Exposure of energy-depleted rat trabeculae to low pH improves contractile recovery: role of calcium.

The beneficial effect of low pH during cardiac ischemia on reperfusion injury has often been attributed to its energy-saving effect due to inhibition of contraction. The role of low pH on Ca2+ accumulation and muscle tension was assessed in energy-depleted tissue by changing the pH of the medium from 7.4 to 6.2 at onset of rigor development during metabolic inhibition (MI), i.e., in the energy-depleted phase. Cytosolic free Ca2+ ([Ca2+]i) and intracellular H+ (pHi) were measured in rat trabeculae at 20 degrees C with fura 2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively, and tension was recorded. The preparations were energy depleted by stimulation at 1 Hz in glucose-free Tyrode solution with 2 mM NaCN. Rigor developed within 20 min, indicating energy depletion. Resting [Ca2+]i was followed during 50 min (group I) or 100 min (group II) of rigor, and recovery was followed for 60 min in glucose-containing Tyrode solution at 0.2-Hz stimulation. Resting [Ca2+]i rose within 50 min (group I) but stabilized in the 50- to 100-min period (group II). All preparations from group I (n = 5) resumed contraction in the recovery period but in group II (n = 10) 70% failed to recover, and [Ca2+]i remained elevated compared with those that recovered. An extracellular pH of 6.2, resulting in similar pHi, from onset of rigor development (group III) led to only a modest rise in [Ca2+]i during the 100-min rigor period, and all preparations resumed contraction after approximately 3 min in normal medium. ATP was very low in all groups at the end of MI but was still significantly lower in group II than in groups I and III. A beneficial energy-sparing effect of low pH during the rigor phase can therefore not be excluded. We conclude that 1) the capacity of trabeculae to recover from MI depends on the time period and magnitude of the [Ca2+]i rise in the energy-depleted phase and 2) low pH in energy-depleted trabeculae protects against Ca overload, improving recovery after normalization of perfusion conditions.

Effect of perfusion pressure on force of contraction in thin papillary muscles and trabeculae from rat heart.

1. Increased coronary perfusion leads to increased myocardial contraction and oxygen consumption (Gregg's phenomenon) even when oxygen supply is presumably sufficient. Previous studies concerned whole hearts, however, in which local hypoxia may play a role. We developed techniques for internal perfusion of thin papillary muscles from rat heart. The influence of perfusion pressure on muscle contraction was studied. We investigated whether Gregg's phenomenon is due to (a) hypoxia, (b) stretch of the muscle fibres, or (c) increased contractility. 2. The effectiveness of the perfusion technique was demonstrated in four ways: (a) the diameter of the capillaries increased with perfusion pressure; (b) 14 +/- 4% (mean +/- S.D., n = 11) increase in muscle diameter was observed on a change of perfusion pressure from 0 to 50 cmH2O; (c) addition of India ink to the perfusate caused rapid staining of the entire muscle; (d) during internal perfusion and external superfusion peak force was mainly determined by the [Ca2+] in the internal perfusate. 3. An increase of perfusion pressure from 0 to 70 cmH2O induced 74 +/- 20% (mean +/- S.D., n = 11) increase in peak force of contraction. In the absence of internal perfusion peak force was not affected by approximately 50% reduction of the PO2 in the bathing solution (from 700 to 350 mmHg). Hence, oxygen supply was not a limiting factor, i.e. the effect of internal perfusion on force was not related to hypoxia. 4. Segment length was measured with markers attached to the surface of the muscle. Perfusion-induced changes in segment length were negligible (-0.2 +/- 1.5%, n = 11). Force-length relationships at different perfusion pressures show that the perfusion-induced increase in force was generally larger than the maximum increase in force that could be induced by stretch. Furthermore, the time course of stretch and perfusion effects on force was different. We conclude that Gregg's phenomenon is not related to changes in fibre length, i.e. the hypothesis of pressure-induced stretch ('garden hose' effect) does not apply to papillary muscles. 5. The pressure-induced changes in the force-length relationship were similar to the changes obtained with interventions that increase contractility, such as increased [Ca2+]. 6. Since hypoxia and length effects were not involved, and the effect of perfusion pressure was similar to that of inotropic interventions, we conclude that Gregg's phenomenon is a change in contractility. Possible explanations include changes in the ionic composition or volume of the interstitium, and inotropic factors produced by the endothelium or intramyocardial neurons.

