Aequorin Light Emission Research Articles

Atrial myocardium is the predominant inotropic target of adrenomedullin in the human heart

Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001-1 micromol/l) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 +/- 8% (at 1 micromol/l; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 +/- 20%; force/light ratio (DeltaF/DeltaL) 0.58 +/- 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/l) proportionally increased force and aequorin light emission (DeltaF/DeltaL 1.0 +/- 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 micromol/l) increased atrial cAMP levels by 90 +/- 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts Ca(2+)-dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles.

Influence of pyruvate on contractile performance and Ca(2+) cycling in isolated failing human myocardium.

Application of pyruvate was shown to improve contractile function in isolated animal myocardium and hemodynamics in patients with congestive heart failure. We assessed the influence of pyruvate on systolic and diastolic myocardial function and its subcellular mode of action in isolated myocardium from end-stage failing human hearts. In muscle strip preparations, concentration-dependent effects of pyruvate on developed and diastolic force (n=6), aequorin light emission reflecting intracellular Ca(2+) transients (n=6), and rapid cooling contractures reflecting sarcoplasmic reticulum (SR) Ca(2+) content (n=11) were measured. Pyruvate resulted in a concentration-dependent increase in developed force and a decrease in diastolic force, with a maximum effect of 155% and 21%, respectively, at 20 mmol/L pyruvate (P<0.05). This was associated with a dose-dependent prolongation of time to peak tension and relaxation time. Pyruvate increased rapid cooling contractures by 51% and aequorin light signals by 85% (at 15 and 20 mmol/L; P<0.05). This indicates increased SR Ca(2+) content and increased intracellular Ca(2+) transients. The inotropic effect of pyruvate was still present after elimination of SR Ca(2+) storage function with 10 micromol/L cyclopiazonic acid and 1 micromol/L ryanodine (n=8). Pyruvate significantly increased intracellular pH from 7.31+/-0.03 to 7.40+/-0.04 by BCECF fluorescence (n=6). The present findings indicate that pyruvate improves contractile performance of failing human myocardium by increasing intracellular Ca(2+) transients as well as myofilament Ca(2+) sensitivity. The former seem to result from increased SR Ca(2+) accumulation and release, the latter from increased intracellular pH.

Influence of mild hypothermia on myocardial contractility and circulatory function.

Myocardial contractility depends on temperature. We investigated the influence of mild hypothermia (37-31 degrees C) on isometric twitch force, sarcoplasmic reticulum (SR) Ca2+-content and intracellular Ca2+-transients in ventricular muscle strips from human and porcine myocardium, and on in vivo hemodynamic parameters in pigs. In vitro experiments: muscle strips from 5 nonfailing human and 8 pig hearts. Electrical stimulation (1 Hz), simultaneous recording of isometric force and rapid cooling contractures (RCCs) as an indicator of SR Ca2+-content, or intracellular Ca2+-transients (aequorin method). In vivo experiments: 8 pigs were monitored with Millar-Tip (left ventricle) and Swan-Ganz catheter (pulmonary artery). Hemodynamic parameters were assessed at baseline conditions (37 degrees C), and after stepwise cooling on cardiopulmonary bypass to 35, 33 and 31 degrees C. Hypothermia increased isometric twitch force significantly by 91 +/- 16 % in human and by 50 +/- 9 % in pig myocardium (31 vs. 37 degrees C; p < 0.05, respectively). RCCs or aequorin light emission did not change significantly. In anesthetized pigs, mild hypothermia resulted in an increase in hemodynamic parameters of myocardial contractility. While heart rate decreased from 111 +/- 3 to 73 +/- 1 min(-1), cardiac output increased from 2.4 +/- 0.1 to 3.1 +/- 0.31/min, and stroke volume increased from 21 +/- 1 to 41 +/- 3 ml. +dP/dtmax increased by 25 +/- 8% (37 vs. 31 degrees C; p < 0.05 for all values). Systemic and pulmonary vascular resistance did not change significantly during cooling. Mild hypothermia exerts significant positive inotropic effects in human and porcine myocardium without increasing intracellular Ca2+-transients or SR Ca2+-content. These effects translate into improved hemodynamic parameters of left ventricular function.

