The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.     

Effect of minor components of virgin olive oil on topical antiinflammatory assays

Interest in the health-promoting effects of virgin olive oil, an important part of the "Mediterranean diet", prompted us to determine the antiinflammatory effects of erythrodiol, beta-sitosterol and squalene, identified as major components of the so-called "unsaponifiable fraction" of virgin olive oil, as well as of the phenolic compounds from the "polar fraction": oleuropein, tyrosol, hydroxytyrosol and caffeic acid. Their activities were compared to those of both, total unsaponifiable and polar fractions. This study was designed to analyse the antiinflammatory effect of these specific compounds from virgin olive oil on edema in mice induced by either arachidonic acid (AA) or 12-O-tetradecanoylphorbol acetate (TPA). The inhibition of the myeloperoxidase (MPO), marker enzyme of the accumulation of neutrophils in the inflamed tissue, was also investigated by the TPA model. The topical application of the olive oil compounds (0.5 mg/ear) produced a variable degree of antiinflammatory effect with both assays. In the auricular edema induced by TPA, beta-sitosterol and erythrodiol from the unsaponifiable fraction of the oil showed a potent antiedematous effect with a 61.4% and 82.1% of inhibition respectively, values not very different to that of the reference indomethacin (85.6%) at 0.5 mg/ear. The four phenolics exerted a similar range of inhibition (33-45%). All compounds strongly inhibited the enzyme myeloperoxidase, indicating a reduction of the neutrophil influx in the inflamed tissues. The strongest inhibitor of AA edema was the total unsaponifiable fraction which inhibition was 34%, similar to that obtained by the reference drug dexamethasone at 0.05 mg/ear. Among the phenolics, oleuropein also produced an inhibition of about 30% with the same dose, but all the other components were found less active in this assay. The anti-inflammatory effects exerted by both unsaponifiable and polar compounds might contribute to the potential biological properties reported for virgin olive oil against different pathological processes.     

Nitric oxide and fusion with prostasomes increase cytosolic calcium in progesterone-stimulated sperm

Spermatozoa must undergo a number of reactions before they are able to fertilize the oocyte. Among these is the acrosome reaction, which is related to an increase in cytosolic Ca2+ concentration ([Ca2+]i). It has been reported in the literature that progesterone may achieve this effect through the intervention of extragenomic receptors. Nitric oxide (NO) has been reported to affect spermatozoa; the nature of the effect depends on the concentration of the radical. In a previous paper, we reported that the fusion of spermatozoa with prostasomes may also produce a transient increase in spermatozoa [Ca2+]i; in addition, this phenomenon causes a long-lasting effect that influences the action of progesterone. In this paper, we test the effects of a NO donor (CysNO) and of fusion of the prostasome to spermatozoa on progesterone-induced [Ca2+]i increase. No effect at all was noticed in the absence of progesterone stimulation. In the presence of the hormone, both CysNO and fusion increased the progesterone effect. This phenomenon was much more evident if the two treatments were used together. We conclude that both NO and fusion with prostasomes act on the progesterone-dependent pathway additively. Probably the effects are independent.     

Activity of olive oil phenols on lymphomonocyte cytosolic calcium

Phenols, present in the Mediterranean diet, have antioxidant properties and are free radical scavengers; however, the molecular mechanisms of their beneficial effects are not yet fully understood. The level of cytosolic calcium ([Ca2+]i) is an important signal also in nonexcitable cells, including immune cells, and regulates fundamental processes. In this paper, we determine [Ca2+]i in human lymphomonocytes incubated with two olive oil phenols: 3,4-(dihydroxyphenyl)ethanol and p-(hydroxyphenyl)ethanol. Both tested phenols increase [Ca2+]i in a dose-dependent way. This effect is antagonized by nifedipine and is noticeable both in the presence and in the absence of calcium in the extracellular medium.     

Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission

The major phenolics from the polar fraction of virgin olive oil (caffeic acid, oleuropein, tyrosol and hydroxytyrosol) have well-established antioxidant activities but their effects on reactive nitrogen species and nitrergic neurotransmission have not been fully investigated. The three catechol compounds were active as scavengers of nitric oxide generated spontaneously from the decomposition of sodium nitroprusside (approximately 50% inhibition achieved at 75 microM), and had similar ability to scavenge chemically generated peroxynitrite, as determined by an alpha1-antiproteinase inactivation assay (67.2%-92.4% reduction when added at 1 mM). Tyrosol was less active in these tests, but does not possess the catechol functionality. Despite their ability to interact with chemically prepared nitric oxide, neither oleuropein nor hydroxytyrosol at 5 microM altered NO*-mediated relaxations of the nerve-stimulated rat anococcygeus preparation, but this may be because the nitrergic transmitter is protected from the effects of externally applied scavengers. In conclusion, the phenolics found in virgin olive oil possess ability to scavenge reactive oxygen and nitrogen species that are implicated in human pathologies, but their impact may be restricted to those species present in the extracellular environment.     