Role of Ica and Na+/Ca2+ exchange in the force-frequency relationship of rat heart muscle.

The inward Ca2+ current, ica, increases with the frequency of stimulation in single ventricular myocytes, but the presence and possible role of this phenomenon in intact heart muscle of mammals has not been studied. The present study addresses the question whether changes in ica play a role in the force-frequency relationship in thin ventricular trabeculae from rat heart. The duration of the action potential at 50% repolarization, APD50, is related to the strength and duration of ica (Mitchell et al., 1984b; Schouten, 1986). APD50 increased with the frequency of stimulation. Peak force of contraction, F, was minimal at 0.1-0.3 Hz and increased at both higher and lower frequencies, suggesting two mechanisms with opposite frequency-dependence. The increase at low frequencies was abolished by drugs that inhibit Ca2+ uptake by the sarcoplasmic reticulum (caffeine, theophylline), but not by Ca2+ antagonists that block ica (nifedipine, Mn2+). This is consistent with the hypothesis that a small net influx of Ca2+ across the sarcolemma during long diastoles was responsible for loading of the reticulum and enhancement of F at low frequencies. The increase of F and APD50 at high frequencies was abolished by Ca2+ antagonists but not by caffeine and theophylline. From this result it is concluded, that frequency-induced enhancement of ica occurs in intact heart muscle and contributes to the increase in F.

Paradox of enhanced contractility in postischemic rat hearts with depressed function.

Depressed function of postischemic hearts may be related to incomplete recovery of coronary perfusion. To circumvent this factor we studied the properties of papillary muscles under controlled extracellular conditions. First, recovery of function was measured in postischemic rat hearts. Next, a muscle was dissected and superfused in a bath. After 40 min of ischemia, recovery of cardiac output was generally zero. Muscles from this group were relaxed or showed small contractures. After 20-30 min of ischemia recovery of coronary flow and cardiac output was 50-100%, and the isolated muscles showed the following properties (compared with Langendorff-perfused controls): 1) increased force at normal or low [Ca2+]; 2) action potential and postextrasystolic potentiation were unchanged, which indicates that Ca2+ influx per beat was unchanged; 3) the decay of potentiation was slowed, indicating a reduced rate of Ca2+ extrusion via Na(+)-Ca2+ exchange. This implies intracellular Ca2+ accumulation and explains the increased force. Postischemic enhancement of contractility (isovolumic pressure) was demonstrated also in whole heart preparations. We conclude that mild injury by preceding ischemia leads to enhanced contractility (Ca2+ accumulation), advanced injury to local contractures, and finally to a general contracture (Ca2+ overload). Recovery of heart function and coronary flow probably depends on the number and size of local contractures.

Interval dependence of force and twitch duration in rat heart explained by Ca2+ pump inactivation in sarcoplasmic reticulum.

