Effect of urine pH on uric acid excretion by manipulating food materials.

A potential utilization of dietary intervention for reducing hyperuricemia was tested by managing food materials. Within the framework of the Japanese Government's health promotion program, we made recipes that consisted of more protein-rich and less vegetable/fruit-rich materials for the acidic diet and others composed of less protein-rich and more vegetable/fruit-rich materials for the alkaline diet. We have shown that urine alkalization facilitates uric acid excretion. In this study, it has been clarified with simultaneous measurements of both serum and urine uric acid concentration that acidic diets increase serum uric acid together with a decrease of uric acid excretion. The ratio (R) of uric acid clearance/creatinine clearance was calculated. On the third experimental day, the relative R, referring to that of the first day for the acidic diet, became smaller than that for the alkaline diet, indicating that in acidic urine, uric acid excretion is limited by more active reabsorption, compared with that in alkaline urine. Taken together, we tentatively conclude that dietary intervention may well be the safest and the most economical way for the prevention of hyperuricemia.

Na+ channel pharmacology and molecular mechanisms of gating.

Electrogenesis of efficiently propagated action potentials requires synchronized opening of transmembrane Na+ channels possessing a sodium selectivity-filter, a high-throughput ion-conductance pathway, and voltage-dependent gating functions. These properties of the Na+ channel have long been the target of molecular analysis. Several toxins and drugs, known to selectively bind to Na+ channels, have been used as pharmacological tools to investigate Na+ channel properties either electrophysiologically or chemically. Recent analyses of the protein crystal structure of bacterial voltage-dependent K+ channels have provided important clues to the identity of mobile structures involved in channel gating. The new information may be applicable to Na+ channels, and may well require a total revision of our understanding of gating mechanisms of sodium channels. Several experiments challenge the emerging view that channel gating by S6 transmembrane segments is triggered by signals from voltage sensors floating in membrane lipid. Herein, we review the various toxin and drug molecules that affect the gating behavior of Na+ channels in this new structural framework, by characterizing the binding sites of these toxins, and assessing the pharmacological effects resulting from changes in the structure of the toxin or sodium channel.

Novel site on sodium channel alpha-subunit responsible for the differential sensitivity of grayanotoxin in skeletal and cardiac muscle.

We searched for sites on the alpha-subunit of the fast Na(+) channel responsible for the difference in GTX (grayanotoxin) sensitivity of the skeletal- and cardiac-muscle Na(+) current. cDNA clones, encoding the skeletal or cardiac isoforms of the alpha-subunit, were inserted into a mammalian expression vector and transiently transfected into human embryonic kidney cells. The expressed channels were measured using whole-cell patch-clamp techniques and examined for GTX sensitivity. As a measure of GTX sensitivity, we used relative chord conductance (ratio of maximum chord conductance of noninactivating GTX-modified Na(+) currents to that of unmodified peak currents). Wild-type channels from skeletal muscle (mu 1) were more sensitive to GTX modification than wild-type cardiac channels (rH1) by a factor of 1.6. To facilitate exploration of alpha-subunit sites determining GTX sensitivity, we used SHHH, a chimera of skeletal muscle (S) domain D1 and heart muscle (H) domains D2D3D4 with supernormal sensitivity to GTX I (1.5-fold of wild-type mu 1). Successive replacement of Ser-251 (D1S4-S5 intracellular loop) and Ile-433 (D1S6 transmembrane segment), with corresponding rH1 residues Ala and Val, reduced, in a stepwise manner, the GTX sensitivity of the chimera and related mutants to that of wild-type rHl. We concluded that, in addition to Ile-433, known as the GTX-binding site, Ser-251 represents a novel site for GTX modification.

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 (I(GTX)) 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.

Novel wasp toxin discriminates between neuronal and cardiac sodium channels.

