Some commonly overlooked properties of calcium buffer systems: a simple method for detecting and correcting stoichiometric imbalance in CaEGTA stock solutions.

When the two-solution approach is used to make CaEGTA buffers, the most critical requirement is that the CaEGTA stock solution contain equimolar quantities of calcium and chelator. The impact on [Ca2+] of errors in the molar ratio is magnified at high buffer ratios, so stoichiometric imbalance is most readily detected there. In this paper we examine some of the properties of calcium buffer systems that illustrate these principles, and describe a simple and sensitive method for detecting and correcting stoichiometric imbalance in the CaEGTA stock solution. The method derives its high sensitivity from the fact that the CaEGTA stock solution itself represents a calcium buffer system with a very high buffer ratio.

Cloning, expression and sequence analysis of cDNA for the Ca(2+)-binding photoprotein, mitrocomin.

The primary structure of mitrocomin consists of 190 amino acid residues, with three Ca(2+)-binding sites and a tyrosine residue at the C-terminus. Mitrocomin shows an amino acid sequence homology of 67.9% and 60.7% when compared with aequorin and clytin, respectively. The amino acid residues Cys152, His58, His169, Trp12, Trp86, Trp108, Trp129 and Trp173 are conserved in all three photoproteins, suggesting that they play a role in light emission.

Inositol trisphosphate (InsP3) causes contraction in skeletal muscle only under artificial conditions: evidence that Ca2+ release can result from depolarization of T-tubules.

It has been proposed that in striated muscle inositol 1,4,5-trisphosphate (InsP3) may serve as a chemical transmitter linking membrane depolarization to Ca(2+)-release from the sarcoplasmic reticulum. Key to that hypothesis of excitation-concentration (EC) coupling was the observation that skinned muscle fibres contract on the application of InsP3. Yet skinned fibres do not always respond in this way, and in our hands intact fibres do not contract when InsP3 (1 microM-1 mM) is microinjected into them. Glycerol-shocked fibres do contract, however, and so do intact fibres that have been depolarized to about -50 mV by increasing [K+]0. These observations and related pharmacological evidence support the hypothesis that InsP3 causes a low-level depolarizing current to cross the T-tubular membrane. This current is sufficient to depolarize the T-tubules to the threshold for contraction only when the tubules are sealed over or when they are already close to the threshold. The InsP3-induced Ca2+ release sometimes observed in skinned muscle fibres and in vesicles derived from junctional sarcoplasmic reticulum probably often results from an action on sealed-over transverse tubules; in such situations it is an artifact of cell disruption. The fact that high concentrations of InsP3 do not cause contraction in normal muscle fibres is strong evidence against the hypothesis that InsP3 plays a central role in EC coupling in skeletal muscle.

Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin.

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.

Use of photoproteins as intracellular calcium indicators.

The calcium-regulated photoproteins, of which aequorin is the best known, continue to be one of the most useful groups of intracellular Ca2+ indicators. They are self-contained bioluminescent systems that emit blue light in the presence of Ca2+ ions, can readily be purified intact, and are nontoxic when introduced into foreign cells. They have been used successfully as Ca2+ indicators in almost every kind of cell, but are most widely used in muscle cells because of their relative freedom from motion artifacts. Photoproteins have also been used in conjunction with microscopic image intensification to localize Ca2+ in cells. Their large molecular size makes them difficult to introduce into cells, but once there, they have the advantage of staying in the cytoplasm. Aequorin can be microinjected satisfactorily into single cells of almost any size, but a number of alternative methods for introducing photoproteins into cells have been developed in recent years. Disadvantages of the photoproteins for some applications include the nonlinear relation between [Ca2+] and light intensity, the modest speed with which they respond to sudden changes in [Ca2+], and the fact the Mg2+ antagonizes the effect of Ca2+. Native photoproteins consist of a mixture of isospecies, and there are differences in Ca2+ sensitivity and in kinetic properties--both among photoproteins and among the isospecies of a given photoprotein. The genes for several of the isospecies of aequorin have been cloned and expressed in E. coli. It seems reasonable to hope that genetic engineering techniques may soon make it possible to consider using, as Ca2+ indicators, rare isospecies or rare photoproteins that have optimal properties for particular applications.