1. The influence of the interstimulus interval on twitch duration was analysed in isolated heart muscle of the rat. When the muscle was in the steady state at interstimulus intervals at 5 s a test interval was interposed and varied. Duration of twitch and action potential, sarcomere length and peak force of the test beats were measured. 2. Twitch force and duration increased when the test interval was increased from 0.4 to 10 s. This effect was abolished by inhibitors of sarcoplasmic reticulum function (ryanodine, caffeine, Sr2+). Hence, the interval dependence is controlled by the sarcoplasmic reticulum. 3. Post-extrasystolic potentiation, variation of [Ca2+]o and [Na+]o and blocking of iCa with nifedipine and Mn2+ led to large variations in force, reflecting variations in the amount of Ca2+ released from the sarcoplasmic reticulum. The effect on twitch duration was small, indicating that twitch duration was rather insensitive to the amount of released Ca2+, and not controlled by iCa and Na(+)-Ca2+ exchange. 4. Action potential duration was much shorter than twitch duration and, depending on the intervention, changes were in the same or in opposite direction. Hence, the action potential did not determine twitch duration. 5. Small variations in sarcomere length amongst test contractions were observed, but these variations could not account for the effects of the test interval. 6. It is proposed that the Ca2+ pump in the sarcoplasmic reticulum is activated during each contraction and inactivates slowly. Thus, after a short interval the pump is still activated and rapidly sequesters much of the released Ca2+ leading to a small twitch and rapid relaxation. This mechanism ensures proper relaxation and diastolic filling of the ventricle. The biochemical basis and implications of the hypothesis are discussed.

Calcium metabolism and depressed contractility in isolated human and porcine heart muscle.

Contractility is often depressed in isolated heart muscle. To analyze this phenomenon, we measured the derivative of left ventricular pressure (dP/dt) in intact and in isolated, blood perfused pig hearts, and peak force (F) or stress (F/mm2) in ventricular trabeculae of man and pig. When the heart was in the steady state at a priming frequency of 2 Hz an extrasystolic interval of 0.3 s was interposed, followed by four postextrasystolic intervals of 0.8 s. In the case of isolated trabeculae the priming frequency was 0.2 Hz, the extra interval 0.4 s, and the post-extrasystolic intervals were 5 s. The exponential decay of potentiation is characterized by the constant D: a low value of D indicates a rapid decay of potentiation. DP/dt was about 1000 mm Hg/s in the intact hearts, but within 1 h after isolation dP/dt decreased to about 700 mm Hg/s, and this was associated with a decrease in D from 0.63 to 0.40. Developed stress in the isolated trabeculae was about 2 mN/mm2 and D was about 0.20 under standard, in vitro conditions (a.o. 1.5 mM Ca2+. 0.2 Hz stimulus frequency). This stress is only 10% of the calculated stress in the intact heart. An increase of priming frequency, or of [Ca2+], or addition of 30 nM isoproterenol to the perfusate caused a marked increase in F and D. Properties of human and porcine trabeculae were quantitatively similar. The strong correlation between dP/dt, or F, and D suggests a causal relationship. This is consistent with the current model of e-c coupling in heart muscle, in which the activity of the Ca2+ pump of the sarcoplasmic reticulum determines the decay of potentiation and the amount of releasable Ca2+ in the reticulum determines force of contraction. Since isoproterenol stimulates the Ca2+ pump in the reticulum, the increase in D and F induced by this drug is consistent with the model. We conclude, that the decreased dP/dt, F, and D in isolated preparations was due to impaired sarcoplasmic reticulum function. The role of this phenomenon in the stunned heart syndrome, species differences and possible causes are discussed.

Sarcolemma, sarcoplasmic reticulum, and sarcomeres as limiting factors in force production in rat heart.

