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The effect of oral administration of β-1,3/1,6-glucans from Saccharomyces cerevisiae on humoral immunity in domestic dogs is not known. In this study, 15 beagle dogs were orally given MacroGard tablets, which contain 150 mg of this β-glucan, daily for 4 weeks. At the end of this period, the total serum immunoglobulin A (IgA) level decreased significantly in the group treated with the glucan compared to that in the control group as well as compared to the concentrations before supplementation. In contrast, the total serum IgM level rose significantly, whereas no effect on the IgG level occurred. Similar changes were seen in Bordetella-specific IgA and IgM titers following vaccination during the supplementation period. The IgA concentration also became significantly lower in the saliva and tears of the glucan group than in the placebo group. The effects disappeared 1 week after the cessation of the supplementation. In conclusion, the results showed a temporary change in the isotype profile during glucan supplementation.

ABSTRACTThe effect of oral administration of ß-1,3/1,6-glucans from Saccharomyces cerevisiaeon humoral immunity in domestic dogs is not known. In this study, 15 beagle dogs were orally given MacroGard tablets, which contain 150 mg of this ß-glucan, daily for 4 weeks. At the end of this period, the total serum immunoglobulin A (IgA) level decreased significantly in the group treated with the glucan compared to that in the control group as well as compared to the concentrations before supplementation. In contrast, the total serum IgM level rose significantly, whereas no effect on the IgG level occurred. Similar changes were seen in Bordetella-specific IgA and IgM titers following vaccination during the supplementation period. The IgA concentration also became significantly lower in the saliva and tears of the glucan group than in the placebo group. The effects disappeared 1 week after the cessation of the supplementation. In conclusion, the results showed a temporary change in the isotype profile during glucan supplementation.

ABSTRACTOne of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia colithermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4+E. colifecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole‐cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1‐5 x 10(‐4) M) over the whole range of membrane potentials tested. 3. In Na(+)‐containing solution half‐maximum Ip activation (K0.5) occurred at 0.4 mM‐Tl+, 1.9 mM‐K+ and 5.7 mM‐NH4+ (holding potential, ‐20 mV). 4. The pump current (Ip)‐voltage (V) relationship of the cells in Na(+)‐containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between ‐120 and ‐20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM‐K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)‐containing solution induced a region of negative slope of the Ip‐V curve at membrane potentials positive to ‐20 mV. 6. The shape of the Ip‐V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)‐containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM‐NH4+ or 2 mM‐Tl+). 7. In Na(+)‐free, choline‐containing solution half‐maximum Ip activation occurred at 0.13 mM‐K+ (holding potential, ‐20 mV). 8. At negative membrane potentials the positive slope of the Ip‐V curve was flatter in Na(+)‐free than in Na(+)‐containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)‐free, choline‐containing solution to 0.05 mM evoked an extended region of negative slope in the Ip‐V relationship at membrane potentials between ‐40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)‐K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip‐V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage‐sensitive steps in the cardiac Na(+)‐K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The Na+ pump current (Ip) of isolated, single rabbit cardiac Purkinje cells in Na(+)‐free solution was measured at 32‐34 degrees C by means of whole‐cell recording. 2. The Ip amplitude was studied as a function of clamp potential (Vc) and external concentration of various monovalent cations known to activate the Na(+)‐K+ pump. 3. Under conditions which strongly activated Ip the Ip‐Vc curve of the cells displayed a positive slope at membrane potentials negative to ‐20 mV and little variation at more positive potentials. 4. The Ip‐Vc relationship showed an extended region of negative slope at positive and negative potentials in solutions containing low concentrations of activator cations which caused little Ip activation. A positive slope of the Ip‐Vc curve was occasionally observed at clamp potentials negative to ‐60 mV under these conditions. 5. The shape of the Ip‐Vc relation was independent of the cation species used as external Ip activator. 6. At zero membrane potential half‐maximum Ip activation (K0.5(Vc = 0 mV) occurred at 0.05 mM Tl+, 0.08 mM K+, 0.4 mM NH4+ and 1.5 mM Cs+. The Hill coefficient derived amounted to 0.9 for Tl+, 1.2 for K+, 1.04 for NH4+ and 1.5 for Cs+. 7. The concentrations of external activator cations required for half‐maximum Ip activation increased with depolarization. The voltage dependence of the K0.5 values could be described by a single exponential function for clamp potentials positive to ‐40 mV. 8. The steepness of the function is determined by a factor alpha, indicating the apparent fraction of an elementary charge which moves in the electrical field across the sarcolemma when external monovalent cations bind to the Na(+)‐K+ pump. 9. The alpha values were calculated to be 0.32 for Tl+, 0.24 for K+, 0.29 for NH4+ and 0.18 for Cs+. Possible interpretations of the alpha values are considered. 10. It is suggested that binding of external monovalent activator cations to the Na(+)‐K+ pump (or a process related to the binding) is voltage dependent. This potential‐dependent process determines mainly the shape of the Ip‐Vc curve in cardiac Purkinje cells superfused with Na(+)‐free media containing low concentrations (< K0.5(Vc = 0 mV)) of K+ or its congeners.