Ca(2+)-dependent and Ca(2+)-independent regulation of contractility in isolated human myocardium.

Changes in contractile force of the myocardium may depend on changes in the intracellular Ca2+ concentration, changes in the responsiveness of the myofibrils for Ca2+, or a combination of both. We investigated in isolated muscle strip preparations from human nonfailing and endstage failing hearts the influence of physical (changes in preload, stimulation rate, or rhythm), and pharmacological interventions (alpha- or beta-adrenoceptor-stimulation, endothelin) on developed force of contraction and the corresponding intracellular Ca2+ transients. Isometric contraction, electrical stimulation, 37 degrees C. Simultaneous registration of force of contraction and intracellular Ca2+ transients (aequorin method). Increases in preload, alpha- and endothelin-receptor stimulation resulted in increases in force of contraction without increasing aequorin light emission. Increasing stimulation rate or increasing rest intervals resulted in parallel increases (nonfailing myocardium) or decreases (failing myocardium) of force of contraction and aequorin light emission. beta-Adrenoceptor-stimulation exerted inotropic and lusitropic effects in human failing myocardium associated with a large, overproportional increase in aequorin light emission. The human heart regulates intrinsic contractility via several subcellular mechanisms. Increases in preload (Frank-Starling-mechanism) and alpha- or endothelin-receptor-stimulation enhance myocardial contractility by increasing the Ca2+ responsiveness of the myofilaments; rate- and rhythm-dependent modulation of the contractile state directly depend on changes in the intracellular Ca(2+)-transients; beta-adrenoceptor stimulation results in an overproportional large increase in intracellular Ca2+ transients, probably due to additional cAMP-dependent Ca(2+)-desensitizing effects on the level of the myofibrils.

Diminished post-rest potentiation of contractile force in human dilated cardiomyopathy. Functional evidence for alterations in intracellular Ca2+ handling.

Post-rest contractile behavior of isolated myocardium indicates the capacity of the sarcoplasmic reticulum (SR) to store and release Ca2+. We investigated post-rest behavior in isolated muscle strips from nonfailing (NF) and endstage failing (dilated cardiomyopathy [DCM]) human hearts. At a basal stimulation frequency of 1 Hz, contractile parameters of the first twitch after increasing rest intervals (2-240 s) were evaluated. In NF (n = 9), steady state twitch tension was 13.7 +/- 1.8 mN/mm2. With increasing rest intervals, post-rest twitch tension continuously increased to maximally 29.9 +/- 4.1 mN/mm2 after 120s (P < 0.05) and to 26.7 +/- 4.5 mN after 240 s rest. In DCM (n = 22), basal twitch tension was 10.0 +/- 1.5 mN/mm2 and increased to maximally 13.6 +/- 2.2 mN/mm2 after 20 s rest (P < 0.05). With longer rest intervals, however, post-rest twitch tension continuously declined (rest decay) to 4.7 +/- 1.0 mN/mm2 at 240 s (P < 0.05). The rest-dependent changes in twitch tension were associated with parallel changes in intracellular Ca2- transients in NF and DCM (aequorin method). The relation between rest-induced changes in twitch tension and aequorin light emission was similar in NF and DCM, indicating preserved Ca(2-)-responsiveness of the myofilaments. Ryanodine (1 microM) completely abolished post-rest potentiation. Increasing basal stimulation frequency (2 Hz) augmented post-rest potentiation, but did not prevent rest decay after longer rest intervals in DCM. The altered post-rest behavior in failing human myocardium indicates disturbed intracellular Ca2- handling involving altered function of the SR.