Electrochemical assay of human haemoglobin S-nitrosylation by nitrosocysteine

Summary.  Oxyhaemoglobin (oxyHb) and methaemoglobin (metHb) react with S-nitrosocysteine (CysNO) to form nitroso derivatives. We test this reaction with a new method for evaluating transnitrosation reaction. The assay exploits an amperometric sensor developed in our laboratory. The results we obtain are in good agreement with those reported by others, although at much higher sensitivity, indicating the suitability of the method for examining high-mass nitroso compounds. The S-nitrosylation of oxyHb at a CysNO/haem ratio of 1 : 1 is about 5% in 60 min. In the same experimental conditions, the nitrosylation of met-Hb reaches 25%. OxyHb and metHb derivatize by 50% in 60 min upon using a CysNO/haem ratio of 10 : 1. The oxidation of haem iron occurs at ratios of haem/CysNO of 1 : 5 or higher. We conclude that CysNO transfers NO⁺ both to metHb and oxyHb. We propose that NO transfer in RBC may occur through transnitrosation reactions between high and low-mass nitrosothiols. Received October 31, 2002 Accepted November 21, 2002 Published online March 17, 2003 Authors' address: Prof. Carlo A. Palmerini, Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, University of Perugia, Via del Giochetto, I-06127, Perugia, Italy, Fax: +39.075.585.7414; E-mail:arienti@unipg.it; crlpal@unipg.it Abbreviations: Hb: haemoglobin; SNO-Hb: S-nitrosohaemoglobin; CysNO: S-nitrosocysteine; metHb: methaemoglobin; oxyHb: oxyhaemoglobin; SNO-metHb: S-nitrosomethaemoglobin; SNO-oxyHb: S-nitrosooxyhaemoglobin     

Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression

Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1–10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.     

The Anti-Botulism Triterpenoid Toosendanin Elicits Calcium Increase and Exocytosis in Rat Sensory Neurons

Toosendanin, a triterpenoid from Melia toosendan Sieb et Zucc, has been found before to be an effective anti-botulism agent, with a bi-phasic effect at both motor nerve endings and central synapse: an initial facilitation followed by prolonged depression. Initial facilitation may be due to activation of voltage-dependent calcium channels plus inhibition of potassium channels, but the depression is not fully understood. Toosendanin has no effect on intracellular calcium or secretion in the non-excitable pancreatic acinar cells, ruling out general toosendanin inhibition of exocytosis. In this study, toosendanin effects on sensory neurons isolated from rat nodose ganglia were investigated. It was found that toosendanin stimulated increases in cytosolic calcium and neuronal exocytosis dose dependently. Experiments with membrane potential indicator bis-(1,3-dibutylbarbituric acid)trimethine oxonol found that toosendanin hyperpolarized capsaicin-insensitive but depolarized capsaicin-sensitive neurons; high potassium-induced calcium increase was much smaller in hyperpolarizing neurons than in depolarizing neurons, whereas no difference was found for potassium-induced depolarization in these two types of neurons. In neurons showing spontaneous calcium oscillations, toosendanin increased the oscillatory amplitude but not frequency. Toosendanin-induced calcium increase was decreased in calcium-free buffer, by nifedipine, and by transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. Simultaneous measurements of cytosolic and endoplasmic reticulum (ER) calcium showed an increase in cytosolic but a decrease in ER calcium, indicating that toosendanin triggered ER calcium release. These data together indicate that toosendanin modulates sensory neurons, but had opposite effects on membrane potential depending on the presence or absence of capsaicin receptor/TRPV 1 channel.     

Role of S-nitrosothiol transport in the cardioprotective effects of S-nitrosocysteine in rat hearts

