bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell.
Authors: Fabiato A
Published: May 2004

A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of a Ca2+ transient. Simultaneously monitoring tension and aequorin bioluminescence provided two methods for estimating the peak myoplasmic [free Ca2+] reached during the spontaneous cyclic Ca2+ release from the SR obtained in the continuous presence of a bulk solution [free Ca2+] sufficiently high to overload the SR. These methods gave results in excellent agreement for the spontaneous Ca2+ release under a variety of conditions of pH and [free Mg2+], and of enhancement of Ca2+ release by calmodulin. Disagreement was observed, however, when the Ca2+ transient was modified during its ascending phase. The experiments also permitted quantification of the aequorin binding within the myofibrils and determination of its operational apparent affinity constant for Ca2+ at various [free Mg2+] levels. An increase of [free Ca2+] at the outer surface of the SR during the ascending phase of the Ca2+ transient induced further release of Ca2+. In contrast, an increase of [free Ca2+] during the descending phase of the Ca2+ transient did not cause further Ca2+ release. Varying [free H+], [free Mg2+], or the [Na+]/[K+] ratio had no significant effect on the Ca2+ transient during which the modification was applied, but it altered the subsequent Ca2+ transient. Therefore, Ca2+ appears to be the major, if not the only, ion controlling Ca2+ release from the SR rapidly enough to alter a Ca2+ transient during its course.
Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell.
Authors: Fabiato A
Published: May 2004

Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.
Intracellular pH-regulating mechanism of the squid axon. Relation between the external Na+ and HCO-3 dependences.
Authors: Boron W F
Published: May 2004

The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3-]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants.
Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270.
Authors: Abramcheck F J , Van Driessche W , Helman S I
Published: May 2004

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells.
Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells.
Authors: Uehara A , Hume J R
Published: May 2004

Inhibition of whole-cell calcium currents in enzymatically dispersed frog atrial myocytes by D-600, diltiazem, and nifedipine was studied using a single-micropipette voltage-clamp technique. The objective of these experiments was to test the applicability of a modulated-receptor hypothesis similar to that proposed for local anesthetic interactions with sodium channels to account for the tonic and frequency-dependent interactions of these organic compounds with myocardial calcium channels. Data consistent with such a hypothesis include: (a) prominent use-dependent block of iCa by D-600 and diltiazem, which are predominantly charged at physiological pH; (b) iCa block by an externally applied, permanently charged dihydropyridine derivative is greatly attenuated; (c) all three antagonists produce large negative shifts in the voltage dependence of iCa availability; (d) block of iCa by these compounds is state-dependent; (e) reactivation of iCa in the presence of all three antagonists is biexponential, which suggests that drug-free channels recover with a normal time course and drug-bound channels recover more slowly; and (f) the kinetics of the drug-induced slow iCa recovery process may be determined largely by factors such as size and molecular weight, in addition to lipid solubility of the compounds. Experiments in which the pH was modified, however, reveal some important differences for the interaction of organic calcium antagonists with myocardial calcium channels. Acidification, in addition to changing the proportion of charged and neutral antagonist in solution, was found to selectively antagonize tonic inhibition of iCa by diltiazem and nifedipine, without changing the kinetics of the drug-induced slow iCa reactivation process. It is concluded that two distinct receptor sites may be involved in block of iCa by some of these compounds: a proton-accessible site and a proton-inaccessible site.
An ion's view of the potassium channel. The structure of the permeation pathway as sensed by a variety of blocking ions.
Authors: French R J , Shoukimas J J
Published: May 2004

We have studied the block of potassium channels in voltage-clamped squid giant axons by nine organic and alkali cations, in order to learn how the channel selects among entering ions. When added to the internal solution, all of the ions blocked the channels, with inside-positive voltages enhancing the block. Cesium blocked the channels from the outside as well, with inside-negative voltages favoring block. We compared the depths to which different ions entered the channel by estimating the "apparent electrical distance" to the blocking site. Simulations with a three-barrier, double-occupancy model showed that the "apparent electrical distance," expressed as a fraction of the total transmembrane voltage, appears to be less than the actual value if the blocking ion can pass completely through the channel. These calculations strengthen our conclusion that sodium and cesium block at sites further into the channel than those occupied by lithium and the organic blockers. Our results, considered together with earlier work, demonstrate that the depth to which an ion can readily penetrate into the potassium channel depends both on its size and on the specific chemical groups on its molecular surface. The addition of hydroxyl groups to alkyl chains on a quaternary ammonium ion can both decrease the strength of binding and allow deeper penetration into the channel. For alkali cations, the degree of hydration is probably crucial in determining how far an ion penetrates. Lithium, the most strongly hydrated, appeared not to penetrate as far as sodium and cesium. Our data suggest that there are, minimally, four ion binding sites in the permeation pathway of the potassium channel, with simultaneous occupancy of at least two.
Electro-osmosis and the reabsorption of fluid in renal proximal tubules.
Authors: McLaughlin S , Mathias R T
Published: May 2004