Inotropic interventions were compared with respect to their maximum effect on force of contraction in rat myocardium to identify limiting steps in calcium handling. Peak force, sarcomere length, and action potentials were measured in thin ventricular trabeculae. Relevant control conditions were stimulation frequency, 0.2 Hz; [Ca2+]o, 1 mM; [K+]o, 5 mM; [Na+]o, 150 mM. The inotropic interventions and results were as follows. 1) The interventions of high [Ca2+]o, low [Na+]o, high [K+]o, addition of tetraethylammonium chloride, or postextrasystolic potentiation resulted in approximately the same (within 5%) maximum force (Fmax). Above the respective optimum doses, force declined and aftercontractions were often observed. Combinations of the different interventions never enhanced force to above Fmax. This suggests that Fmax is determined by a maximum level of Ca2+ in the sarcoplasmic reticulum, above which spontaneous release occurs. 2) Sr2+ (10 mM) caused an increase of force to 1.3 X Fmax and lengthening of contraction and action potentials. The force-sarcomere length relation was, then, similar to that in skinned fibers at maximum activation. Hence, 1.3 X Fmax reflects saturation of the sarcomeres. We postulate that a large influx of Sr2+ during the long action potential can circumvent the reticulum and activate the sarcomeres directly. When the reticulum was blocked with ryanodine, maximum force of tetanic contractions was about 1.1 X Fmax. This result supports the above conclusions. 3) Isoproterenol increased force to a maximum that was 20% below Fmax and shortened the contraction. This may be due to a decreased sensitivity of the sarcomeres to Ca2+ or to stimulation of the Ca2+ pump in the reticulum, that is, an increasing fraction of the released Ca2+ is sequestered before it can activate the sarcomeres. Thus, three factors that limit force production were identified, depending on the inotropic stimulus.

Influence of electrogenic Na/Ca exchange on the action potential in human heart muscle.

STUDY OBJECTIVE: The plateau of the action potential in heart muscle is largely due to the inward Ca2+ current, ica; however, Ca2+ extrusion via Na+/Ca2+ exchange may also generate a significant current, ina/ca. The aim was to assess the influence of ina/ca on the action potential in isolated human heart muscle. DESIGN: Action potentials and force of isometric contractions were recorded in ventricular trabeculae. The muscle was subjected to various stimulus patterns, Ca2+ antagonists, and variations in the ionic composition of the extracellular medium. PATIENTS: From 49 patients, aged 0.5 to 14 years, small right ventricular trabeculae were obtained during open heart surgery. The operations concerned corrections of ventricular septal defects. Data presented in this paper were from nine preparations in which action potentials were recorded during several hours. MEASUREMENTS AND MAIN RESULTS: The results confirmed that: (1) the amplitude of the early part of the plateau was depressed by low [Ca2+] and by Ca2+ antagonists, showing that ica dominates this early part; and (2) that low [Na2+] and post-extrasystolic potentiation also depressed the early component of the plateau of the action potential, which can be explained by inactivation of ica due to increased levels of intracellular Ca2+. A novel observation was that post-extrasystolic potentiation led to an increase in action potential duration (APD). An explanation is that the potentiated contraction follows from an increased amount of intracellular Ca2+ which also activates an inward current, possibly ina/ca. This assumption is strengthened by the finding that lengthening of APD after extrasystoles was abolished: (a) at low [Ca2+], ie, when force was small and there was little Ca2+ to be extruded; and (b) at low [Na+], ie, when force was large but the driving force of the Na+ gradient for extrusion of Ca2+ was small. CONCLUSIONS: The early part of the plateau is dominated by ica, whereas ina/ca is relatively more important during the later part, and tends to lengthen the action potential.

Altered calcium handling at normal contractility in hypertrophied rat heart.

Left or right ventricular hypertrophy was induced by banding of the aorta or pulmonary artery in different groups of rats. After 5 to 10 weeks the degree of hypertrophy was about 15% in left and 80-160% in right ventricles, as determined by weight of the ventricle or by myocyte size. Action potentials and force-interval relationships were measured in papillary muscles isolated from either ventricle. As compared to muscles from control and SHAM-operated rats, hypertrophied papillary muscles showed: (1) Marked prolongation of the action potential and greater degree of post-extrasystolic potentiation. This indicates enhanced influx of Ca2+ probably via Ica; (2) Delayed relaxation of isometric force and faster decay of potentiation, which indicates reduced sequestration of Ca2+ by the sarcoplasmic reticulum; (3) Minor changes in steady-state peak force under standard conditions, which is explained from the opposite inotropic effects of enhanced Ca2+ influx and impaired function of the reticulum. Myocyte volume in the normal left ventricle was almost two times larger than in the normal right ventricle, and this was associated with a longer action potential and greater degree of post-extrasystolic potentiation in left as compared with right ventricular muscles. The rate of decay of potentiation, however, was not different. This might indicate that depressed function of the sarcoplasmic reticulum occurs with pressure-overload hypertrophy and not with normal age-dependent growth.