Pompilidotoxins (PMTXs), derived from the venom of solitary wasp has been known to facilitate synaptic transmission in the lobster neuromuscular junction, and a recent further study from rat trigeminal neurons revealed that the toxin slows Na+ channel inactivation without modifying activation process. Here we report that beta-PMTX modifies rat brain type II Na+ channel alpha-subunit (rBII) expressed in human embryonic kidney cells but fails to act on the rat heart alpha-subunit (rH1) at similar concentrations. We constructed a series of chimeric mutants of rBII and rH1 Na+ channels and compared modification of the steady-state Na+ currents by beta-PMTX. We found that a difference in a single amino acid between Glu-1616 in rBII and Gln-1615 in rH1 at the extracellular loop of D4S3-S4 is crucial for the action of beta-PMTX. PMTXs, which are small peptides with 13 amino acids, would be a potential tool for exploring a new functional moiety of Na+ channels.

Novel mechanism of blocking axonal Na(+) channels by three macrocyclic polyamine analogues and two spider toxins.

1. The mechanism of Na(+) channel block by three macrocyclic polyamine derivatives and two spider toxins was studied with voltage clamp and internal perfusion method in squid axons. 2. All these chemicals specifically block Na(+) channels in the open state only from the internal surface, and do not affect K(+) channels. 3. The blocking effect is enhanced as the depolarizing pulse becomes larger. Blocked channels are unable to shift to the inactivated state. 4. In the case of cyclam and guanidyl-side armed cyclam (G-cyclam), quick release of these chemicals from the binding sites is proven by the increase in the tail current and prolongation of the time course of the off gating current. On the other hand, in the presence of N-4 and the spider toxins, their detachment was delayed significantly. 5. Molecular requirements for the block of Na(+) channels by these molecules are the presence of positive charge and hydrophobicity.

Rapid cardiac adaptation to exercise demand signal and execution of maximal leg muscle contraction.

We investigated the neural regulation of the cardiac interval to an exercise demand signal and to a repeated exercise in 20 healthy human subjects. Electrocardiogram (ECG), muscle torque, and electromyogram (EMG) were simultaneously measured and their time relationships compared before and during the exercise. The R-R interval of ECG was directly increased by the exercise demand signal itself before the onset of EMG but not reflexly by muscle contraction. The cardiac interval decreased at the onset of exercise. Under the condition of repeated maximum eccentric training, the resting cardiac interval decreased prior to the exercise, whereas the brief increase in cardiac interval to the exercise demand signal remained unchanged. These results suggested that when autonomic nerve activity to the pacemaker is activated by the exercise demand signal, an initial effect of vagal nerve activity appears, and an effect of vagal nerve withdrawal and/or sympathetic nerve activity then appears. The responses of the heart and leg skeletal muscle at the onset of exercise are not synchronized, and the cardiac interval is controlled by vagal and sympathetic nerve activities to effect a transition to a high heart rate as quickly and smoothly as possible.

An analysis of the variations in potency of grayanotoxin analogs in modifying frog sodium channels of differing subtypes.

Responses of tetrodotoxin-sensitive (TTX-s) and insensitive (TTX-i) Na(+) channels, in frog dorsal root ganglion (DRG) cells and frog heart Na(+) channels, to two grayanotoxin (GTX) analogs, GTX-I and alpha-dihydro-GTX-II, were examined using the patch clamp method. GTX-evoked modification occurred only when repetitive depolarizing pulses preceded a single test depolarization; modification, during the test pulse, was manifested by a decrease in peak Na(+) current accompanied by a sustained Na(+) current. GTX-evoked modification of whole-cell Na(+) currents was quantified by normalizing the conductance for sustained currents through GTX-modified Na(+) channels to that for the peak current through unmodified Na(+) channels. The dose-response relation for GTX-modified Na(+) channels was constructed by plotting the normalized slope conductance against GTX concentration. With respect to DRG TTX-i Na(+) channels, the EC(50) and maximal normalized slope conductance were estimated to be 31 microM and 0.23, respectively, for GTX-I, and 54 microM and 0.37, respectively, for alpha-dihydro-GTX-II. By contrast, TTX-s Na(+) channels in DRG cells and Na(+) channels in ventricular myocytes were found to have a much lower sensitivity to both GTX analogs. In single-channel recording on DRG cells and ventricular myocytes, Na(+) channels modified by the two GTX analogs (both at 100 microM), had similar relative conductances (range, 0.25-0.42) and open channel probabilities (range, 0.5-0.71). From these observations, we conclude that the differences in responsiveness of DRG TTX-i, and ventricular whole cell Na(+) currents to the GTX analogs studied are related to the number of Na(+) channels modified.

Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes.

The effects of sulfhydryl (SH) reagents on the L-type Ca current (ICa) were studied in frog ventricular myocytes using the whole-cell patch-clamp method. Methanethiosulfonate ethylammonium (MTSEA+) was found to enter the cell through the membrane and cause a remarkable increase in Ica from the intracellular side. Methanethiosulfonate ethyltrimethylammonium (MTSET+) and methanethiosulfonate ethylsulfonate (MTSES-) could not penetrate the membrane and were effective only when directly applied to the intracellular side. In addition, suppressive effects on ICa of these MTS reagents were indicated by the following observation. A progressive decay in the peak amplitude of ICa after establishing maximal ICa, stimulated by intracellular MTSET+, was prevented by adding extracellular dithiothreitol (DTT). The SH-oxidizing agents N-ethylmaleimide (NEM), chloramine-T (CL-T), 2,2'-dithiodipyridine (DTDP) and 2,2'-dithio-bis-5-nitropyridine (DTBNP) also exerted a stimulatory effect on Ica. The effect of SH reagents persisted even when cAMP production was inhibited with Rp-cAMP-S, or when G-protein was inhibited with 1 mM GDPbetaS, indicating that the effect is not due to cAMP production or G-protein stimulation. It is concluded that there are sites on the Ca channels that are subject to direct modification by SH reagents.

Augmentation of somato-sympathetic reflex in the ischemic hindlimb of anesthetized rats.

Activities of the visceral sympathetic nerve increase with noxious mechanical stimulation. This study investigated the effects of noxious mechanical stimulation of hindlimb on arterial blood pressure, heart rate (HR) and renal sympathetic nerve activities (RNA) in anesthetized rats intact or under ischemic conditions. This study utilized two methods of noxious mechanical stimulation. One was pinching of the hindpaw; the other, strangulation of the ankle joint. Twenty-three male Wistar rats were used in the experiment. Pinching and strangulation of the intact hindlimb caused an increase in RNA. The increased RNA was greater during strangulation than during pinch stimulation (p < 0.0001). In the ischemic hindlimb produced by occlusion of the left common iliac artery, pinching and strangulation caused a further increase in RNA (p < 0.0001). Arterial blood pressure and HR significantly increased during strangulation with ischemic condition (p < 0.05). These results suggest that the reflex effects of somatosensory input on RNA were augmented in the ischemic hindlimb.

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 micro1. 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.

Point-mutations related to the loss of batrachotoxin binding abolish the grayanotoxin effect in Na(+) channel isoforms.

The effect of grayanotoxin (GTX) on site-specific mutants of the alpha-subunit of rat skeletal muscle Na(+) channels (micro1) (micro1-I433K, micro1-N434K and micro1-L437K), which are resistant to batrachotoxin (BTX) (Wang and Wang (1998) Proc Natl Acad Sci USA, 95, 2653-2658) was studied using a whole-cell patch-clamp method. The GTX modification of the Na(+) channels was detected as a characteristic-sustained Na(+) current flow with repetitive pulses. We also studied the GTX action on mutants of the alpha-subunit of rat heart Na(+) channels (RH1) (RH1-V406K and RH1-L410K) which match with micro1-I433 and micro1-L437. All the mutants lost their sensitivity to GTX. This finding indicates that GTX may share a binding site with BTX in transmembrane segment I-S6 of two different Na(+) channel isoforms, micro1 and RH1.

Regulation of L- and N-types of Ca2+ channels by intracellular ATP4- in frog dorsal root ganglion neurons.