The objective of this study was to determine if prior exposure of rat hearts to S-nitrosocysteine (CysNO) was able to provide protection against reperfusion injury. We probed NO release using the extracellular NO scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid transport system L (L-AT), a known transporter of CysNO, using the L-AT competitor, L-leucine (L-Leu). Isolated (9- to 12-week-old Wistar male) rat hearts (six to eight per group) were perfused with CysNO (10 microM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of ischemia. Cardiac function was assessed before, during, and after treatment and during 120 min of reperfusion after ischemia. Functional recovery (rate-pressure product) was significantly improved in the CysNO group compared to hearts in the CysNO+L-Leu group and the control group (p<0.05). Necrosis, measured by triphenyltetrazolium chloride staining, was significantly reduced in CysNO hearts (p<0.05) and this improvement was reversed by L-Leu. The NO scavenger oxyHb (20 microM) was perfused either concomitant with CysNO or just before ischemia. In neither case did oxyHb affect the cardioprotection afforded by CysNO. OxyHb alone, given in either time window, did not alter the course of ischemia-reperfusion injury. When nitrite was used in place of CysNO, no protective effects were observed. Perfusion with CysNO increased tissue S-nitrosothiol (RSNO) levels from an unmeasurable background to a value of about 15.7+/-4.1 pmol RSNO/mg protein, as measured by triiodide-based chemiluminescence in the presence and absence of mercury(II) chloride. In the presence of L-Leu, this value dropped to 0.4+/-0.3 pmol RSNO/mg protein. This study demonstrates that exposure to CysNO before ischemia increases tissue S-nitrosothiol levels, improves postischemic contractile dysfunction, and attenuates necrosis. The mechanism of cardioprotection requires the uptake of CysNO via the L-AT and does not seem to involve NO release either during CysNO exposure or during ischemia. This suggests that the protective effects of CysNO are mediated through the posttranslational modification of cellular proteins through an NO-independent transnitrosation mechanism.     

Effect of extracts obtained from olive oil mill waste waters on Bacillus megaterium ATCC 33085

n -Propanol was the most effective solvent for extracting antibacterial substances from olive oil mill waste water ('alpechines'). Several phenolics were detected in propanol extracts that had bactericidal effects on Bacillus megaterium ATCC 33085, inhibiting sporulation and germination at 5.6 mmol/l total phenolics (expressed as syringic acid). The biological effect was increased in the presence of high glucose and NaCl concentrations and after β-glucosidase hydrolysis.     

Olive oil phenolics are dose-dependently absorbed in humans

Olive oil phenolic constituents have been shown, in vitro, to be endowed with potent biological activities including, but not limited to, an antioxidant action. To date, there is no information on the absorption and disposition of such compounds in humans. We report that olive oil phenolics, namely tyrosol and hydroxytyrosol, are dose-dependently absorbed in humans after ingestion and that they are excreted in the urine as glucuronide conjugates. Furthermore, an increase in the dose of phenolics administered increased the proportion of conjugation with glucuronide.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Pathways for plasmalemmal repair mediated by PKA, Epac, and cytosolic oxidation in rat B104 cells in vitro and rat sciatic axons ex vivo

Plasmalemmal repair (sealing) is necessary for survival of damaged eukaryotic cells. Ca²⁺ influx through plasmalemmal disruptions activates pathways that initiate sealing, which is commonly assessed by exclusion of extracellular dye. These sealing pathways include PKA, Epac, and cytosolic oxidation. In this article, we investigate whether PKA, Epac, and/or cytosolic oxidation, activate specific proteins required to produce a plasmalemmal seal. We report that toxin cleavage of proteins required for neurotransmitter release (SNAP‐25), inhibition of Golgi trafficking (with Brefeldin A: Bref A) or inhibition of N‐ethylmaleimide sensitive factor (NSF) all decrease sealing of rat B104 hippocampal cells with transected neuritis in vitro. Epac, but not PKA or cytosolic oxidation, partly overcomes the decrease in sealing produced by cleavage of SNAP‐25. PKA and increased cytosolic oxidation, but not Epac, can partly overcome the decrease in sealing due to Bref A. PKA, Epac, and/or cytosolic oxidation cannot overcome NSF inhibition. Substances that affect plasmalemmal sealing of B104 neurites in vitro have similar effects on plasmalemmal sealing in rat sciatic axons ex vivo. From these and other data, we propose a model of plasmalemmal sealing having three redundant, evolutionarily conserved, parallel pathways that all converge on NSF. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Increase of cytosolic calcium induced by trichosanthin suppresses cAMP/PKC levels through the inhibition of adenylyl cyclase activity in HeLa cells

Increase of cytosolic free calcium played a pivotal role in apoptotic cells induced by trichosanthin. However, little is known about the influence of cytosolic calcium increase on adenylyl cyclase activity and intracellular cAMP signaling pathway in HeLa cells. The present study showed that an influx of extracellular Ca²⁺ initiated by trichosanthin was required for the suppression of adenylyl cyclase activity and decrease of intracellular cAMP level. Furthermore, this inhibition was abolished by activation of PKC rather than PKA. Therefore, our results suggested that increase of cytosolic calcium induced by trichosanthin inhibits cAMP levels via suppression of adenylyl cyclase activity.     

