K channels are ubiquitous in animal cells, where they are involved in a variety of physiological functions. In epithelial cells of the kidney, K channels are primarily involved in maintaining membrane potential, recycling and secreting K and regulating cell volume. As many renal K channels have now been studied or identified at the molecular level by means of a variety of approaches, including patch-clamp recordings, cDNA cloning and immunohistochemistry, the purpose of this review is to summarize what is presently known about the molecular identity of renal K channels with an emphasis on their regulatory properties.

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That the cell is a gel is broadly acknowledged. Textbooks begin with this assertion—and then proceed with great abandon to derive mechanisms based on free diffusion, as though the gel concept were groundless and cell was an aqueous solution. This disconnect emerges in part because the behavior of gels is not well understood, particularly among most biologists. Recently, great strides have been made in the understanding of gel behavior. It has become clear, for example, that a central mechanism in gel function is the phase-transition—a qualitative structural change prompted by a subtle change of environment, not unlike the transition from ice to water. Phase-transitions are capable of doing work. If the cell is a gel, then a logical approach to understanding cell function is to understand gel function—especially whether some role may be played by the phase-transition. Here we pursue this approach. We first consider the dichotomy of the cell as a gel and the cell as an aqueous solution. We then set up a gel-based foundation for cell behavior, in which the gels' physical chemical features are used to explore how the cell achieves its everyday tasks. If there is a common underlying mechanism of cell function, it appears that the polymer gel phase-transition could well be a candidate.

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This study investigated whether the sarcoplasmic reticulum Ca²⁺ content of rat skeletal muscle fibers affected contractile responses obtained by an application of 4-chloro-m-cresol (4-CmC) and caffeine. Contractures were elicited on saponin-skinned fibers under different Ca²⁺ loading conditions. The amplitude of 4-CmC and caffeine contractures of fast-twitch muscle fibers (edl, extensor digitorum longus) differed between the different loading conditions, and this is associated with a greater change in sensitivity to 4-CmC. When the sarcoplasmic reticulum was loaded with a low Ca²⁺ concentration for a short period, the 4-CmC concentration providing half-maximal response was tenfold higher than with a larger sarcoplasmic reticulum Ca²⁺ loading for a longer period, whereas for caffeine this concentration was only twofold higher in the same conditions. These findings indicate that 4-CmC contractile responses of edl muscle fibers are more dependent on luminal Ca²⁺ activity than those of caffeine are. Thus 4-CmC would appear to be of greater interest than caffeine for the study of muscle contractile responses where variations in intracellular Ca²⁺ activity exist.

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We studied the buffer mechanism of human whole blood by means of a titration experiment under the open condition in which blood is exposed to carbon dioxide gas. Van Slyke proposed the theory of a blood buffer mechanism in 1922 (J Biol Chem 52: 525-570). However, his theory is not applicable to our experiments because it did not take into consideration the effects of changes in volume and the concentration of bicarbonate ion in the buffer as a result of titration. We studied the blood buffer mechanism theoretically by using graphical representation and developed a theory, which we then applied to our experiments. Buffer value, as defined by Van Slyke, is the quantity of the base δn_OH/V in the sample without volume change (in gram equivalent per liter) required to change one unit in pH. Based on our blood buffer theory, we obtain the experimental buffer value β′, which is the reciprocal slope of the dpH-bdV/V (δn_OH in the sample with volume change) relation during a titration experiment at a constant carbon dioxide pressure (pCO₂) with volume change dV and an isotonic NaOH concentration of b = 0.15 M. The true buffer value β under physiological conditions is estimated to be β = δn_OH/VdpH = β′ (0.15 − [HCO₃⁻])/0.15. Here, [HCO₃⁻] is the concentration of bicarbonate ion in blood that can be calculated by using the Henderson-Hasselbalch equation. We determined the agreement of our experimental results with the theoretical results and estimated the quantity m_A of the buffer component, except for the bicarbonate buffer, and the dissociation constant K_A of the buffer reaction in a neutral pH range. The true buffer value of whole blood can be estimated by using our theory and the results of precise experiments.