The roles of free Mg2+ ions, ATP4- ions and Mg-ATP complexes in the regulation of N- and L-types of Ca2+ channels were studied in frog dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. Because Mg2+ ions interact with ATP4- ions to form Mg-ATP complexes, addition of one species can influence the concentrations of the other two. In this study their concentrations were carefully controlled by varying the concentrations of two constituents at a time while keeping the third constant. The effects of each of the three species on barium currents through L-type (IBaL) and N-type (IBaN) Ca2+ channels were plotted against its concentrations. The dose-response curves for ATP4- show that IBaL and IBaN proportionally increased with ATP4- concentrations up to 1 mM at three different Mg2+ concentrations. At a fixed concentration of ATP4-, IBaL and IBaN remained unchanged even when pMg changed from 3 to 5. Dose-response curves for IBaL and IBaN plotted against Mg-ATP concentration did not show a consistent pattern. H-7 and Mg2+ ions did not exert any blocking effect on the activity of either Ca2+ channel type, and neither dibutyryl-cAMP nor NKH-477 had any stimulating effect, suggesting that phosphorylation is not likely to be involved in ATP-induced potentiation. From these observations, it is concluded that L-type and N-type Ca2+ channels in frog DRG neurons are regulated by ATP4- ions alone, and that the neuronal Ca2+ channels are regulated by mechanisms that are different from those regulating the cardiac Ca2+ channels.

Characteristics of two slow inactivation mechanisms and their influence on the sodium channel activity of frog ventricular myocytes.

Inactivation of the fast Na+ current of heart muscle occurs in two kinetically distinct phases: a fast process operating on a millisecond time scale and a considerably slower process, the kinetic properties of which have not been explored fully. In this study, we analysed the slow inactivation process in isolated frog ventricular myocytes using the whole-cell variation of the patch-clamp method. Slow inactivation of the Na+ current followed a double-exponential time course, corresponding to slow and ultraslow components of Na+ channel inactivation. The individual time constants were 2-7 s (slow component) and 40-560 s (ultraslow component). Recovery from these slow inactivation processes also followed a double-exponential time course, but was characterized by significantly briefer time constants than those for the inactivation process. The relationship between transmembrane potential and steady-state slow or ultraslow inactivation was well described by the Boltzmann equation. The membrane potential at which half the Na+ channels are inactivated (V1/2) and the slope factor were estimated to be -48.1 and 13.6 mV, respectively, for the slow component alone. Under conditions in which the slow and ultraslow inactivation components were both present, these parameters were -53.1 and 8.7 mV respectively. When the fast and the two slow inactivation processes occurred concomitantly, the resultant steady-state inactivation curves were shifted to more negative potentials and the slope factor was decreased. Treatment with 1 mM Cd2+ externally did not affect the time course of slow inactivation, but produced a 3-7 mV depolarizing shift in its steady-state voltage dependency by virtue of cadmium's known effect on the cell surface potential. This study has thus identified two components of slow Na+ inactivation in heart muscle, operating on a time scale of seconds (slow inactivation) and minutes (ultraslow inactivation).

Phosphorylation modulates L-type Ca channels in frog ventricular myocytes by changes in sensitivity to Mg2+ block.

The effect of phosphorylation on the intracellular Mg2+ concentration-dependent change in Ca channel activity was examined using the patch clamp technique. The kinetic changes in Ca channels induced either by phosphorylation [1 muM forskolin (FSK) plus 50 muM isobutylmethylxanthine (IBMX)] or by lowering intracellular [Mg2+] ([Mg2+]i) are qualitatively identical: an increase in both the open probability and availability of channels, as well as a decrease in the closed time without a change in the mean open time. This suggests that the mechanism for the increase in activity of Ca channels shares a common pathway of kinetic change. The concentration/response curve for the Mg2+-evoked modification of calcium channels was altered greatly by channel phosphorylation. In the external medium containing 1 muM FSK + 50 muM IBMX, Ca channels recorded with pipettes containing okadaic acid (OA) lost their sensitivity to Mg2+ in the range 1 x 10(-6) M-1 x 10(-3) M and remained in a fully active state. On the contrary, under basal conditions, the activity of Ca channel was strongly dependent on the internal Mg2+ over the same range of [Mg2+]. Similarly, phosphorylation of Ca channels eliminated the blocking action of guanosine triphosphate observed under basal conditions. A model is proposed in which Ca channels are equipped with regulatory gates for opening and closing the channels, and their regulation is dependent on [Mg2+]i and the degree of phosphorylation.