Dietary oils high in oleic acid, but with different non-glyceride contents, have different effects on lipid profiles and peroxidation in rabbit hepatic mitochondria

The influence on the lipid profile and lipid peroxidation in rabbit-liver mitochondria exerted by different edible oils high in oleic acid but different non-glyceride phenolic fractions was studied. High-phenolic virgin olive oil from the variety "Picual", the same oil submitted to an exhaustive process of washing to eliminate the phenolic fraction without altering the lipid profile and high-oleic sunflower oil (poor in phenolic compounds) were added to rabbit diets. The results reveal the importance of the different oleic: linoleic ratio of the lipid sources on the lipid profile of mitochondrial membranes. This is highlighted by the greater proportion of saturated fatty acids and the lower content in oleic acid (p < 0.05) shown by the rabbits fed on high-oleic sunflower oil. The group fed on the fat rich in phenolics exhibited the highest level of antioxidants (alpha-tocopherol, ubiquinone 10) and the highest activity of glutathione peroxidase as well as the lowest content in hydroperoxides and TBARS. The study provides evidences in vivo about the considerable antioxidant capacity of the phenolic fraction of virgin olive oil in rabbit-liver mitochondria and the important role that this non-glyceride fraction can play in the overall antioxidant benefits attributed to this oil.     

Long-chain fatty alcohols from pomace olive oil modulate the release of proinflammatory mediators

Pomace olive oil is a by-product of olive oil extraction that is traditionally produced and consumed in Spain. The nonglyceride matter of this oil is a good source of interesting minor compounds, like long-chain fatty alcohols, which are present free or as part of waxes. In the present study, long-chain fatty alcohols were isolated from the nonglyceride fraction of pomace olive oil, and the composition was identified and quantified. The major components of long-chain fatty alcohols were tetracosanol, hexacosanol and octacosanol. We investigated the ability of long-chain fatty alcohols from pomace olive oil to inhibit the release of different proinflammatory mediators in vitro by cells involved in inflammatory processes. Long-chain fatty alcohols significantly and dose-dependently decreased nitric oxide production by RAW 264.7 murine macrophages stimulated with lipopolysaccharide. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression. Long-chain fatty alcohols also reduced tumor necrosis factor-alpha and prostaglandin E(2) production, although the potency of inhibition for the latter was lower. On the other hand, long-chain fatty alcohols significantly reduced thromboxane A(2) production in rat peritoneal neutrophils stimulated with the calcium ionophore A-23187. The reduction of eicosanoid release was related to the inhibition of phospholipase A(2) enzyme activity by long-chain fatty alcohols, reaching an inhibitory concentration 50% value of 6.2 microg/ml. These results showed that long-chain fatty alcohols may have a protective effect on some mediators involved in the inflammatory damage development, suggesting its potential value as a putative functional component of pomace olive oil.     

Olive oil increases the hepatic triacylglycerol content in mice by a distinct influence on the synthesis and oxidation of fatty acids

Diet supplementation with olive oil exerts beneficial effects on an organism, even if an increase in the level of hepatic lipids has been concomitantly observed. This study was therefore designed to investigate whether the stimulation of lipogenesis was responsible for the olive oil-induced hepatic fat accumulation. In mice fed for 8 weeks with an olive oil-enriched diet, an increase of about 2.6 fold in the level of liver triglycerides was found in comparison to animals fed with a corn oil-containing diet. Despite that, no increase in the activities of cytosolic lipogenic enzymes or of the mitochondrial tricarboxylate carrier was found; on the contrary, a decrease in the activity of carnitine palmitoyltransferase I was observed. This impairment of fatty acid oxidation, which was not apparent in corn oil-fed animals, may have had a role in the increase of hepatic lipid content found in the olive oil-fed mice.     

Regulation of the ATP dependent calcium uptake activity of heart sarcolemmal vesicles by endogenous cytosolic proteins

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by sarcoplasmic reticulum; further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53-66, 1983). The present study examined the effects of the endogenous cytosolic Ca2+ transport inhibitor and its antagonist on ATP-dependent Ca2+ uptake by sarcolemmal vesicles isolated from rat and canine heart. The cytosolic inhibitor caused strong inhibition (up to 97%) of Ca2+ uptake by sarcolemma (SL); this inhibition could be reversed by the cytosolic inhibitor antagonist. Studies on the characteristics of inhibition revealed the following: a) Inhibition was dependent on the concentration of the inhibitor (50% inhibition with approximately 80 micrograms inhibitor protein). b) The inhibitor reduced the velocity of Ca2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca2+ but caused greater than 2-fold decrease in its apparent affinity for ATP. c) The rates of unidirectional passive Ca2+ release from actively Ca2+ loaded SL vesicles were not altered by low concentrations of the inhibitor (less than 100 micrograms/ml) which were effective in producing marked inhibition of Ca2+ uptake; at higher concentrations (greater than 100 micrograms/ml), the inhibitor caused increase in the rates of passive Ca2+ release. These findings demonstrate that the activity of the ATP-driven Ca2+ pump of cardiac SL can be regulated in vitro by endogenous cytosolic proteins.