Taurine's possible protective role in age-dependent response to calcium paradox

When hearts were reperfused with Ca++ after a short period of Ca++-free perfusion, irreversible loss of electrical and mechanical activity was observed. This phenomenon, first described by Zimmerman and Hulsmann, was termed the "calcium paradox". Chizzonite and Zak recently reported that rat hearts exhibited an age-dependent response in a calcium paradox model. The taurine (2-aminoethanesulfonic acid) content of hearts in the newborn animal is high, and decreases rapidly during the first few days of life. The present experiments were performed to test whether the myocardial taurine content was closely linked to an age-dependent response in the calcium paradox model, using post-hatched chicks. The mechanical dysfunction of the heart was much more severe in 9-day-old post-hatched chicks than in 2-day-old chicks when the hearts were subjected to the calcium paradox. Myocardial taurine content was lower in the 9-day-old chicks than in the 2-day-old chicks. The age-related response to the calcium paradox was partially protected by oral pretreatment with taurine, and there was a small increase in myocardial taurine level. It is proposed that myocardial taurine is one factor in the protection against the calcium paradox phenomenon.     

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.     

Effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa in vitro

The effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa were investigated in vitro. The addition of taurine, within the range of 0-5 mmol l(-1), did not appreciably affect the motility of intact fowl spermatozoa. Motility remained almost negligible at 40 degrees C, while vigorous movement was observed at 25 degrees C. Even with the addition of Ca2+ before the addition of taurine, neither stimulation nor inhibition of motility was observed compared with the control (no addition of taurine). Similar results were obtained by the addition of taurine and calyculin A, a specific inhibitor of protein phosphatases. There were no changes in intracellular free Ca2+ concentrations, measured by a fluorescent Ca2+ indicator, fura-2, in taurine-treated spermatozoa. These results suggest that taurine is not involved in the regulation of fowl sperm motility and metabolism by intracellular Ca2+ mobilization in vitro.     

Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat

The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

The histo- and cytochemical localization of Ca++-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca++-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca++ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg++ in different concentrations were substituted for Ca++ in the incubation medium the reaction was always reduced. Both Ca++ and Mg++ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca++-ATPase activity. The function of the Ca++-ATPase activity is discussed in relation to the regulation of the extracellular Ca++ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Mechanisms of enhanced taurine release under Ca2+ depletion

The sulfur-containing amino acid taurine is an inhibitory neuromodulator in the brain of mammals, as well as a key substance in the regulation of cell volumes. The effect of Ca(2+) on extracellular taurine concentrations is of special interest in the context of the regulatory mechanisms of taurine release. The aim of this study was to characterize the basal release of taurine in Ca(2+)-free medium using in vivo microdialysis of the striatum of anesthetized rats. Perfusion of Ca(2+)-free medium via a microdialysis probe evoked a sustained release of taurine (up to 180 % compared to the basal levels). The Ca(2+) chelator EGTA (1mM) potentiated Ca(2+) depletion-evoked taurine release. The substitution of CaCl(2) by choline chloride did not alter the observed effect. Ca(2+)-free solution did not significantly evoke release of taurine from tissue loaded with the competitive inhibitor of taurine transporter guanidinoethanesulfonate (1mM), suggesting that in Ca(2+) depletion taurine is released by the transporter operating in the outward direction. The volume-sensitive chloride channel blocker diisothiocyanostilbene-2,2'-disulfonate (1mM) did not attenuate the taurine release evoked by Ca(2+) depletion. The non-specific blocker of voltage-sensitive Ca(2+) channels NiCl(2) (0.65 mM) enhanced taurine release in the presence of Ca(2+). CdCl(2) (0.25 mM) had no effect under these conditions. However, both CdCl(2) and NiCl(2) attenuated the effect of Ca(2+)-free medium on the release of taurine. The data obtained imply the involvement of both decreased influx of Ca(2+) and increased non-specific influx of Na(+) through voltage-sensitive calcium channels in the regulation of transporter-mediated taurine release in Ca(2+) depletion.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca++ +Mg++)-ATPase of sarcoplasmic reticulum.

