higher in the acidic group than in the alkaline group, while uric acid in the urine in the acidic group was lower than that in the alkaline group. These changes of uric acid in acidic urine and in serum were reflected in the reduction of its clearance. From these observations, it is considered that uric acid may be reabsorbed more actively in acidic urine than in alkaline urine.ConclusionWe conclude that alkalization of urine by eating nutritionally well-designed alkaline -prone food is effective for removing uric acid from the body.

Urine alkalization facilitates uric acid excretion

BackgroundIncrease in the incidence of hyperuricemia associated with gout as well as hypertension, renal diseases and cardiovascular diseases has been a public health concern. We examined the possibility of facilitated excretion of uric acid by change in urine pH by managing food materials.MethodsWithin the framework of the Japanese governments health promotion program, we made recipes which consist of protein-rich and less vegetable-fruit food materials for H+-load (acid diet) and others composed of less protein but vegetable-fruit rich food materials (alkali diet). Healthy female students were enrolled in this consecutive 5-day study for each test. From whole-day collected urine, total volume, pH, organic acid, creatinine, uric acid and all cations (Na+,K+,Ca2+,Mg2+,NH4+) and anions (Cl-,SO42-,PO4-) necessary for the estimation of acid-base balance were measured.ResultsUrine pH reached a steady state 3 days after switching from ordinary daily diets to specified regimens. The amount of acid generated ([SO42-] +organic acid-gut alkai) were linearly related with those of the excretion of acid (titratable acidity+ [NH4+] - [HCO3-]), indicating that H+ in urine is generated by the metabolic degradation of food materials. Uric acid and excreted urine pH retained a linear relationship, where uric acid excretion increased from 302 mg/day at pH 5.9 to 413 mg/day at pH 6.5, despite the fact that the alkali diet contained a smaller purine load than the acid diet.ConclusionWe conclude that alkalization of urine by eating nutritionally well-designed food is effective for removing uric acid from the body.

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes.

Under conditions of low intracellular [Mg2+] ([Mg2+]i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), l,2-bis(2-aminophenoxy)ethane-N, N, N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (ICa) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β, γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenyl-hydrazone (CCCP) did not inhibit the increase in ICa in the Mg2+-deficient medium. Thus, the involvement of phosphorylation process in the increase in ICa was not likely. Effective suppression of this enhancement of ICa was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC50) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of ICa is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed ICa similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg2+ and GTP on ICa. GTP is therefore assumed to bind directly to the Ca2+ channel. Interaction of Mg2+ and GTP with the Ca2+ channel activated in the Mg2+-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg2+]. The IC50 for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg2+ and GTP independently bind and control Ca2+ channels.

State-dependent action of grayanotoxin I on Na + channels in frog ventricular myocytes

1 Distinct properties of grayanotoxin (GTX) among other lipid‐soluble toxins were elucidated by quantitative analysis made on the Na+ channel in frog ventricular myocytes. 2 GTX‐modified current (IGTX) was induced strictly in proportion to the open probability of Na+ channels during preconditioning pulses irrespective of its duration, amplitude or partial removal of inactivation by chloramine‐T. This confirms that GTX binds to the Na+ channel exclusively in its open state, while batrachotoxin (BTX) was reported to be capable of modifying slow‐inactivated Na+ channels, and veratridine exhibited voltage‐dependent modification. 3 The GTX‐modified channel did not show any inactivation property, which is different from reported results with veratridine and BTX. 4 Estimated unbinding rates of GTX were in reverse proportion to the activation curve of GTX‐modified Na+ channels. This was not the previously reported case with veratridine. 5 A model including unbinding kinetics of GTX and slow inactivation of unmodified Na+ channels in which GTX was permitted to bind only to the open state of Na+ channels indicated that unbinding reactions of GTX occur only in the closed state.