1. The antagonistic effect of extracellular potassium ions (K+o) and dihydro‐ouabain (DHO) on the Na(+)‐K+ pump current (Ip) was studied in isolated ventricular cells. 2. The myocytes were isolated from rats and guinea‐pigs, two species with different sensitivity towards cardiac glycosides. Ip measurements were performed at 32‐34 degrees C by means of whole‐cell recording. The membrane potential was held at ‐20 mV throughout. 3. The DHO concentration ([DHO]) required for half‐maximal Ip inhibition (apparent KD value, KD') amounted to 2.4 x 10(‐3) and 1.4 x 10(‐5) M for rat and guinea‐pig myocytes, respectively, at 5.4 mM K+o. 4. The data suggest one‐to‐one binding of DHO to the Na(+)‐K+ pump and a smaller association rate constant, as well as a larger dissociation rate constant, for binding of DHO in the rat cells. 5. Ip activation by K+o was nearly identical in myocytes of both species and was measured to be half‐maximal at approximately 1 mM K+o. Half‐maximal Ip activation by K+o remained essentially unchanged, but Ip decreased in media containing [DHO] near the respective KD' at 5.4 mM K+o. 6. The concentration‐response curve of Ip inhibition by DHO was shifted to higher [DHO] at higher [K+]o. KD' increased correspondingly. The slope of the curve was unaffected. 7. Ip and KD' displayed a similar dependence on [K+]o. 8. KD' was larger in Na(+)‐free than in Na(+)‐containing media under conditions in which the activation of Ip by K+o was nearly the same. 9. It is concluded that the antagonism between K+o and DHO, with regard to the activation of Ip, is non‐competitive. A possible mechanism of the antagonism is discussed. The mechanism implies binding of K+o and DHO to different conformational states of the Na(+)‐K+ pump which are temporarily exposed to the external face of the sarcolemma in the pump cycle. The DHO‐bound states do not participate in the generation of Ip.

The ionic components of the pacemaker current are quantitatively analysed in sheep cardiac Purkinje fibres by simultaneous measurements of the intracellular Na activity (aNai) and the membrane current under voltage clamp. The pacemaker current is operationally defined as the Cs inhibited membrane current (ICs) in Ba containing media at clamp potentials negative to -60 mV. At these potentials solutions containing CsCl (0.2–5 mM) shift the holding membrane current into the outward direction and simultaneously decreaseaNai. The Cs effects on membrane current andaNai display a similar voltage dependence. A Cs inhibited Na influx contributes toICs. The ratioICs/(Cs inhibited Na influx in electrical units) is <1 at membrane potentials positive to the potassium equilibrium potentialEK and >1 at potentials negative toEK. The ratio is close to 1 atEK suggesting Na ions to be the only carriers of the current atEK whereas K ions contribute toICs at potentials different fromEK. The effects of Cs on the Cs inhibited Na influx andICs show a very similar dose dependence. The effect is half maximum at ~0.2 mM CsCl (in 21.6 mM K; clamp potential: -85 mV). An increase of the external K concentration augmentsICs and the Cs inhibited Ca influx. Na and K ions carryingICs probably cross the membrane via an identical channel. The permeability of the channel for K+ is about 10–20 times larger than for Na+. TheICs reversal potential of a fibre bathed in a medium containing 5.4 mM K is estimated to be -50 to -60 mV.

The cell membrane of sheep Purkinje fibres hyperpolarizes transiently on returning to K containing media after several minutes in K free solution. To analyse this ‘K activated response’ voltage clamp experiments and measurements of the internal Na activity (aNai) are performed in fibres bathed in solution containing 0.5–2 mM BaCl2. Compared to the response in Tyrode solution the transient hyperpolarization beyond the resting potential is increased in Ba containing media. The response is blocked by 10-4 M dihydroouabain. During the response, in Ba treated fibres, aNai and a transient outward current decline with the same time constant at all clamp potentials tested. The transient outward current is most probably due to a temporary increase in electrogenic Na pumping. Both the amplitude and the time constant of the pump current show little voltage dependence. The electrogenic fraction of the active Na efflux is estimated to be about 39% and is independent of aNai. Ba ions facilitate the analysis of the pump current in voltage clamped fibres because K depletion is reduced and changes of the I–V relationship by K depletion are minimized. It is concluded that activation of the electrogenic Na pump is mainly responsible for the K activated response of fibres in Ba containing media.