The lateral intercellular spaces (LIS) are believed to be the final common pathway for fluid reabsorption from the renal proximal tubule. We postulate that electrogenic sodium pumps in the lateral membranes produce an electrical potential within the LIS, that the lateral membranes bear a net negative charge, and that fluid moves parallel to these membranes because of Helmholtz-type electro-osmosis, the field-induced movement of fluid adjacent to a charged surface. Our theoretical analysis indicates that the sodium pumps produce a longitudinal electric field of the order of 1 V/cm in the LIS. Our experimental measurements demonstrate that the electrophoretic mobility of rat renal basolateral membrane vesicles is 1 micron/s per V/cm, which is also the electro-osmotic fluid velocity in the LIS produced by a unit electric field. Thus, the fluid velocity in the LIS due to electro-osmosis should be of the order of 1 micron/s, which is sufficient to account for the observed reabsorption of fluid from renal proximal tubules. Several experimentally testable predictions emerge from our model. First, the pressure in the LIS need not increase when fluid is transported. Thus, the LIS of mammalian proximal tubules need not swell during fluid transport, a prediction consistent with the observations of Burg and Grantham (1971, Membranes and Ion Transport, pp. 49-77). Second, the reabsorption of fluid is predicted to cease when the lumen is clamped to a negative voltage. Our analysis predicts that a voltage of -15 mV will cause fluid to be secreted into the Necturus proximal tubule, a prediction consistent with the observations of Spring and Paganelli (1972, J. Gen. Physiol., 60:181).
First-order kinetics of muscle oxygen consumption, and an equivalent proportionality between QO2 and phosphorylcreatine level. Implications for the control of respiration.
Authors: Mahler M
Published: May 2004

In frog sartorius muscle, after a tetanus at 20 degrees C, during which an impulse-like increase occurs in the rate of ATP hydrolysis, the rate of O2 consumption (QO2) reaches a peak relatively quickly and then declines monoexponentially, with a time constant not dependent on the tetanus duration (tau = 2.6 min in Rana pipiens and 2.1 min in Rana temporaria). To a good approximation, these kinetics are those of a first-order impulse response, and the scheme of reactions that couple O2 consumption to extramitochondrial ATP hydrolysis thus behaves as a first-order system. It is first deduced and then demonstrated directly that while QO2(t) is monoexponential, it changes in parallel with the levels of creatine and phosphorylcreatine, with proportionality constants +/- 1/tau p, where p is the P/O2 ratio in vivo. From this, it is further deduced that the mitochondrial creatine kinase (CK) reaction is pseudo-first order in vivo. The relationship between [creatine] and QO2 predicted by published models of the control of respiration is markedly different from that actually observed. As shown here, the first-order kinetics of QO2 are consistent with the hypothesis that respiration is rate-limited by the mitochondrial CK reaction; this has as a corollary the "creatine shuttle" hypothesis.
Neurophysiological and biophysical evidence on the mechanism of electric taste.
Authors: Herness M S
Published: May 2004