Alterations in Intracellular Calcium Handling Associated With the Inverse Force-Frequency Relation in Human Dilated Cardiomyopathy

The present study was performed to test the hypothesis that the altered force-frequency relation in human failing dilated cardiomyopathy may be attributed to alterations in intracellular calcium handling. The force-frequency relation was investigated in isometrically contracting ventricular muscle strip preparations from 5 nonfailing human hearts and 7 hearts with end-stage failing dilated cardiomyopathy. Intracellular calcium cycling was measured simultaneously by use of the bioluminescent photoprotein aequorin. Stimulation frequency was increased stepwise from 15 to 180 beats per minute (37 degrees C). In nonfailing myocardium, twitch tension and aequorin light emission rose with increasing rates of stimulation. Maximum average twitch tension was reached at 150 min-1 and was increased to 212 +/- 34% (P < .05) of the value at 15 min-1. Aequorin light emission was lowest at 15 min-1 and was maximally increased at 180 min-1 to 218 +/- 39% (P < .01). In the failing myocardium, average isometric tension was maximum at 60 min-1 (106 +/- 7% of the basal value at 15 min-1, P = NS) and then decreased continuously to 62 +/- 9% of the basal value at 180 min-1 (P < .002). In the failing myocardium, aequorin light emission was highest at 15 min-1. At 180 min-1, it was decreased to 71 +/- 7% of the basal value (P < .01). Including both failing and nonfailing myocardium, there was a close correlation between the frequencies at which aequorin light emission and isometric tension were maximum (r = .92; n = 19; P < .001). Action potential duration decreased similarly with increasing stimulation frequencies in nonfailing and end-stage failing myocardium. Sarcoplasmic reticulum 45Ca2+ uptake, measured in homogenates from the same hearts, was significantly reduced in failing myocardium (3.60 +/- 0.51 versus 1.94 +/- 0.18 (nmol/L).min-1.mg protein-1, P < .005). These data indicate that the altered force-frequency relation of the failing human myocardium results from disturbed excitation-contraction coupling with decreased calcium cycling at higher rates of stimulation.

30] Photoprotein-mediated measurement of calcium ion concentration in mitochondria of living cells

Publisher Summary This chapter discusses the expression of the transfected complementary DNA (cDNA), the intracellular localization of the recombinant photoprotein, the reconstitution of the active, Ca 2+ measuring holoprotein, the measurement of aequorin light emission, and the conversion of luminescence data into estimates of [Ca 2+ ]. It describes the development of a new methodology for measuring [Ca 2+ ] m in living cells. By this approach the chapter demonstrates that the in vivo local domains of [Ca 2+ ] c , sufficiently high to induce fast mitochondrial Ca 2+ accumulation, are transiently generated close to plasmalemmal or intracellular Ca 2+ channels. Two appealing applications appear to be an important goal for the future: single-cell imaging and the generation of transgenic animals. As to the first, the low-light emission of photoprotein represents a major problem, and work needs to be done, both in the development of suitable apparatus designed for low-light imaging and in the enhancement of the levels of aequorin expression. As to transgenic animals, there is the fascinating possibility of studying mitochondrial Ca 2+ homeostasis in situ (e.g., in brain slices).

Changes in Free Calcium in the Endoplasmic Reticulum of Living Cells Detected Using Targeted Aequorin

The Ca2+-activated photoprotein aequorin has been engineered with the endoplasmic reticulum (ER)-targeting sequence from calreticulin at the N-terminus and the KDEL sequence at the C-terminus so that it locates in the ER of living cells. Targeting of apoaequorin to the ER of COS7 cells was demonstrated by immunolocalization. Selective permeabilization of cells expressing the modified protein suggested that targeting was highly efficient. Functional photoprotein was reconstituted in live cells by incubating them with coelenterazine. Light emission from cells expressing ER aequorin showed that the estimated free Ca2+ within the ER of live cells at 37°C was 0.3-1.0 μM, some 10 times that in the cytosol. An increase in the rate constant for aequorin light emission was demonstrated when the cells were warmed from 4°C. This increase could be in part, but not wholly, explained by an increase in rate constants for aequorin at higher temperatures and a change in kinetics as a result of the ER targeting of aequorin. The increase in rate constants in the cells was inhibited by thapsigargin and occurred in the presence or absence of extracellular Ca2+. These results highlight the importance of converting aequorin light emission to rate constants and of calibrating any variants if qualitative and quantitative conclusions are to be drawn about free Ca2+ in intracellular compartments.