Actions of sympathomimetic amines on the Ca2+ transients and contractions of rabbit myocardium: reciprocal changes in myofibrillar responsiveness to Ca2+ mediated through alpha- and beta-adrenoceptors.

The effects of sympathomimetic amines on Ca2+ transients and isometric contractions were assessed in isolated rabbit papillary muscles in which multiple superficial cells had been microinjected with the calcium-sensitive bioluminescent protein aequorin. In the presence of beta-adrenoceptor blockade, the alpha-receptor agonist phenylephrine increased both the amplitude of the aequorin signals and the force of contraction in a concentration-dependent manner. However, the maximum increase in the aequorin signals was less than 10% of that produced by the beta-receptor agonist isoproterenol, while the maximum increase in force of contraction produced by alpha-stimulation was about 50% of that elicited via beta-adrenoceptors. For a given increase in the force of contraction, stimulation of alpha-adrenoceptors produced much less change in the amplitude of the aequorin signals than did elevation of the extracellular Ca2+ concentration; we interpret this to mean that the positive inotropic effect of alpha-adrenoceptor stimulation is in large part the result of an increase in myofibrillar sensitivity to Ca2+. Stimulation of alpha-adrenoceptors produced little change or a slight decrease in the duration of the aequorin signal and an increase in the duration of contraction, while stimulation of beta-adrenoceptors significantly decreased the time to peak and duration of both the aequorin signals and the contractions. For a given level of inotropic effect, high concentrations of isoproterenol often increased the aequorin signals more than did elevations of Ca2+, which is consistent with other evidence that the cyclic AMP-dependent phosphorylation of troponin I leads to a decrease in myofibrillar Ca2+ sensitivity. However, concentrations of isoproterenol that did not produce evidence of this sort of desensitization also abbreviated the contractions much more than they did the aequorin signals. This suggests that the traditionally accepted mechanisms--a decrease in the Ca2+ affinity of troponin C and an acceleration of Ca2+ uptake by the sarcoplasmic reticulum--may not be sufficient to account for the actions of beta-receptor stimulation on the time course of contraction. In the absence of blocking agents, the naturally occurring catecholamines norepinephrine, epinephrine, and dopamine appear to influence the function of the rabbit papillary muscle through both alpha- and beta-adrenoceptors. Dopamine has a relatively greater effect on alpha-adrenoceptors than the other catecholamines.

Modification of myofibrillar responsiveness to Ca++ as an inotropic mechanism.

Intracellular Ca++ transients were monitored in preparations of mammalian heart muscle in which multiple superficial cells had been microinjected with the Ca++-sensitive bioluminescent protein aequorin. The relationship between changes in intracellular [Ca++] and changes in isometric tension development were studied under a variety of conditions in which experiments with "skinned" cardiac muscle preparations have indicated that alterations take place in the sensitivity of the myofilaments to Ca++. In each case (changes in muscle length, changes in intracellular pH, exposure to caffeine or theophylline, exposure to catecholamines, exposure to sulmazole), the relationship between the aequorin signal and the developed tension changed in ways that suggest that alterations in myofibrillar Ca++ sensitivity play an important role in the responses of intact myocardial cells to these inotropic interventions. Studies of the positive inotropic response of the rabbit papillary muscle to alpha-adrenoceptor stimulation indicate that an increase in myofibrillar responsiveness to Ca++ must play an important role in the genesis of this response as well.

Effects of new inotropic agents on cyclic nucleotide metabolism and calcium transients in canine ventricular muscle.