The phenomenon of electric taste was investigated by recording from the chorda tympani nerve of the rat in response to both electrical and chemical stimulations of the tongue with electrolytes in order to gain some insight into its mechanism on both a neurophysiological and biophysical basis. The maximum neural response levels were identical for an individual salt (LiCl, NaCl, KCl, or CaCl2), whether it was presented as a chemical solution or as an anodal stimulus through a subthreshold solution. These observations support the idea that stimulation occurs by iontophoresis of ions to the receptors at these current densities (less than 100 microA/cm2). Electric responses through dilute HCl were smaller than the chemically applied stimulations, but the integrated anodal responses appeared similar to chemical acid responses, as evidenced by an OFF response to both forms of stimuli. Hydrogen may be more permeant to the lingual epithelium and would thus be shunted away from the taste receptors during anodal stimulation. When the anion of electric taste was varied via subthreshold salt solutions, the response magnitude increased as the mobility of the anion decreased. The transport numbers of the salts involved adequately explains these differences. The physical aspects of ion migration occurring within the adapting fluid on the tongue are also discussed. Direct neural stimulation by the current appears to occur only at higher current densities (greater than 300 microA/cm2). If the taste cells of the tongue were inactivated with either iodoacetic acid (IAA) or N-ethyl maleimide (NEM), or removed with collagenase, then responses from the chorda tympani could be obtained only at these higher current densities. Latency measurements before and after IAA or NEM treatment corroborated these findings. The results are discussed in terms of several proposed mechanisms of electric taste and it is concluded that an ion accumulation mechanism can adequately explain the data.
Olfactory stimulation variables. Which model best predicts the olfactory nerve response?
Authors: Kurtz D B , Mozell M M
Published: May 2004

Mozell et al. (1984. J. Gen. Physiol. 83:233-267) have examined the traditional manner in which olfactory stimulus-response relationships have been addressed. They developed a model that describes the olfactory nerve response as a function of three factors, viz., the number of odorant molecules (N), the stimulus duration (T), and the stimulus volume (V). In addition, two models derived from this three-variable model were also found to predict the response well. These were the [F, N] model involving flow rate (F = V/T) and, ranking closely behind, the [C, T] model involving concentration (C = N/V). A model involving the delivery rate (D = N/T) and volume was found to predict the response poorly. These models imply very different stimulus-response relationships. The present study was designed to assess the validity of this earlier approach by testing specific predictions drawn from each of the models. Because of the excellence of the [F, N] model, one would predict that the response will not change when F and N are held constant in spite of proportional increases in V and T. Similarly, one would predict from the [C, T] model that the response will be constant when C and T are held constant in spite of proportional increases in N and V. Because of the poor showing of the [D, V] model, one would predict changes in the response even when D and V are held constant while N and T are increased proportionately. It was observed that when F and N were held constant, the response was, in fact, constant. When D and V were held constant, the response increased dramatically. When C and T were held constant, there was a statistically significant, but small, change in the response. These results support the approach taken by Mozell et al. (op. cit.) and highlight the applicability of the [F, N] model to peripheral olfactory processing. The results are discussed in terms of their impact on the traditional manner in which olfactory stimulus-response relationships are conceived.
Intracellular topography of rhodopsin regeneration in vertebrate rods.
Authors: Williams T P , Penn J S
Published: May 2004

The vertebrate visual pigment of rods, rhodopsin, bleaches in light and regenerates in darkness. When the bleaching and regeneration are carried out in vivo, it is found that the regeneration takes place at nonuniform rates along the rod outer segment (ROS): toads and frogs regenerate rhodopsin faster in the proximal ends of the ROS than in the distal ends. Rats do the reverse. These patterns of regeneration persist whether the bleaching is done with flashes or with steady light. They are also independent of the extent to which the retinal pigment epithelium contains melanin. Furthermore, the dichotomy of patterns (proximal faster vs. distal faster) does not seem to depend upon the presence of an excess of stored retinoid in the eye. Instead, it is suggested that the villous processes of the epithelial cells may play an important role in the regeneration patterns. These processes in amphibia extend nearly to the rod inner segment but in the rat they surround only the apical end of the outer segment. If they "funnel" the retinoids back to the ROS, their location and morphology could explain the two different kinds of patterns seen.
Voltage dependence of the Na/Ca exchange in voltage-clamped, dialyzed squid axons. Na-dependent Ca efflux.
Authors: Dipolo R , Bezanilla F , Caputo C , Rojas H
Published: May 2004