We have shown that a Ca++-ionophore activity is present in the (Ca++ +Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A. E. Shamoo & D. H. MacLennan, 1974. Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca++ +Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential -SH groups. However, it appears that there are no essential -SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential -SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity.     

Calcium dependence of the stimulating action of cadmium on organic acid transport in the frog kidney

The stimulatory effect of cadmium ions on the Na-dependent fluorescein transport into the frog renal proximal tubules ceased with decreasing Ca++ concentration in solution on both the sides of the cell layer down to micromolar level. The decrease in Ca++ concentration per se stimulated fluorescein uptake during short-term incubations. A further diminution of Ca++ concentration in the tubular lumen with the aid of EGTA resulted in a sharp inhibition of the organic acid transport. Amiloride, which prevented the stimulatory effect of cadmium, inhibited the fluorescein transport at both millimolar and micromolar levels of Ca++ concentration, but it failed to affect the transport process after introducing EGTA into the tubular lumen. The results are discussed within the frames of a model regarding extracellular Ca++ as an allosteric inhibitor, and intracellular Ca++ as an allosteric activator of sodium channels in the apical membrane. Cd++ is assumed to compete with Ca++ for binding to centers of the allosteric inhibition, thereby accelerating the sodium ion flux across the cells of the proximal tubules.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.     

Ca++-ATPase in the central nervous system: an EM cytochemical study

Ca++-ATPase plays an important role in regulation of the intracellular Ca++ concentration. Biochemical studies of brain have demonstrated that Ca++-ATPase co-purifies with synaptosomes, with synaptic plasma membrane and synaptic vesicle fractions. To better understand the role of this enzyme in normal brain function, we used an electron microscopic (EM) cytochemical method to determine the localization of Ca++-ATPase in rat brain. Reaction product occurred along cytoplasmic membranes. Specific areas of increased reaction product were seen at many but not all post-synaptic densities. Intracellular Ca++-ATPase reaction product was associated with all synaptic vesicles examined and with the Golgi and smooth endoplasmic reticulum (SER). Unlike the situation in peripheral nerve, Ca++-ATPase at the node of Ranvier in the CNS localized preferentially to the nodal axolemma. The localization of Ca++-ATPase at synaptic vesicles agrees with the biochemical evidence for its localization and with the cytochemical evidence for Ca++-ATPase sequestration in those vesicles. The restricted localization at postsynaptic densities suggests that it may be involved in extrusion of Ca++ at synapses where neurotransmitter release causes Ca++ influx.     

Role of taurine in osmoregulation in brain cells: Mechanisms and functional implications

Summary All cells including neurons and glial cells are able to keep their volume within a very limited range. The volume regulatory mechanism involves changes in the concentration of osmolytes of which taurine appears to be of particular importance in brain cells. Swelling in brain cells may occur as a result of depolarization or small fluctuations in osmolarity. In isolated brain cells these conditions will always lead to a release of taurine, the time course of which is superimposable on that of the volume regulatory decrease which follows the initial cell swelling. The mechanism responsible for taurine release associated with swelling has not been fully elucidated but a large body of evidence seems to exclude participation of the taurme high affinity carrier. Using a number of inhibitors of anion exchangers it has been demonstrated that both volume regulation and taurine release in brain cells are inhibited by these drugs, implicating an anion channel in the process. It has be controversial issue as to whether or not taurine release is Ca++ dependent. Recent evidence appears to suggest that the release process is not associated with Ca⁺⁺ or Ca⁺⁺ channels. It is, however, quite possible that the swelling process may involve the Ca⁺⁺ calmodulin system or other second messengers. Taurine also contributes to volume regulation after shrinkage of brain cells, in this case by increasing its intracellular concentration. This change is accomplished byan upregulation of the Na+/taurine cotransporter, together with reduced passive fluxes and increased endogenous synthesis.     