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We examined the possibility of Ca²⁺ permeation through cardiac Na channels ("slip mode conductance") by an analysis of the voltage-dependent block of Na channels by Ca²⁺. A Ca²⁺ block of Na channels was evident in rat and guinea pig ventricular myocytes during cell-attached single channel recordings with a physiological ionic environment (140 mM Na⁺ and 1 to 10 mM Ca²⁺ in the pipette solution). Increasing external Ca²⁺ concentration ([Ca²⁺]_o) in the pipette solution reduced the unitary current amplitude predominantly at negative potentials. With [Ca²⁺]_o > 1 mM, unitary current amplitude did not increase at potentials negative to −40 mV in spite of augmented driving forces. The application of 5 &mgr;M isoproterenol potentiated the single channel activity elicited by depolarizing pulses from the holding potential of −120 mV, indicating that the channels in the patch under examination were modified by protein kinase A (PKA) stimulation. Increased activity was also confirmed with veratridine-modified Na channels, where channel openings were markedly prolonged. In either case, isoproterenol-induced potentiation neither reduced nor altered the properties of Ca²⁺ block of cardiac Na channels, as evidenced by the stable unitary current amplitudes at potential levels from −60 to −20 mV. These results indicate that interactions among Na⁺, Ca²⁺, and the channel molecule were not modified with respect to permeation properties. They therefore argue against the "slip mode" concept of classical cardiac Na channel if a general concept of ion permeation through "multi-ion pores" is applicable to determine the ionic selectivity of Na channels.

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L-Glutamate has been considered to be a neurotransmitter in the nucleus tractus solitarius (NTS) of the afferent baroreflex pathway, though this has not yet been decisively shown. A bolus injection of a neurotransmitter candidate amino acid L-proline into the cisterna magna and that of L-glutamate shows the same pressor action in the freely moving rat, but the actual nuclei responding L-proline remain undetermined. Besides L-glutamate, L-proline might be another candidate amino acid in the NTS. The present study was therefore performed to characterize the circulatory action of L-proline injected into the NTS where responses to glutamate in the anesthetized rat had already been shown. The NTS was first determined as a site on the dorsal surface of the medulla where a microinjection of L-glutamate decreased arterial pressure and heart rate. Microinjected L-proline (1.65 to 13.2 nmol, 33 nl) into the NTS decreased arterial pressure and heart rate in a dose-dependent manner. The injection of a mixed solution (66 nl) of kynurenate, an ionotropic excitatory amino acid receptors antagonist (1.32 nmol), and L-proline (6.6 nmol) into the NTS abolished the depressor and bradycardic actions with L-proline alone (6.6 nmol, 66 nl). However, a mixture of an increased concentration of kynurenate (6.6 nmol) with glutamate augmented the actions seen with glutamate alone (0.66 nmol, 66 nl). D-Proline (13.2 nmol, 66 nl), the optic isomer of L-proline, produced no change in arterial pressure or heart rate, suggesting that the actions of L-proline in the NTS were optically specific. The results indicate that L-proline but not D-proline induces its depressor and bradycardic actions through ionotropic excitatory amino acid receptors in the NTS of the anesthetized rat. L-Proline may become a candidate transmitter of baroreceptor information in the NTS.

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In circular smooth muscle tissues of the guinea pig gastric fundus, transmural nerve stimulation (TNS) evoked an atropine-sensitive cholinergic excitatory junction potential (e.j.p.) and, after inhibiting the e.j.p. with atropine, an apamin-sensitive nonadrenergic noncholinergic (NANC) inhibitory junction potential (i.j.p.). The amplitude of e.j.p.s was similar when the frequency of TNS was low (<0.5 Hz), but it decreased successively (depression phenomenon) when the frequency was high (>1 Hz). The depression phenomenon was attenuated after inhibiting the production of nitric oxide (NO) with N^ω-nitro-L-arginine (NOLA), but was not altered by inhibiting the i.j.p. with apamin. The e.j.p.s were increased in amplitude by the inhibition of cholinesterase activity, but they were decreased by NO produced from SNP with no alteration of their depression phenomenon. Isometric twitch contractions were depressed during high-frequency TNS. NOLA caused an increase in the amplitude of twitch contractions and the attenuation of their depression that changed the transient contraction produced by high-frequency TNS (1 Hz) to a tetanic one. SNP reduced the amplitude of twitch contractions, with no alteration of the depression phenomena. Contractions produced by low concentrations of acetylcholine, but not by high concentrations, were attenuated by SNP, with no alteration of the membrane depolarization. The results suggest that NO produced during TNS has inhibitory actions on cholinergic transmission; the depression of e.j.p.s is mainly prejunctional events, and the depression of mechanical responses is mainly postjunctional events.