1. The efflux of radioactive K and Cl ions was measured in cow Purkinje fibres and ventricular preparations of cow, cat and frog. The effect of K and Cl was studied by changing the extracellular K concentration between zero and 54 mM, and by substituting Cl ions by acetylglycinate, isethionate, benzenesulphonate, propionate and nitrate. 2. In the absence of Cl the rate coefficient for 42K efflux showed a pronounced fall, which was more pronounced the higher the K concentration. This effect was not related to the change in membrane potential. The rate coefficient for 42K efflux increased in the presence of higher extracellular K concentrations. 3. 36Cl efflux increased in the presence of reduced as well as increased extracellular K concentrations. 4. The calculated permeability coefficient for K (PK) was maximal at 5‐4 mM‐K, decreased slightly at higher K concentrations, but fell markedly in K‐free solutions, (to about 1/5 of the value in 5‐4 mM‐K). In Cl‐free medium PK reduced to 0‐67 of the value in the Cl medium, irrespective of the K concentration. 5. The calculated PCl was greater in K‐free and 16‐2 mM‐K than in 5‐4 mM‐K. 6. The ratio PK/PCl showed important changes as a function of extracellular K concentration: the value was 5 in 5‐4 mM‐K and fell to 2 and 0‐5 respectively in 16‐2 and K‐free solutions. 7. The results suggest that part of the changes in membrane resistance measured by electrical methods in Cl‐free media is due to a simultaneous decrease in K conductance.

1. The influence of sarcolemmal Na(+)‐H+ exchange on intracellular Na+ activity (aiNa), intracellular pH (pHi) and membrane holding current (Ih) was investigated in rabbit cardiac Purkinje fibres. pHi and aiNa were measured with liquid sensor ion‐selective microelectrodes. A two‐microelectrode voltage clamp was used while recording pHi or aiNa.pHi was varied by alternating a nominally CO2‐free HEPES buffer and a CO2‐HCO3‐buffer. 2. The intrinsic buffer capacity was calculated from the decrease in pHi after addition of CO2. The most accurate estimate was obtained when transmembrane pHi regulation was blocked and equalled 18.4 +/‐ 1.0 mequiv H+/pH unit (mean +/‐ S.E.M.). 3. aiNa started to rise when pHi fell below 7.0. A hyperpolarization paralleled the increase in aiNa. The magnitude of the rise in aiNa and the hyperpolarization were steeply dependent on pHi. 4. Inhibition of the Na(+)‐K+ pump by K(+)‐free superfusion increased aiNa. The rate of rise in aiNa was highly dependent on pHi. The rates of rise in 5% (pHi = 7.00 +/‐ 0.03), 7% (pHi = 6.89 +/‐ 0.04) and 15% CO2 (pHi = 6.74 +/‐ 0.02) at constant external pH (pHo) relative to the rate in HEPES solution (pHi = 7.24 +/‐ 0.02) were: 1.5 +/‐ 0.2, 2.4 +/‐ 0.1 and 3.1 +/‐ 0.2. The acid‐induced rise in aiNa was abolished by 2 mM‐amiloride. 5. Extracellular acidosis slowed down the recovery of pHi and depressed the rate of rise in aiNa upon intracellular acidification. When both pHi and pHo were decreased to 6.7 the acid‐dependent rate increase fell to about 10% of the value found at pHo 7.4. 6. The tetrodotoxin (TTX)‐sensitive Na+ current was not influenced by the change in pHi in the range 6.7‐7.2. 7. Intracellular acidosis was associated with an early aiNa‐independent depolarization and an inward shift in Ih. Current‐voltage plots revealed that the initial inward current shift reversed at ‐81.5 mV on average, showed inward rectification and was largely depressed in the presence of 1 mM‐Ba2+. These observations indicate a decrease in K+ conductance when pHi falls. 8. The increase in aiNa elicited a Na(+)‐K+ pump‐dependent outward current which could override the initial aiNa‐independent current shift. At a pHo of 6.7 the initial fall in Ih remained, while the secondary outward current was largely depressed. 9. The rate of active Na+ extrusion and Na(+)‐K+ pump current were suppressed by about 30% at pHi 6.7 compared to pHi 7.2.(ABSTRACT TRUNCATED AT 400 WORDS)