In isolated canine ventricular trabeculae and papillary muscles driven electrically at 0.5 Hz at 37 degrees C, the positive inotropic effects of milrinone, amrinone, MDL 17,043, MDL 19,205, OPC-8212, and forskolin were all associated with simultaneous elevations in tissue cyclic AMP levels. After the administration of sulmazole (AR-L 115 BS), the increase in force preceded that of cyclic AMP levels. Bay k 8644 increased the force of isometric contractions without producing any change in cyclic AMP levels. None of these agents affected the cyclic GMP level. Intracellular calcium transients were determined in similar preparations in which multiple superficial cells had been microinjected with the Ca++-sensitive bioluminescent protein aequorin. The aequorin signals increased in parallel with increases in force in response to cumulative administration of milrinone, amrinone, MDL 17,043, OPC-8212, forskolin, and low concentrations of sulmazole (less than or equal to 3 X 10(-4) M). Forskolin and sulmazole produced larger increases in the amplitude of the calcium transients than the other agents, and their inotropic effects were less variable in magnitude. High concentrations (greater than 3 X 10(-4)M) of sulmazole decreased the amplitude of the calcium transients while increasing further the force. The increases in cyclic AMP levels, calcium transients, and force produced by all of the inotropic agents except Bay k 8644 and high concentrations of sulmazole were effectively antagonized by carbachol. These results indicate that the accumulation of cyclic AMP resulting from the inhibition of cyclic AMP phosphodiesterase plays an important role in the action of new positive inotropic agents, milrinone, amrinone, MDL 17,043, MDL 19,205, OPC-8212, and sulmazole (less than or equal to 3 X 10(-4)M), on dog ventricular muscle.

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.

Active shortening retards the decline of the intracellular calcium transient in mammalian heart muscle.

When active shortening of the cat papillary muscle was allowed at any time during a contraction, the intracellular concentration of free calcium ions, detected with the calcium-sensitive bioluminescent protein aequorin, was higher than at comparable times in isometric twitches. The difference was not attributable to the differences of length involved or to motion artifacts, and must have been related to the act of shortening or the difference in force development in the two types of contractions. This observation and the phenomenon of shortening deactivation are both consistent with the hypothesis that attachment of cross bridges increases the affinity of the myofilaments for calcium ions.

Influence of Ca++-channel blocking agents on calcium transients and tension development in isolated mammalian heart muscle.

The Ca++-sensitive bioluminescent protein aequorin was used to detect cytoplasmic Ca++ transients in dog Purkinje strands and cat papillary muscles. Isometric tension development was recorded in both preparations, and transmembrane potential was monitored in the Purkinje strands. Voltage-clamp studies were carried out on some of the Purkinje strands. The aequorin signal of the Purkinje strand normally consists of two readily discernible components, which we have provisionally attributed to the entry of Ca++ through the surface membrane, and to the release of Ca++ from intracellular stores. D600 and verapamil reduced or eliminated both of these components, as they did the net inward current in fibers depolarized from a holding potential of -45 mV. In cat papillary muscles D600, verapamil, nifedipine, diltiazem, and perhexiline all had qualitatively similar effects on the aequorin signals and contractions. The effects of these agents differed from those of reduced [Ca++] in two important respects: the relation between light intensity and tension development was altered, and the mechanical contraction was abbreviated by the Ca++ channel blockers. These changes persist when the depressant effects of the Ca++ channel blockers are offset by elevations of [Ca++]o; they probably reflect a reduction in the Ca++ sensitivity of the myofilaments.

Contractile basis of ameboid movement. VII. Aequorin luminescence during ameboid movement, endocytosis, and capping.

Aequorin luminescence has been utilized to determine the spatial and temporal fluctuations of the free calcium ion concentration [Ca++] in Chaos carolinensis during ameboid movement, pinocytosis, and capping. The [Ca++] increases above approximately 10(-7) M during normal ameboid movement. Three types of luminescent signals are detected in cells: continuous luminescence, spontaneous pulses, and stimulated pulses. Continuous luminescence is localized in the tails of actively motile cells, and spontaneous pulses occur primarily over the anterior regions of cells. We are sometimes able to correlate the spontaneous pulses with extending pseudopods, whereas stimulated pulses are induced by mechanical damage, electrical stimulation, concanavalin A-induced capping, and pinocytosis. The localization of both distinct actin structures and sites where [Ca++] increases suggests cellular sites of contractile activity. The independent evidence from localizing actin structures and the distribution of [Ca++] can also be viewed in relation to the solation-contraction coupling hypothesis defined in vitro.

Stimulant and depressant effects of beta-adrenoceptor blocking agents on isolated heart muscle. A positive inotropic effect not mediated through andrenoceptors.