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes: (aequorin/calcium/oocyte maturation/1-methyladenine/starfish).

A transient increase in intracellular Ca2+ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca2+ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca2+ transient followed by GVBD. Even when the transient increase of Ca2+ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca2+ increase is neither necessary nor sufficient for maturation of the starfish oocyte. When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca2+ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca2+ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.

Changes in internal ionized Ca 2+ and H + in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na +, K +, and some inert cation, at either physiological or zero bath Ca 2+ concentration ([Ca 2+] o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca 2+ level, ([Ca 2+] i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K +] o (i.e. ≈ −15 mV). In the absence of [Ca 2+] o a significant steady depolarization brought about by current flow increased [Ca 2+] i (and acidified the axoplasm). Changes in internal hydrogen activity, [H +] i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca 2+] i could be made. However, there are effects on [Ca 2+] i that can be ascribed to membrane potential. Thus, in the absence of [Ca 2+] o, hyperpolarization can reduce [Ca 2+] i, implying that a Ca 2+ efflux mechanism is enhanced. It is also observed that [Ca 2+] i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na + for Ca 2+ in squid giant axon.

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca 2+ concentration ([Ca 2+] o) containing 150mM each of Na +, K + and an inert cation such as Li +, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca 2+ level, [Ca 2+] i. The effect of membrane voltage on [Ca 2+] i was found to depend on the concentration of internal Na + ([Na +] i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca 2+] i. For depolarizing pulses a relationship between [Ca 2+] i gain and [Na +] i indicates that Ca 2+ entry is sigmoid with a half maximal response at 22 mM Na +. This Ca 2+ entry is a steep function of [Na +] i suggesting that 4 Na + ions are required to promote the influx of 1 Ca 2+. There was little change in Ca 2+ entry with depolarizing pulses when [Ca 2+] o is varied from 1 to 10mM, while at 50mM [Ca 2+] o calcium entry clearly increases suggesting an alternate pathway from that of Na + Ca 2+ exchange. This entry of Ca 2+ at high [Ca 2+] o, however, was not blocked by Cs + o. The results obtained lend further support to the notion that Na + Ca 2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.

Release of Free Ca²⁺ from the Nematocysts of Aiptasia mutabilis during the Discharge

Although the mechanism of discharge of nematocysts is still controversial, most authors agree that bound calcium plays an definite role in stabilizing the resting state of capsules. Therefore, the kinetics of the release of Ca²⁺ during the discharge is expected to provide useful information about the mechanism of discharge itself. The investigation was performed on microbasic mastigophores extruded in 1 M citrate from the acontia of Aiptasia mutabilis. The release of free Ca²⁺ during the discharge of a population of nematocysts induced by treatment with hypoosmotic medium was revealed by the light emission of aequorin. The time course of light emission was related to the discharge curve. The recorded light signal had two different components: a short-latency one preceding the discharge and a longer one paralleling the discharge. The effectiveness of the hypoosmotic medium in inducing the discharge was not constant in various samples. Unresponsive nematocysts gave rise to a different light signal. These data suggest that in microbasic mastigophores of Aiptasia some change in the outer layer of the capsule wall is associated with the discharge. In conclusion, in the discharge of the microbasic mastigophores of Aiptasia mutabilis, the unbinding of calcium from protein molecules of the capsule fluid could be involved. However, also in these nematocysts, the osmotically generated energy for discharge is not effective unless the "stopper" is opened.