A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors

Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection.     

Oxalate transfer across the membranes of sarcoplasmic reticulum during the uptake of Ca++

The kinetic profile of Ca++ uptake in the presence of oxalate is biphasic. An initial phase independent on oxalate is followed by an oxalate-dependent phase delayed in time. The ionophore X-537A only abolishes the net Ca++ uptake if added before the onset of the oxalate phase. However, during this phase, X-537A suddenly releases an amount of Ca++ similar in quantity to that released in the initial phase. The delay of the oxalate-dependent phase is a function of pH. At pH of about 5.5, the oxalate phase and simultaneous calcium oxalate precipitation would theoretically start at the beginning, with no delay. Ejection of protons during Ca++ uptake is strongly depressed by oxalate, but not by other organic anions which do not trap Ca++. It is suggested that oxalate is transferred to the inside of the vesicles as a monoprotonated species at expense of protons ejected by the Ca++-pump during the uptake of Ca++.     

Anticonvulsant activity of 2-phthalimidoethanesulphonamides: New derivatives of taurine

A number of 2-phthalimidoethanesulphonamides, new derivatives of the inhibitory neuromodulator taurine, were tested for their anticonvulsant activity in maximal electroshock seizure and pentetrazole seizure threshold tests in mice. Certain lower N-alkylamides showed activity, methylamide, dimethylamide and isopropylamide derivatives and the unsubstituted amide being pharmacologically most promising. Possible interferences with the intracellular uptake, release and membrane binding of taurine and GABA were assessed in an attempt to elucidate their mode of action. Since the uptake and release processes were only minimally affected, but the sodium-independent binding of GABA and/or taurine to synaptic membranes strongly reduced, the compounds studied may preferentially act as taurine or GABA receptor agonists.     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.     

Phosphorylation of the red blood cell membrane during the active transport of C++

The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca++ transport was investigated. When red cell membrane fragments are incubated with [gamma-32P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg++ without Ca++. When Ca++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca++. These data suggest that the enzymatic sequence of the Ca++ transport mechanism may be very similar to that of the Na+ transport mechanism.     

Influence of calcium and magnesium deprivation on ovulation and ovum maturation in the perfused rabbit ovary

The role of calcium (Ca++) and magnesium (Mg++) in the ovulation process was studied using in vitro perfused rabbit ovaries. Ovaries were perfused with or without human chorionic gonadotropin (hCG) in Ca++/Mg++-free medium (M199) alone or combined with standard M199 to yield varying concentrations of Ca++ and/or Mg++. In all ovaries perfused with hCG, ovulatory efficiency was similar regardless of the concentration of Ca++ and/or Mg++. In ovaries perfused in Ca++/Mg++-free medium without hCG, ovulatory efficiency was similar to that in ovaries perfused with hCG. As Ca++/Mg++ levels were increased without hCG, ovulatory efficiency declined. Ovulation time was significantly accelerated in ovaries perfused in Ca++/Mg++-free medium with or without hCG. Most ovulated ova from ovaries perfused without hCG were immature. With hCG, degree of ovum maturity was directly related to ovulation time. Ovarian smooth muscle contractions were undetectable in 3 ovaries perfused in Ca++/Mg++-free M199 despite occurrence of ovulation. Smooth muscle contractions were recorded in 2 of 3 ovaries perfused in standard M199 with hCG. These results indicate: 1) Ca++/Mg++ exclusion results in rapid follicle rupture and immature ova; 2) oocyte maturation appears to be gonadotropin-dependent; 3) ovulation occurs in the absence of ovarian smooth muscle contractions during perfusion with Ca++/Mg++-free medium.     

cAMP-dependent protein kinase substrates in platelets. Evidence that thrombolamban, a 22,000 dalton substrate, and the Ca++-ATPase are not associated proteins

Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.