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The purpose of this study was to investigate red blood cell (RBC) behavior during an increase in local cerebral blood flow (LCBF). We measured changes in RBC behavior by using laser-Doppler flowmetry (LDF) in &agr;-chloralose-anesthetized rats. An increase in LCBF was carried out by approximately 2.5 and 4.0% CO₂ inhalation and activation of the somatosensory cortex. The activation of the cortex was induced by electrical stimulation of the hind paw with 1.5-mA pulses (0.1 ms) applied at frequencies of 0.2, 1, 5, and 10 Hz for a 5 s duration. The increases in LCBF and RBC velocity during both CO₂ inhalations were larger than that in RBC concentration (p < 0.05). LCBF and RBC velocity during 4.0% CO₂ inhalation were larger than those during 2.5% CO₂ inhalation (p < 0.05), though there was no significant difference in RBC concentration between the two conditions, suggesting a limitation of capillary volume. During somatosensory stimulation, the evoked LCBF increased with increasing stimulus frequency up to 5 Hz and decreased at 10 Hz. The responses of RBC concentration at 0.2 and 10 Hz were greater than those of RBC velocity (p < 0.05), but no significant differences in response magnitude were found at 1 and 5 Hz between RBC concentration and RBC velocity. These results suggest that the increase in LCBF during neuronal activity is different from that of controlling the LCBF as induced by CO₂, and that the regulation of RBC concentration and RBC velocity is controlled by independent mechanisms.

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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.

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C-kit immunoreactive cells are known to be interstitial cells of Cajal (ICCs), and they generate pacemaker activity of the gastrointestinal tract. Recently a large number of special smooth muscle cells corresponding to c-kit immunoreactive cells were found in the proximal colon of the guinea pig. We learned that the rat proximal colon showed tetrodotoxin-insensitive regular rhythmic spontaneous contractions (RSCs) and hypothesized that RSCs are generated and/or regulated by ICCs. To prove our hypothesis, we investigated whether RSCs are absent in homozygous Ws/Ws mutant rats, since c-kit positive ICCs along the submucosal surface of the circular muscle (ICC_SM) and myenteric plexus (ICC_MY) are lacking. In contrast to our hypothesis, we found that RSCs were still present in the proximal colon of the Ws/Ws mutant rats. A recent study has reported that c-kit negative ICC_SM remains in Ws/Ws mutant rats. Taken together, RSCs may be generated by c-kit negative ICC_SM in the rat proximal colon. The blockade of sarcoplasmic reticulum Ca²⁺-ATPase by cyclopiazonic acid (CPA) (10⁻⁶M) or by thapsigargin (10⁻⁶M) increased the frequency of RSCs. The increasing effects of CPA on the frequency of RSCs were more prominent in Ws/Ws mutant rats than in +/+ rats. We concluded that the functional coordination between c-kit negative ICC_SM and other mutationally impaired c-kit positive ICC_MY and ICC_SM may be required for moderate regulation in the frequency of spontaneous activity.

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The properties of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels were studied in rat hippocampal CA1 pyramidal neurons by using the patch-clamp technique in the excised-inside-out-patch configuration. The lowest [Ca²⁺]_i in which BK_Ca channel activities were observed was 0.01 μM with the membrane potential of +20 mV and the [Ca²⁺]_i at which P_O of the channel is equal to 0.5 was 2 μM. The unitary conductance of the single BK_Ca channel was 245.4 pS with symmetrical 140 mM K⁺ on both sides of the excised membrane. With a fixed [Ca²⁺]_i of 2 μM, P_O increased e-fold with a 17.0 mV positive change in the membrane potential. Two exponentials, with time constants of 2.8 ms and 19.2 ms at the membrane potential of +120 mV with 2 μM [Ca²⁺]_i, were required to describe the observed open time distribution of BK_Ca channel, suggesting the existence of two distinct open channel states with apparently normal conductance. A BK_Ca channel occasionally entered an apparent third open channel state with the single channel current amplitude about 45% of the normal amplitude. The properties of BK_Ca channel, which were found in this study to be more steeply dependent on voltage and more sensitive to [Ca²⁺]_i in adult hippocampal neurons than in cultured or immature hippocampal neurons, may be responsible for the shortened duration of action potential in hippocampal CA1 pyramidal neurons of adult rat.

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Myocardial Ca²⁺ handling in excitation-contraction coupling is the second primary determinant of energy or O₂ demand in a working heart. The intracellular and extracellular routes remove myocardial Ca²⁺ that was released into the sarcoplasma with different Ca²⁺: ATP stoichiometries. The intracellular route is twice as economical as the extracellular route. Therefore the fraction of total Ca²⁺ removed via the sarcoplasmic reticulum, i.e., the recirculation fraction of intracellular Ca²⁺ (RF), determines the economy of myocardial Ca²⁺ handling. RF has conventionally been estimated as the exponential decay rate of postextrasystolic potentiation (PESP). However, we have found that PESP usually decays in alternans, but not exponentially in the canine left ventricle beating above 100 beats/min. We have succeeded in estimating RF from the exponential decay component of an alternans PESP. We previously found that the Frank-Starling mechanism or varied ventricular preload did not affect the economy of myocardial Ca²⁺ handling. Then, to account for this important finding, we hypothesized that the Frank-Starling mechanism would not affect RF at a constant heart rate. We tested this hypothesis and found its supportive evidence in 11 canine left ventricles. We conclude that RF at a constant heart rate would remain constant, independent of the Frank-Starling mechanism.