Effects of halothane on transmembrane potentials, Ca2+ transients, and papillary muscle tension in the cat.

The effects of halothane on cat papillary muscle were studied in vitro at different pacing rates (60, 75, 100, and 150 beats/min). Simultaneous measurements of transmembrane potentials, intracellular Ca2+ transients, and isometric tension were made before and after introduction of 1 and 2 minimal alveolar concentration halothane. Anesthetic concentrations in the superfusion bath were measured using a gas chromatography procedure. The Ca2+-sensitive photoprotein, aequorin, was microinjected into papillary muscle cells. Aequorin light emission is a function of free intracellular [Ca2+] [( Ca2+]i). The most prominent effects of halothane were a dose-dependent decrease in the contractile force and a decrease in [Ca2+]i, at all pacing rates. The negative inotropic effect of halothane was associated with only small changes in the papillary muscle action potential duration. Increasing the extracellular [Ca2+] produced an increase in [Ca2+]i and counteracted the negative inotropic effects of halothane. Addition of the Ca2+ channel blocker, verapamil, and halothane, produced an additive decrease in [Ca2+]i compared with the control.

Dependence of ionized and total Ca in squid axons on Nao-free or high-Ko conditions.

The level of intracellular Ca in squid axons (both ionized and total Ca) was studied as a function of the experimental variables [Na]i, [Na]o, pHi, cyanide, and depolarization. Ionized Ca was measured by following the light emission of aequorin while total Ca was measured by the atomic absorption analysis of samples of axoplasm. Aequorin glow is known to be increased either by the application of Nao-free solutions or by depolarization produced by external solutions containing greater than normal K concentrations. The present results show that if [Na]i is low, the depolarization that is brought about by solutions with elevated [K] leads to a resting light emission that is decreased rather than increased, as is the case when [Na]i is high. In axons where [Na]i is varied, a comparison of the increments in light emission produced by the application first of Na-free and then of high-K solutions shows that they have an identical dependence on [Na]i, with a half-activation of Ca entry produced by an [Na]i of 25-30 mM. Changes in pHi affect the aequorin signal produced by depolarization, with acidification reducing and alkanization increasing the response. Cyanide did not greatly affect the size of the signal resulting from either Nao removal or that from depolarization.

Calcium‐force relationships as detected with aequorin in two different vascular smooth muscles of the ferret.

The bioluminescent calcium indicator aequorin was chemically loaded into isolated strips of ferret portal vein and ferret aorta. Aequorin light emission (a function of [Ca2+]i) was recorded simultaneously with tension. Assuming an [Mg2+]i of 0.5 mM, [Ca2+]i was 1.8 X 10(-7) M in the unstimulated portal vein at 22 degrees C where there was negligible resting tone. In contrast, in the unstimulated aorta at 22 degrees C where there was significant basal tone, the [Ca2+]i was 2.7 X 10(-7) M. In both portal vein and aorta, potassium depolarization caused a monophasic rise in intracellular Ca2+ in parallel with the rise in tension, whereas phenylephrine caused an initial spike of light during the period of the force development which then fell to a much lower plateau level during the period of force maintenance. Calcium-force curves were generated by plotting calibrated aequorin light against force while intracellular [Ca2+] was made to change either by increasing degrees of potassium depolarization or decreasing extracellular [Ca2+]. The steady-state calcium-force curve in the presence of phenylephrine was shifted to the left of the curve in the presence of potassium depolarization in both the portal vein and aorta. In the aorta there was a counter-clockwise hysteresis in the calcium-force relationship. In contrast, in the portal vein there was no demonstrable hysteresis, indicating that the apparent change in calcium sensitivity of the contractile apparatus in the presence of phenylephrine must be caused by a second messenger other than calcium.

Influence of deuterium oxide on calcium transients and myofibrillar responses of frog skeletal muscle.