The kinetics of the inhibition by dihydroouabain (DHO) of the Na pump current were studied in isolated rabbit cardiac Purkinje cells by means of whole-cell recording. A fast exchange of the extracellular solution at the membrane of the cell studied was performed via two multibarrelled pipettes nearby. Judging from the steady-state inhibition of the Na pump current at various DHO concentrations half maximal inhibition occured at 1 × 10-5 mol/l DHO in a medium containing 2 mmol/1 K+ and at 3.5 × 10-5 mol/l DHO in a solution containing 10.8 mmol/1 K+. Assuming a reversible one-to-one binding reaction, the time course of DHO binding and unbinding was analysed under a variety of conditions. It was shown that the antagonistic effect of K+ on the inhibition of the pump current was entirely due to a decrease of the association rate constant of DHO binding to the sodium pump at higher K+ concentrations. The association rate constant amounted to 1 × 104 (mol/l)-1 · s-1 at 2 mmol/1 K+ and to 2.8 × 103 (mol/l)-1 · s-1 at 10.8 mmol/l K+. The dissociation rate constant of DHO unbinding remained unchanged (~0.06 s-1). The equilibrium dissociation constant KD for the inhibition of the pump current by DHO decreased by a factor 3 to 5 if the Na+ concentration of the patch pipette solution was augmented from 5 to 50 mmol/l. Increasing DHO concentrations inhibited the pump current increasingly faster and to a larger extent. The KD values derived showed little variation with the concentration of DHO applied. It is concluded that the inhibition by DHO of the pump current in cardiac Purkinje cells can be adequately described by an one-to-one binding reaction between the cardioactive steroid and the pump molecules. The reaction is modulated by external K and internal Na ions.

1. Whole‐cell recording from isolated rat and guinea‐pig ventricular myocytes revealed a change of the cardiac Na+ pump current (Ip)‐voltage (V) relationship by cardiac glycosides, specific inhibitors of the Na(+)‐K+ pump. 2. Dihydro‐ouabain (DHO) diminished Ip in rat ventricular cells at 0 mV in a concentration‐dependent manner. 3. The concentration‐response curve of Ip inhibition caused by DHO was shifted to higher [DHO] at higher extracellular K+ concentrations ([K+]o) or at more negative membrane potentials. 4. In rat myocytes, DHO immediately flattened the normalized cardiac Ip‐V curve and evoked or enhanced a region of negative slope. 5. Ouabain, at concentrations which caused a comparable inhibition of Ip, exerted DHO‐like effects on the Ip‐V relationship of rat ventricular myocytes. However, the effects developed more slowly. 6. A slowly developing alteration of the Ip‐V curve was also observed upon application of DHO to guinea‐pig ventricular cells. The range of [DHO] used was about 100‐fold lower than that applied to rat ventricular cells, but was equally effective for Ip inhibition. 7. Increasing the K+ concentration of DHO‐containing media affected the existing equilibrium of DHO binding to the cardiac Na(+)‐K+ pump. A new equilibrium was reached within about 3 s in rat ventricular myocytes, but only within about 50 s in guinea‐pig ventricular cells under the experimental conditions chosen. 8. It is concluded that the changes of the cardiac Ip‐V curve induced by cardiac glycosides are mediated by voltage‐dependent variations of the local [K+]o at the K+ binding sites of the Na(+)‐K+ pump in an ‘access channel’. The variations were estimated by means of the Boltzmann equation. The estimations agreed with those derived from the measured DHO binding to the Na(+)‐K+ pump at various [K+]o. A new equilibrium of glycoside binding to the pump is established at the altered [K+]o. The time necessary to reach the new binding equilibrium varies with the cardioactive steroid, its concentration and the glycoside sensitivity of the cardiac cells.

The Na pump current (Ip) was measured at 30–33 °C as a function of the extracellular K+ or Cs+ concentrations ([K]0; [Cs]0) by means of whole cell recording from cardioballs isolated from sheep Purkinje fibres. The results show that the magnitude of Ip is an s-shaped, saturating function of [K]0 or [Cs]0, respectively. Half maximal Ip activation occurs at 1.2 mM K0 or 3.2 mM Cs0 (clamp potential: -20 mV). These values are appceciably lower than earlier measurements on sheep Purkinje fibres indicated. The Hill coefficients for Ip activation by external K+ or Cs+ were calculated to be 1.94 and 1.73, respectively. The numbers suggest that Ip activation requires at least two external activator cations.

Accumulation of long-chain acylcarnitines in the cytoplasm of ischemic myocardium has been associated with a changed ion transport resulting in a decline of contractile force and an increased susceptibility to arrhythmias (Katz & Messineo, 1981). To elucidate the underlying biochemical mechanism for the impaired calcium availability, we studied the influence of acylcarnitines on :