Regulation of Ca2+ current in frog ventricular myocytes by the holding potential, c-AMP and frequency.

The whole-cell patch-clamp technique was used to study the effects of holding potential and frequency on the Ca2+ current in frog ventricular myocytes. INa was blocked by TTX, and ica was activated with depolarizing clamps from different holding potentials. Variation of the holding potential revealed three new effects on ica: (1) At -40 mV iCa declined with a time constant of 15 min, while at -90 mV, this irreversible decline (run down) in iCa did not occur. (2) The decline of iCa at -40 mV was biphasic: run down was preceeded by a slow inactivation with a time constant of 40 s, which was reversible upon returning the holding potential to -90 mV. (3) Increasing the frequency of the clamp pulses from 0.1 to 1 Hz led to a rapid decline of iCa when the holding potential was positive to -60 mV, but at -90 mV had either no effect or increased iCa by 35%, if c-AMP was included in the dialyzing solution. On the other hand, c-AMP did not alter the time course of the run down and the slow inactivation. Replacement of extracellular Ca2+ by Ba2+ markedly slowed iCa kinetics, but did not change the very slow inactivation or the frequency-induced enhancement of iCa. Injection of c-AMP led to a transient increase of iCa. The phosphodiesterase inhibitor theophylline enhanced the amplitude of the transient and slowed its decay. This effect was mimicked by increased frequency. It is concluded that frequency-induced enhancement of iCa is highly dependent on the holding potential, independent of Ca2+, and may involve elevation of the intracellular level of c-AMP via inhibition of phosphodiesterase activity. The new type of very slow inactivation is probably under direct voltage control and independent of Ca2+ and c-AMP.

Excitation-contraction coupling in myocardium: implications of calcium release and Na+-Ca2+ exchange.

In this paper, we present evidence in support of the hypothesis that electrogenic Na+-Ca2+ exchange is responsible for three phenomena in rat cardiac muscle: the slow repolarization phase of the action potential, the time course of the mechanical recovery process, and the development of triggered arrhythmias. It was shown that the duration of the slow phase of repolarization of the action potential varies in proportion to the Na+ concentration gradient and inversely with the Ca2+ concentration gradient over the cell membrane. This suggested that Na+-Ca2+ exchange can generate a current of sufficient magnitude to maintain the membrane depolarized at a level of -60 mV. The mechanical restitution process of rat cardiac trabeculae was shown to exhibit three phase. The first phase, alpha, probably reflects rapid transport of calcium in the sarcoplasmic reticulum from the uptake sites to the release sites. After the initial increase of force during alpha, force rises further during phase beta and then declines during phase gamma. During all phases, force increases with the extracellular calcium concentration. beta is accelerated by preceding extrasystoles, while an increase of the heart rate causes force to increase at approximately the same rate but to a higher level during phase beta. These observations are compatible with a model in which the sarcoplasmic reticulum sequesters calcium from the cytosol, while the membrane of the sarcoplasmic reticulum is assumed to exhibit also a small leak of calcium into the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Force-interval relationship in heart muscle of mammals. A calcium compartment model.

A mathematical model was derived that describes peak force of contraction as a function of stimulus interval and stimulus number in terms of Ca2+ transport between three hypothetical Ca2+ compartments. It includes the conventional uptake and release compartments and recirculation of a fraction r of the activator Ca2+. Peak force is assumed to be proportional to the amount of activator Ca2+ released from the release compartment into the sarcoplasm. A new extension is a slow exchange of Ca2+ with the extracellular space via an exchange compartment. Six independent parameters were necessary to reproduce the different effects of postextrasystolic potentiation, frequency potentiation, and post-rest potentiation in isolated heart muscle from the rat. The normalized steady state peak force (F/Fmax) under standard conditions varied by a factor of ten between preparations from rat heart. Analysis with the model indicated that most of this variation was caused by two variables: the Ca2+ influx per excitation and the recirculating fraction of activator Ca2+. The influence of the Ca2+ antagonist nifedipine of the force-interval relationship was reproduced by the model. It is concluded that the model may serve to analyze the variability of contractile force and the mode of actions of drugs in heart muscle.