A combination of the voltage-clamp and the intracellular dialysis techniques has been used to study the membrane potential dependence of the Nao-dependent Ca efflux in squid giant axons. In order to improve axon survival, experiments were carried out using internal solutions prepared with large impermeant organic anions and cations, which did not affect the operation of the Na/Ca exchange mechanism. In axons dialyzed with solutions prepared without internal Na, the Nao-dependent Ca efflux had a small sensitivity to membrane potential changes. For a 25-mV membrane displacement in the hyperpolarizing direction, the basal Ca efflux increased by only 7.4% (n = 13). When the dialysis medium contained Na (from 20 to 55 mM), the efflux increased 32.3% (n = 25) for the same membrane potential change. The K1/2 for this effect is approximately 5 mM Na, and saturation appears to occur at a Na concentration above 20 mM. Adding ATP to the dialysis medium increased the magnitude of the Nao-dependent Ca efflux without changing its voltage sensitivity. Wide changes in the intracellular ionized Ca concentration (from 0.1 to 230 microM) did not modify the voltage sensitivity of the exchange system. Elimination of the reversal of Na/Ca exchange (Nai-dependent Ca influx) by removing Cao did not modify the voltage sensitivity of the Nao-dependent Ca efflux. When the axon membrane potential was submitted to prolonged changes, the corresponding changes in the Ca efflux were not sustained, but declined exponentially to intermediate values. This effect may indicate a slow inactivation process in the Na/Ca exchange mechanism. Voltage-clamp pulse experiments revealed: (a) the absence of a fast inactivation process in the Na/Ca exchange, and (b) that the activation of the carrier for hyperpolarizing pulses occurs as rapidly as 1 ms.
Volume-regulatory responses of Amphiuma red cells in anisotonic media. The effect of amiloride.
Authors: Siebens A W , Kregenow F M
Published: May 2004

Amphiuma red cells were incubated for several hours in hypotonic or hypertonic media. They regulate their volume in both media by using ouabain-insensitive salt transport mechanisms. After initially enlarging osmotically, cells in hypotonic media return toward their original size by losing K, Cl, and H2O. During this volume-regulatory decrease (VRD) response, K loss results from a greater than 10-fold increase in K efflux. Cells in hypertonic media initially shrink osmotically, but then return toward their original volume by gaining Na, Cl, and H2O. The volume-regulatory increase (VRI) response involves a large (greater than 100-fold) increase in Na uptake that is entirely blocked by the diuretic amiloride (10(-3) M). Na transport in the VRI response shares many of the characteristics of amiloride-sensitive transport in epithelia: (a) amiloride inhibition is reversible; (b) removal of amiloride from cells pretreated with amiloride enhances Na uptake relative to untreated controls; (c) amiloride appears to act as a competitive inhibitor (Ki = 1-3 microM) of Na uptake; (d) Na uptake is a saturable function of external Na (Km approximately 29 mM); (e) Li can substitute for Na but K cannot. Anomalous Na/K pump behavior is observed in both the VRD and the VRI responses. In the VRD response, pump activity increases 3-fold despite a decrease in intracellular Na concentration, while in the VRI response, a 10-fold increase in pump activity is observed when only a doubling is predicted from increases in intracellular Na.
Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin.
Authors: Jones G S , Knauf P A
Published: May 2004

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.
Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux.
Authors: Stephenson E W
Published: May 2004

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.
Effects of caffeine, tetracaine, and ryanodine on calcium-dependent oscillations in sheep cardiac Purkinje fibers.
Authors: Nieman C J , Eisner D A
Published: May 2004

Membrane current and tension were measured in voltage-clamped sheep cardiac Purkinje fibers. Elevating the intracellular calcium concentration ([Ca2+]i) results in oscillations of membrane current and tension both at rest and during stimulation. During stimulation, an oscillatory transient inward current and an after contraction follow repolarization. We have examined the effects on the oscillations of changing the extracellular calcium concentration ([Ca2+]o) and of adding various drugs. In agreement with previous work, high concentrations of drugs that affect the sarcoplasmic reticulum, namely caffeine (10-20 mM), tetracaine (1 mM), and ryanodine (10 microM), abolish the oscillations. However, at lower concentrations, these three drugs have different effects on the oscillations. Caffeine (1-2 mM) decreases the oscillation amplitude but increases the frequency. Tetracaine (100-500 microM) has little effect on the magnitude of the oscillations but decreases their frequency. Ryanodine, at all concentrations used (0.1-10 microM), eventually abolishes the oscillations but, in doing so, decreases the magnitude, leaving the frequency unaffected. When [Ca2+]o was changed in order to vary [Ca2+]i, both the frequency and the magnitude of the oscillations always changed in the same direction. This suggests that these three drugs have effects in addition to just changing [Ca2+]i.
Properties of two types of calcium channels in clonal pituitary cells.
Authors: Matteson D R , Armstrong C M
Published: May 2004