Differential effects of lipid-soluble toxins on sodium channels and L-type calcium channels in frog ventricular cells.

The effect of grayanotoxin I (GTX I), veratridine and aconitine with either an external or internal concentration of 100 microM on L-type calcium (Ca) channels was studied using the whole cell patch clamp and internal dialysis methods. The experimental conditions for the modification of sodium (Na) channels induced by the internal application of these toxins was determined by showing sustained inward currents with depolarizing repetitive pulses. These toxins failed to generate any change in Ca channels under the same experimental protocol as for Na channels. However, external application of these toxins caused a moderate block of the Ca channels without changing the kinetics.

Kinetics of grayanotoxin evoked modification of sodium channels in squid giant axons.

Kinetics of modification of the sodium channel by alpha-dihydrograyanotoxin II (GTX) were studied with voltage-clamped squid giant axons. GTX modified the channel to generate sustained inward current, only when the membrane was kept depolarized to levels more positive than -80mV, in a voltage-dependent manner, increasing the depolarization. Repetitive depolarizing pulses suppressed rather than increased the degree of GTX-evoked modification. GTX-evoked modification proceeded with a dual exponential time course, regardless of the presence or absence of the inactivation gate, but the elimination by pronase of the inactivation gate accelerated GTX-evoked modification. GTX unbound from the sodium channel with a time constant of 30 s at -150 mV in a manner independent of the concentration. The effective concentration that produced a half-maximal sustained sodium current (EC50), which represents GTX-modified channel activity, was estimated to be about 10 microM with one-to-one stoichiometry. The activation/voltage relationship for the sustained sodium current was shifted in the hyperpolarizing direction by as much as 63-94 mV compared with that of peak sodium current. At a GTX concentration of 100 microM and at +20mV, 64% of the sodium channel population was modified. A kinetics model is proposed to account for the behavior of GTX -modified sodium channels.

Identification of ATP-sensitive potassium channel in frog ventricular myocytes.

ATP-sensitive potassium channels were found in frog ventricular myocytes using the inside-out patch-clamp technique. The channel was selectively permeable to K+. Single-channel conductance was 32.6 pS at 3.0 mM of [K+]o and 132 mM [K+]i and 77.3 pS at 114 mM [K+]o and 132 mM [K+]i. ATP did not affect single-channel conductance. The open probability of the channel was decreased by intracellular application of ATP in both the presence and absence of 2 mM MgCl2. The coexistence of Mg2+ with ATP shifts the dose-response curve for the open probability of ATP-sensitive K+ channel against ATP rightward. The shift of the curve indicates that Mg-ATP is less effective than free ATP in inhibiting the channel. An open-time histogram was fitted by a single exponential function with a time constant of 1.63 +/- 0.17 msec (n = 5) in an ATP-free medium. Mean open time (1.57 +/- 0.10 msec; n = 5) was not altered but the inter-burst time (closed time between bursts) lengthened in 10 microM ATP.

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), 1,2-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 beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (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 microM 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 microM at pMg 6 and 6.9 microM at pMg 5. The results suggest strongly that Mg2+ and GTP independently bind and control Ca2+ channels.

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells.

Ca2+-activated K+ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg2+ decreased channel activity in a dose-dependent manner. In the absence of Mg2+, administration of adenosine 5'-trisphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg2+, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg2+, the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca2+ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP) adenylyl [beta,gamma-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5'-O-(3-thiotriphosphate) (ATP[gammaS]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca2+-activated K+ channels are regulated by Mg2+ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.