The influence of substituting D2O for H2O on calcium transients and on contraction was studied in intact single skeletal muscle fibres injected with aequorin and in mechanically skinned fibres from frogs. Most experiments were carried out at 10 degrees C. Experiments performed in vitro established that the calcium concentration-effect curve for aequorin is depressed and the rate of change of aequorin light emission after sudden changes of [Ca2+] is decreased when D2O is substituted for H2O. D2O substitution greatly reduces the amplitude of the aequorin signals of intact fibres both in twitches and in tetani. This is partly the result of the influence of D2O on aequorin, but the amplitude of the calcium transients is reduced as well. In both H2O and D2O Ringer solutions, the amplitude and time course of the sarcoplasmic calcium transient during a tetanus vary greatly with the stimulus frequency. In H2O, frequencies high enough to produce mechanical fusion normally produce cytoplasmic calcium concentrations high enough to saturate the myofibrils, with the result that stimulus frequency has very little influence on the mechanical response. Saturation does not occur in D2O, and the amplitude and form of fully fused tetani are greatly influenced by the frequency of stimulation. Aequorin was used as a calcium indicator to estimate the influence of D2O substitution on the apparent equilibrium association constant for the calcium-EGTA complex. The constant was reduced by more than one order of magnitude in D2O at pD = pH = 7.0. Experiments on mechanically skinned muscle fibres showed that D2O substitution decreased the apparent sensitivity of the myofibrils to calcium, but increased the force of contraction at saturating calcium concentrations. The latter effect is probably responsible for the potentiation of tetanic tension that is observed in certain D2O/H2O mixtures or in H2O solutions immediately after a switch from D2O. In intact muscle fibres the potentiation of twitch or tetanic tension after a switch from D2O to H2O declines with a half-time of about 25 s; this probably reflects the time course of exchange of intracellular D2O for H2O. The effects of D2O on the intracellular calcium transient appeared and disappeared with a half-time of less than 1 s; this time is of the same order of magnitude as that calculated for the exchange of H2O and D2O in the T-tubules. We conclude that D2O affects calcium release at a site in the T-tubule which is more accessible to the extracellular space than to the intracellular space.

Sensitivity to injected cholera toxin of the sodium efflux in single barnacle muscle fibers

1. A study has been made of the effect of microinjected cholera toxin (CT) on the Na efflux in single barnacle muscle fibers. Characteristically, injected CT causes sustained stimulation of the ouabain-insensitive Na efflux but only after a lag phase. An effect is seen with as little as a 10 −7 M-solution of CT. Sustained stimulation after a lag phase is also seen following injection of subunit A fragment. 2. Enrichment of fibers with NAD + fails to enhance the response to CT. 3. Prior injection of GTP or its non-hydrolyzeable analogue, Gpp(NH)p, markedly reduces the response to CT, whilst prior injection of CT reduces the response to guanine nucleotides. 4. Evidence is also brought forward that omission of external Ca 2+ reversibly reduces the response to CT and that pre- or postinjection of EGTA markedly reduces the response to CT. In addition, fibers preinjected with CT show increased aequorin light emission. 5. Whereas verapamil and Cd 2+ are ineffective, both Mg 2+ and trace metals, e.g. Fe and Zn, reverse the response to CT following injection. 6. Prior injection of protein kirtase inhibitor reduces the response to CT. 7. As for calmodulin inhibitors, e.g. chlorpromazine, imipramine and mepacrine, they are effective in reducing the response to CT but not calmodulin antibody (IgG). 8. Collectively, the above results are compatible with the view that sustained stimulation of the ouabain-insensitive Na efflux by injected CT is due to persistent activation of adenylate cyclase by the toxin and that a fall in myoplasmic pCa facilitates or augments this activation mechanism.

The effects of changes of pH on intracellular calcium transients in mammalian cardiac muscle.