The negative correlation between action potential duration and force of contraction during restitution in rat myocardium.

Action potential duration at 50% of the amplitude (APD50) and peak force of isometric contractions were recorded from right ventricular trabeculae of rat heart at 26 degrees C. When the preparation was in the steady state at 0.2 Hz, a varied test interval was interposed. The premature contraction after a test interval of 0.25 s showed reduced force and increased APD50. Gradual prolongation of the interval from 0.25 to 80 s led to a gradual increase of force and shortening of APD50. At longer intervals force decreased and APD50 increased. This gave a negative APD50-force correlation. In the presence of the Ca2+ antagonists nifedipine or Mn2+, force was reduced to about 70%. APD50 was shortened to about 50% and was almost independent of the test interval. The slope of the negative APD50-force relationship was only 20% of that under control conditions. It is concluded that the inward Ca2+ current, isi, is a prerequisite for the interval-dependent changes in APD50 and for the negative APD50-force relationship. A reduction of the stimulation frequency from 0.2 to 0.003 Hz resulted in a gradual reduction of force and prolongation of APD50. Both force and APD50 were then almost independent of interposed test intervals. In low [Na+], at 0.2 Hz, force was almost maximal and APD50 was short at all test intervals. Thus, throughout these two interventions all changes in force were associated with opposite changes in APD50. The causal factor underlying the negative APD50-force relationship is probably an intracellular release of Ca2+ which activates the contractile filaments and inactivates isi.

The force-frequency relationship in rat myocardium. The influence of muscle dimensions.

Peak force and membrane potential were recorded from papillary muscles and trabeculae excised from the ventricles of adult rat hearts. Experiments were performed at 2.5 mM Ca2+ and 26 degrees C. In thick preparations (diameter 0.2-1.2 mm) an increase of stimulation frequency caused a reduction of peak force and action potential duration as has been found in many studies previously. In thin preparations (diameter less than 0.2 mm) both peak force and action potential duration were almost independent of stimulation frequency. When the flow of Tyrode solution through the muscle bath was reduced an increase of stimulation frequency caused a reduction of peak force and action potential duration in thin preparations. We conclude that the reduced peak force and action potential duration in papillary muscles at high stimulation frequencies is due to insufficient exchange of metabolites and oxygen between the medium and the core of the muscle. The results indicate that the critical diameter for the preparations is about 0.2 mm.

The slow repolarization phase of the action potential in rat heart.

Intracellular action potentials and isometric force were measured from thin trabeculae of the right ventricle of rat heart. Characteristic for the action potential of rat myocardium is a short plateau and a slow final repolarization phase. We have studied the influence of ionic composition of the medium and of stimulation frequency on the slow phase of repolarization and its relation to peak force. The results confirmed a positive correlation between peak force and the duration of the slow phase of repolarization, as has been reported for other species. An increase of [Ca2+]o caused a shortening of the slow phase of repolarization when peak force was kept constant. In low [Na+]o peak force was increased and the slow phase of repolarization was shortened. Reperfusion with normal medium after a period in low [Na+]o induced a transient prolongation of the slow phase of repolarization and reduction of peak force. The transient lasted about 20 min. In the presence of the Ca2+ entry blocker nifedipine the action potential duration and peak force were reduced. Low [Na+]o caused less shortening of the slow phase of repolarization and a greater increase of peak force. The slow phase of repolarization was prolonged transiently following reperfusion at normal [Na+]o, but only during a few beats. These results are in agreement with the hypothesis that the slow phase of repolarization is due to an inward current generated by Na+-Ca2+ exchange, as latter mechanism is known to be sensitive to the intracellular and extracellular concentrations of both Na+ and Ca2+.