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We investigated the capacity of newborn rats to excrete an acute potassium load to understand the development of a renal potassium excretion system. Three groups of the rats (7-14 d) were used to collect urine periodically over 6 h after oral infusion of potassium: control (no potassium loading) and low- and high-potassium-loaded rats. In the low-potassium-loaded group, infused with about 0.6 μEq of potassium chloride/g body wt., the rate of renal potassium excretion increased from 0.08 ± 0.02 (7 d) to 0.13 ± 0.02 (10 d) and 0.21 ± 0.03 (14 d) μEq/h/g body wt. The high-potassium-loaded rats (1.5-2.8 μEq/g body wt. potassium load) excreted potassium at a higher rate of 0.18 ± 0.05 (7 d), 0.30 ± 0.02 (10 d), and 0.45 ± 0.10 (14 d) μEq/h/g body wt. They excreted 77% (7 d), 76% (10 d), and 95% (14 d) of the potassium load. These values were much larger than the rate of 0.026 μEq/h/g body wt. of the control rats and of 0.08 μEq/h/g body wt., a mean potassium excretion rate during development from 7 to 14 d calculated from the data in the previous study (Kanno T et al.: J. Pediatr. Gastr. Nutr. 24: 242-252, 1997). In the same period, serum potassium concentration in the newborn rats decreased significantly (p < 0.01) from 7.2 ± 0.1 (7 d) to 6.7 ± 0.1 mEq/l (14 d). All these results suggest that a renal potassium excretion system in the rat develops at least in the second week of life, and its capacity is high enough to excrete the daily potassium intake.

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The effects of 17β-estradiol (10⁻⁵ M), an active estrogen, on the tension and fatigue responses of single fiber or fiber bundle prepared from frog skeletal muscle were investigated. The administration of 17β-estradiol caused a transient potentiation of tetanus tension by field stimulation at every minute. This potentiation was not affected by the presence of nicardipine, suggesting that the action of 17β-estradiol would place the excitation-contraction (E-C) coupling beyond T-tubule depolarization. Fatigue was produced by repeated tetanic stimulation every second until tension declined to approximately 40% of the initial level. Fibers were then allowed to recover by having tetani given to them every minute. In the normal Ringer solution, the time to 50% of the initial tetanus tension was 41.7 s. With the presence of 17β-estradiol, the time to 50% tension was faster than that of control. The presence of 17α-estradiol, a stereoisomer, caused no potentiation of tetanic tension to be stimulated every minute, and the rate of decline of fatigued response was almost the same as that of control, suggesting the existence of specific estrogen receptors in the frog muscle. In fatigued muscle with or without estrogen, the tension to field stimulation was transient and not sustained. When the fatigued muscle was again treated with field stimulation at every minute after the more-frequent stimulation, the recovery rate was increased in 17β-estradiol. A prompt reduction in temperature to 5°C, from 20°C, in the presence of caffeine elicited the tension response, a rapid cooling contracture (RCC). The presence of 17β-estradiol inhibited peak tension and maximum rate of rise of the RCC only after the repetitive electrical stimuli. These results suggest that the potentiation of contraction upon the electrical stimulation by 17β-estradiol was induced by the increase of myoplasmic-free Ca²⁺ concentration via an activation of some E-C coupling process. The 17β-estradiol-induced facilitation of fatigue response to repetitive tetanus stimuli with high frequency may be due to an increase in the imbalance of Ca²⁺ turnover in the cytoplasm.

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The purpose of this study was to analyze the effects of caffeine ingestion on the slow component of oxygen uptake (ΔVO₂) during high-intensity endurance exercise. Nine subjects (8 male and 1 female; age: 21 ± 1 years; VO_{2 max}: 57.9 ± 1.5 ml kg⁻¹ min⁻¹) performed two 9-min tests on a treadmill at a running velocity eliciting 90% of their VO_{2 max}, 60 min after ingesting either a placebo capsule (PLAC) or a capsule containing a caffeine dose of 5 mg (kg body mass)⁻¹ [CAFF]. The mean values of ΔVO₂ were significantly lower in CAFF than in PLAC (83 ± 31 ml min⁻¹ vs. 167 ± 26 ml min⁻¹, respectively; p < 0.05). These findings suggest that the ergogenic effect of caffeine in a high-intensity endurance exercise shown in previous research may be partly mediated by a possible attenuation of the VO₂ slow component.

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