The calcium currents of GH3 cells have been studied using the whole cell variant of the patch-clamp technique. Under conditions that eliminate sodium and potassium currents, we observed inward currents that activated within a few milliseconds, and deactivated with two time constants, approximately 150 microseconds and 3 ms at -80 mV, 18-20 degrees C. The components are called FD and SD (fast deactivating and slow deactivating). Both components are calcium currents, and are greatly reduced when magnesium is substituted for most of the calcium in the bath. In addition to (a) their different rates of deactivation, the two components differ in a number of other properties. (b) The SD component inactivates almost completely, with a time constant of 23 ms at 20 mV, 19 degrees C. The FD component, on the other hand, shows little or no sign of inactivation, and is almost the same in amplitude from 10 to 100 ms. The components thus seem quite independent of each other, and must arise from two independent sets of channels. (c) The FD channels activate more rapidly than SD at 20 mV, by a factor of approximately 2 as is shown in several ways. (d) In 10 Ca or 10 Ba, the activation curve for SD channels is approximately 20 mV more negative than for FD or Na channels. (e) FD channels conduct barium ions more effectively than calcium by a ratio of approximately 2. (f) FD channels "wash out" within minutes after the patch electrode breaks into a cell, whereas SD channel current remains relatively stable. It is argued that SD channels, because of their negative activation threshold, are involved in electrical events near threshold, and that FD channels are best suited for calcium injection once a spike has been initiated.
Voltage-dependent gating of veratridine-modified Na channels.
Authors: Leibowitz M D , Sutro J B , Hille B
Published: May 2004

Na channels of frog muscle fibers treated with 100 microM veratridine became transiently modified after a train of repetitive depolarizations. They open and close reversibly with a gating process whose midpoint lies 93 mV more negative than the midpoint of normal activation gating and whose time course shows no appreciable delay in the opening or closing kinetics but still requires more than two kinetic states. Like normal activation, the voltage dependence of the modified gating can be shifted by changing the bathing Ca2+ concentration. The instantaneous current-voltage relation of veratridine-modified channels is curved at potentials negative to -90 mV, as if external Ca ions produced a voltage-dependent block but also permeated. Modified channels probably carry less current than normal ones. When the concentration of veratridine is varied between 5 and 100 microM, the initial rate of modification during a pulse train is directly proportional to the concentration, while the rate of recovery from modification after the train is unaffected. These are the properties expected if drug binding and modification of channels can be equated. Hyperpolarizations that close modified channels slow unbinding. Allethrin and DDT also modify channels. They bind and unbind far faster than veratridine does, and their binding requires open channels.
Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts.
Authors: Kennedy B G , Lunn G , Hoffman J F
Published: May 2004

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.
The initial response of Limulus ventral photoreceptors to bright flashes. Released calcium as a synergist to excitation.
Authors: Payne R , Fein A
Published: May 2004

The leading edge of the response of Limulus ventral photoreceptors to brief flashes was investigated using a voltage clamp. The leading edge of responses increases linearly with flash intensity when dim flashes produce less than one photoisomerization per square micron of cell surface. Brighter flashes accelerate the initial portion of the response, resulting in a fourth-power relationship between the magnitude of the response at brief times after the flash and the flash intensity. The onset of this nonlinearity with increasing flash intensity is determined by the local density of photoisomerizations within the receptor. Responses to bright 10-15-mum-diam spots therefore rise faster than responses to diffuse flashes producing the same number of photoisomerizations within the receptor. Background illumination shortens the response latency and suppresses the initial nonlinearity. These phenomena can be explained by a model of transduction in which light activates two parallel cascades of reactions. Particles released by the first of these cascades open ionic channels, while the second produces an agent that accelerates the rate of production of particles by the first. Injection of the calcium buffer EGTA slows the initial portion of the response to bright flashes and suppresses its nonlinearity, which suggests that the accelerating agent released by the second cascade is calcium.
Two modes of gating during late Na+ channel currents in frog sartorius muscle.
Authors: Patlak J B , Ortiz M
Published: May 2004