The calcium-sensitive photoprotein aequorin was micro-injected into cells of rat, ferret, rabbit and cat papillary muscles. Aequorin light emission is a function of free intracellular calcium concentration. The changes in intracellular calcium concentration [( Ca2+]i) and tension accompanying changes of pH have been studied. When the solution perfusing the papillary muscle was changed from Tyrode solution equilibrated with 5% CO2 to Tyrode solution equilibrated with 15% CO2, developed tension showed a rapid fall followed by a slower rise to a steady state which was less than the control. However the calcium transient associated with each contraction increased monophasically to a new steady state. When the external pH was held constant during exposure to 15% CO2 (by increasing the [HCO3-]), the initial fall of tension was reduced and the slow recovery of tension was greater than when CO2 alone was changed. The amplitude of the calcium transient increased monophasically to a new steady state which was greater than control, but less than when [CO2] alone was increased. If [HCO3-] was decreased while maintaining [CO2] at 5%, there was a slow monophasic decline in developed tension, and a small increase in peak light. Alkaloses produced by changing the [HCO3-]/[CO2] ratio produced similar results but the changes observed were in the opposite direction to those described above. The effects of changes of pHo can be explained if pHi affects tension by two mechanisms. The first mechanism, which is responsible for the rapid change in tension, is not associated with a change in [Ca2+]i. The second mechanism leads to a slower and smaller change in tension, in the opposite direction to the first, and is due to a change in the intracellular calcium transient.

Transient increase of intracellular Ca2+ during excitation of tonoplast-freeChara cells

The photoprotein aequorin was introduced into tonoplast-freeChara cells. Aequorin light emission transiently increased when the cell generated an action potential, which consisted of initial rapid depolarization and a subsequent maintenance of this at a plateau level. The light emission change also consisted of two components. First was a rapid rise followed by a rapid return to nearly the initial level. This rapid component corresponded to the initial rapid depolarization of the action potential and seemed to reflect the Ca2+ release from the plasmalemma. The second component of light emission was a gradual increase of luminescence that continued for the prolonged depolarization phase of the action potential. When the action potential was terminated, the luminescence instantly began to decrease. This component probably reflects the Ca2+ influx through the plasmalemma which may become highly permeable to ions during excitation. Free Ca2+ concentrations of tonoplast-freeChara cells containing 1 m MEGTA are estimated to be about 2 μM at the resting state and about 4 μM at the initial phase of the light emission change.

CALCIUM BUFFERING IN SQUID AXONS

This review discusses recent work on the intracellular calcium buffering systems found in the axoplasm of the giant nerve fiber of squid. The probable existence of such systems was inferred some years ago (8) when it was noted that poisoning squid axons with 2 mM cyanide in the seawater led, after a variable delay, to a fiveto tenfold increase in calcium outflux. Analysis of this observation led to the conclusion that it was due to conversion of the internal calcium from a nondif­ fusible to a diffusible form. Several years later, Baker, Hodgkin & Ridgway (4), on the basis of changes in aequorin light emission during cyanide poisoning, concluded that even though the bulk of the intracellular calcium became diffusible, it still remained un-ionized. A reasonable hypothesis to explain the findings was that in the unpoisoned axon, the intracellular calcium held within the mitochondria was released when mitochon­ drial respiration was inhibited, and was largely bound to diffusible anions. However the nature of the diffusible moiety was never determined, nor has the distribution of intracellular calcium between mitochondria, mitochondrial buffers, and free calcium in intact axons been investigated. These questions, as well as the related one of isotopic exchangeability between the various compartments, are of considera­ ble practical importance in research on calcium transport in squid axons, because control of the concentration of free calcium in the axoplasm requires control of calcium in the other compartments. Questions concerning calcium compartmentalization within the giant axon have an even more general interest to those concerned with calcium metabolism and homeostasis. Because of the large size of the squid axon, it is possible to study certain aspects of the buffering systems in situ without isolating them from the cell, whereas the requirement for prior isolation of most cellular buffering systems for in vitro study raises serious questions about the relation of the in vitro measure-