The relationship between action potential duration and force of contraction in rat myocardium.

Intracellular action potentials and peak force (F) of isometric contractions were recorded from ventricular trabeculae of adult rats at 26 degrees C. The duration of the action potentials was measured at 50% of the amplitude (APD50) and at 10% of the amplitude (APD10). The effect of a variety of experimental interventions on the three variables was studied. APD50 reflects the duration of the plateau phase which is typically about 30 ms in rat myocardium. APD10 reflects the sum of the duration of the plateau phase and of the slow final repolarization phase (phase 3') and is about 80 ms. During paired-pulse stimulation, strong contractions coincided with shortened APD50 and prolonged APD10, i.e. a negative APD50-F and a positive APD10-F relationship was obtained. Addition of Sr2+ to the medium caused an increase of F and prolongation of APD50 and APD10. Nifedipine caused a decrease of F and a shortening of APD50 and APD10. Substitution of extracellular NaCl by LiCl caused an increase of F but a shortening of APD50 and APD10. An increase of [Ca2+]o caused an increase of F whereas APD50 was almost constant and APD10 increased. A simple model is proposed which explains the results, assuming that: the effect of paired-pulse stimulation is related to time dependent intracellular Ca2+ transport; Sr2+ and nifedipine act mainly on isi; NaCl substitution inhibits Na+/Ca2+ exchange; and [Ca2+]o affects both isi and Na+/Ca2+ exchange.

Cardiac action potential duration and contractility in the intact dog heart.

The maximum rate of rise of left ventricular pressure (DP) and action potential duration (a.p.d.) were measured in closed-chest anaesthetized dogs with atrioventricular dissociation and beta-adrenergic blockade. Right ventricular stimulation was carried out with protocols consisting of a conditioning 'priming' period and a test period. When a single test stimulus was introduced at varying intervals after the priming period, DP was found to be maximal at 800-1000 ms. With this single test stimulus fixed at the optimum, DP was found to be a variable inverse function of the a.p.d. of the same beat; no positive correlation could be found between DP and a.p.d. When a second test stimulus at the optimum interval was introduced after the first, the DP (DP2) was found to be strongly dependent on that elicited by the first test stimulus (DP1); the relationship was positive, linear, independent of the method used to vary DP, and independent of whether DP1 was depressed or potentiated. The slope of the relationship was less than 1.0 and the line passed through the point where DP2 = DP1; this is the point of continuous stimulation at the optimum interval in a steady state. This result is consistent with the hypothesis that the coefficient relating DP1 to DP2, at constant a.p.d. of the first test pulse (AP1), is an index of the proportion of the activator of contraction stored during relaxation of test beat 1 which is released again on beat 2. In order to test the hypothesis that the remaining contractility depended on the action potential of test beat 1, AP1 was varied by changing the intervals between the priming stimuli. In order to determine the relationship between DP2 and AP1 it was necessary to carry out multiple regression analysis because DP2 was already known to be strongly dependent on DP1 (point 3 above), i.e. DP2 = BDP(DP1) + BAP(AP1 - D). This analysis yielded highly significant positive values for the coefficients BDP and BAP. This result is compatible with the postulate that a.p.d. influences the amount of the activator of contraction entering the intracellular store, but that this activator is not available for release to the contractile proteins until the next depolarization.

Absence of cell communication for fluorescein and dansylated amino acids in an electrotonic coupled cell system.

Quantitative evaluation of the diffusion process of sodium fluorescein and dansylated amino acids in the salivary gland of the larvae of Drosophila hydei reveals that the differences in specific permeability between the junctional and nonjunctional membranes, as found for small ions, do not apply to the fluorescent probes. There are no significant differences between the permeability properties for the different dansylated amino acids tested, and the same properties are found for sodium fluorescein.