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.
Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium.
Authors: Worley J F , French R J , Krueger B K
Published: May 2004

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.
Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium.
Authors: Hanrahan J W , Wills N K , Phillips J E , Lewis S A
Published: May 2004

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.
Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux.
Authors: Cox T C , Helman S I
Published: May 2004

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.
Catecholamine-induced transport systems in trout erythrocyte. Na+/H+ countertransport or NaCl cotransport?
Authors: Borgese F , Garcia-Romeu F , Motais R
Published: May 2004

It has previously been shown (Baroin, A., F. Garcia-Romeu, T. Lamarre, and R. Motais. 1984a, b. Journal of Physiology. 350:137, 356:21; Mahé, Y., F. Garcia-Romeu, and R. Motais. 1985. European Journal of Pharmacology. 116:199) that the addition of catecholamines to an isotonic suspension of nucleated red blood cells of the rainbow trout first stimulates a cAMP-dependent, amiloride-sensitive Na+/H+ exchange. This stimulation seems to be transient. It is followed by a more permanent activation of a coupled entry of Na+ and Cl-, which is inhibited by amiloride but also by inhibitors of band 3 protein (DIDS, furosemide, niflumic acid). The coupled entry of Na+ and Cl- could therefore result from the parallel and simultaneous exchange of Na+out for H+in (via the cAMP-dependent Na+/H+ antiporter) and Cl- out for HCO3- in (via the anion exchange system located in band 3 protein). However, in view of the following arguments, it had been proposed that NaCl uptake does not proceed by the double-exchanger system but via an NaCl cotransport: (a) Na+ entry requires Cl- as anion (in NO3- medium, the Na uptake is strongly inhibited, whereas NO3- is an extremely effective substitute for Cl- in the anion exchange system); (b) Na uptake is not significantly affected by the presence of HCO3- in the suspension medium despite the fact that in red cells, Cl-/HCO3- exchange occurs more readily than the exchanges of Cl- for basic equivalents in a theoretically CO2-free medium (the so-called Cl-/OH- exchanges). The purpose of the present paper was a reassessment of the two models by using monensin, an ionophore allowing Na+/H+ exchange. From this study, it appears that NaCl entry results from the simultaneous functioning of the Na+/H+ antiporter and the anion exchange system. The apparent Cl dependence is explained by the fact that, in these erythrocytes, NO3- clearly inhibits the turnover rate of the Na+/H+ antiporter. As Na+/H+ exchange is the driving component in the salt uptake process, this inhibition explains the Cl requirement for Na entry. The lack of stimulation of cell swelling by bicarbonate is explained by the fact that the rate of anion exchange in a CO2-free medium (Cl-/OH- exchange) is roughly equivalent to that of Na+/H+ exchange and thus in practice is not limiting to the net influx of NaCl through the two exchangers.(ABSTRACT TRUNCATED AT 400 WORDS)
Decline of electrogenic Na+/K+ pump activity in rod photoreceptors during maintained illumination.
Authors: Shimazaki H , Oakley B
Published: May 2004

Light-evoked changes in membrane voltage were recorded intracellularly from rod photoreceptors in the isolated retina preparation of the toad, Bufo marinus, during superfusion with a solution containing pharmacological agents that blocked voltage-dependent conductances. Under these conditions, the amplitude of the hyperpolarizing photoresponse became much greater than under control conditions. The results of several experiments support the conclusion that this increase in photoresponse amplitude was due primarily to a voltage that was produced when the electrogenic current from the rods' Na+/K+ pump flowed across an increased membrane resistance (Torre, V. 1982. Journal of Physiology. 333:315). At the onset of a period of continuous illumination, the rod membrane first hyperpolarized and then began to repolarize, and after 180 s of illumination, the membrane voltage had recovered by 60-72% of its initial hyperpolarization. There did not appear to be any significant decrease in rod membrane resistance associated with this repolarization. Both the enhanced hyperpolarization at light onset and the slow repolarization during maintained illumination were blocked by superfusion with 10.0 microM strophanthidin. These data support the hypothesis that the activity of the rods' Na+/K+ pump declines progressively during maintained illumination. It is likely that the decline in pump activity produces significant changes in [K+]o in the subretinal space during maintained illumination.