The chronotropic and inotropic effects of fourteen beta-adrenoceptor blocking agents were studied in vitro on preparations of isolated heart muscle from kittens and guinea pigs. Most of the agents exert negative inotropic and chronotropic effects that increase rapidly with concentration above 10(-6) M. Exceptions are the closely related compounds practolol and atenolol, which have minimal depressant effects in concentrations as high as 2 X 10(-3) M. Dose-response relations for the depressant effects are similar for pacemaker frequency and for tension development in atrial and papillary muscle. The cardiodepressant effects of beta-blockers are non-stereospecific and are apparently unrelated to the actions of the drugs at beta-adrenoceptors. Many beta-blockers exert positive inotropic and chronotropic effects at concentrations lower than those that depress. The stimulant effects are slow in onset and do not fade. In most instances these effects are blocked by propranolol and may therefore be considered to be mediated throught beta-adrenoceptors; (-)-enantiomers are more potent than (+)-enantiomers as adrenoceptor stimulants. Adrenoceptor-mediated inotropic effects are usually more pronounced in atrial than in ventricular muscle. Certain beta-blockers, notably INPEA and sotalol, exert positive inotropic effects that are not blocked by propranolol or phenyoxybenzamine. The effects are very slow in onset and offset, and are accompanied by the development of contractions with a late tonic component which coincides with a greatly prolonged action potential. Unlike the adrenoceptor-mediated effect, the non-sympathomimetic inotropic effect is more pronounced in ventricular than in atrial muscle, and is not stereospecific. Pindolol also causes the development of a late tonic component, but the accompanying positive inotropic effect is overcome by the simultaneous development of the depressant action of the drug.

Comparative assessment of beta-adrenoceptor blocking agents as simple competitive antagonists in isolated heart muscle: similarity of inotropic and chronotropic blocking potencies against isoproterenol.

The pattern of antagonism between isoproterenol and various beta-adrenoceptor blocking agents was explored in spontaneously beating right atria and in driven left atrial strips from kittens and guinea pigs. The onset of beta-adrenoceptor blockade is usually very slow in such preparations; incubation periods of up to an hour may be required for equilibrium conditions. The speed of onset of the blocking action is directly related to the concentration of the antagonist, and therefore, for a given degree of blockade, inversely related to its potency. beta-Adrenoceptor blocking agents were found to interact with isoproterenol in a manner consistent with a simple competitive antagonism provided that (1) the antagonist had little intrinsic stimulant action on the preparation under study, (2) the concentrations of antagonist used had no direct depressant action on the preparation, (3) precautions were taken to assure that the pattern of antagonism was not distorted by loss of agonist into tissue sinks, and (4) dose-response curves were normalized for changes in the baseline frequency or force in successive curves. Corrections for desensitization were necessary only in inotropic dose-response curves. Estimates of the equilibrium dissociation constants (KB) derived from the antagonism of the chronotropic and inotropic effects of isoproterenol were determined for fifteen beta-adrenoceptor blocking agents of widely differing potency. In no case was there a substantial difference between the inotropic and chronotropic values. Published estimates of binding constants for beta-blockers determined on cardiac membrane particles are more variable than those determined on intact tissues, and tend to be slightly (adenylyl cyclase measurements) or considerably (radioligand binding studies) lower than the values obtained in intact tissues. These differences raise the possibility that the properties of the beta-adrenoceptor may sometimes be altered during the isolation and partial purification of membrane fragments.

beta-Adrenoceptor blocking agents as partial agonists in isolated heart muscle: dissociation of stimulation and blockade.

The cardiac stimulant actions of nine beta-adrenoceptor blocking agents were examined in kitten papillary muscles and in isolated atria of kittens and guinea pigs to determine to what extent these drugs behaved as classical partial agonists. In many ways the agents do appear to comprise a spectrum of partial agonists with widely differing efficacies. However, in one respect the actions of some of the beta-blockers did not fit into the classical mold. Several beta-blockers were found to exert stimulant effects only in concentrations appreciably higher than those required for substantial beta-adrenoceptor blockade. These observations suggest that more than one type of beta-adrenoceptor may be involved in the production of sympathomimetic effects on cardiac muscle.