Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes.

Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

Apoptosis suppression by bcl-2 is correlated with the regulation of nuclear and cytosolic Ca2+.

Bcl-2 expression is associated with the progression of prostate cancer from androgen-dependence to androgen-independence. Bcl-2 is an integral membrane protein which localizes to mitochondria, endoplasmic reticulum, and the nuclear envelope. Using spectrofluorometry and laser confocal microscopy, the ability of bcl-2 to modulate intracellular Ca2+ was examined in the Dunning G prostate carcinoma cell line following apoptosis induction by adriamycin. Adriamycin and thapsigargin, an endoplasmic reticulum Ca2+-pump inhibitor, were effective inducers of apoptosis in control, but not bcl-2 transfected, cells. Treatment with adriamycin was accompanied by a sustained rise in cytoplasmic Ca2+ in control and bcl-2 transfected cells. An increase in intranuclear Ca2+ was observed in control cells only. Apoptosis induction by thapsigargin was associated with an increase in cytoplasmic Ca2+ in control cells that was not detected in the resistant bcl-2 transfectants. Ca2+ was excluded from nuclei isolated from bcl-2 expressing cells, but was sequestered in control nuclei, following the addition of ATP. These findings suggest that bcl-2 may regulate levels of intranuclear Ca2+ independently of cytosolic Ca2+ levels. The ability of bcl-2 to modulate, directly or indirectly, sustained increases in both cytosolic and intranuclear Ca2+ may provide a common basis for bcl-2 function in different subcellular compartments.

Altered adrenergic receptor density in myocardial hibernation in humans: A possible mechanism of depressed myocardial function.

BACKGROUND: Alterations in adrenergic receptor densities can potentially contribute to myocardial dysfunction. Their relevance to myocardial hibernation in humans is unknown. METHODS AND RESULTS: Accordingly, 22 transmural myocardial biopsies were obtained in 11 patients with ischemic ventricular dysfunction during bypass surgery, guided by transesophageal echocardiography. Patients underwent dobutamine echocardiography (DE) and rest scintigraphic studies before revascularization and DE at 3 to 4 months. alpha- and ss-receptor density (ARD and BRD) and extent of fibrosis were quantified from the myocardial biopsies. Of the 22 segments, 16 had abnormal rest function and 6 were normal. Severely hypokinetic or akinetic segments showed a 2.4-fold increase in ARD with a concomitant 50% decrease in BRD compared with normal segments. An increase in ARD, a decrease in BRD to a lesser extent, and thus an increase in ARD/BRD ratio were seen in dysfunctional segments with contractile reserve compared with normal segments and were most pronounced in those without contractile reserve (P:<0.001). Similar findings were observed if recovery of function or scintigraphic uptake was analyzed as a marker for viability. No significant relation between either ARD or BRD and percent myocardial fibrosis was noted (r=0.37 and -0.39, respectively). CONCLUSIONS: Thus, graded and reciprocal changes in alpha- and ss-adrenergic receptor densities occur in viable, hibernating myocardium and may account in part for the observed depression in resting myocardial function and preserved contractile reserve in this entity.

Relation of tissue Doppler derived myocardial velocities to myocardial structure and beta-adrenergic receptor density in humans.

OBJECTIVES: We sought to evaluate the relation of segmental tissue Doppler (TD) velocities to both the regional amount of interstitial fibrosis and the myocyte beta-adrenergic receptor density in humans. BACKGROUND: The systolic myocardial velocity (Sm) and early diastolic myocardial velocity (Em) acquired by TD are promising new indexes of left ventricular function. However, their structural and functional correlates in humans are still unknown. METHODS: Ten patients with coronary artery disease underwent echocardiographic examination including TD imaging, along with transmural endomyocardial biopsy at the time of coronary bypass surgery (two biopsies per patient for a total of 20 specimens). The specimens were analyzed for percent interstitial fibrosis and beta-adrenergic receptor density. RESULTS: Normal segments (n = 8) had a higher beta-adrenoceptor density (2,280 +/- 738 vs. 1,373 +/- 460, p = 0.03) and a lower amount of interstitial fibrosis (13 +/- 3.3% vs. 28 +/- 11.5%, p = 0.002) than dysfunctional segments (n = 12). Myocardial systolic velocity and Em were also significantly higher (9.5 +/- 2.7 vs. 5.9 +/- 1.8 cm/s, p = 0.025 and 11.3 +/- 2.8 vs. 6.4 +/- 2.1 cm/s, p = 0.002, respectively) in normal segments. A significant relationship was present between Em and the beta-adrenergic receptor density (r = 0.78, p < 0.001) and percent interstitial fibrosis (r = -0.7, p = 0.0026), which together accounted for 81% of the variance observed in Em. Likewise, a significant relationship was present between Sm and the beta-adrenergic receptor density (r = 0.68, p < 0.001) and the percent interstitial fibrosis (r = -0.66, p = 0.004) and together accounted for 62% of the variance observed in Sm. CONCLUSIONS: Systolic myocardial velocity and Em are strongly dependent on both the number of myocytes and the myocardial beta-adrenergic receptor density.

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal and adult cardiomyocytes.

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.

Cytokines increase neonatal cardiac myocyte calcium concentrations: the involvement of nitric oxide and cyclic nucleotides.

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

Improved left ventricular function after chronic left ventricular unloading.

BACKGROUND: This study assessed the effect of prolonged left ventricular unloading on native ventricular function. METHODS: We reviewed data from 31 patients (30 men, 1 woman) supported more than 30 days (mean, 137 days; range, 31 to 505 days) with the HeartMate left ventricular assist system. The patients' mean age was 46 years (range, 22 to 64 years); 17 had idiopathic and 14 had ischemic cardiomyopathy. Data (anatomic, physiologic, hemodynamic, histologic, and biochemical) were collected at the time of HeartMate implantation, during support with the device temporarily off, and at the time of device explantation. RESULTS: Routine chest roentgenogram showed improvement in cardiothoracic ratio (0.62 +/- 0.04 to 0.55 +/- 0.03; p < 0.0001). Echocardiography performed with the pump off showed a significant decrease in left ventricular end-diastolic dimension (6.81 +/- 0.87 cm to 5.39 +/- 1.08 cm; p < 0.0005) and a significant improvement in ejection fraction (0.11 +/- 0.05 to 0.22 +/- 0.17; p < 0.02). Cardiac index increased (1.96 +/- 0.52 L.min-1.m-2 to 2.93 +/- 0.73 L.min-1.m-2; p < 0.0001), mean aortic pressure increased (71.40 +/- 10.63 mm Hg to 76.33 +/- 16.84 mm Hg; p = 0.48), pulmonary capillary wedge pressure decreased (24.18 +/- 6.27 mm Hg to 14.48 +/- 3.01 mm Hg; p < 0.0001), and pulmonary vascular resistance decreased (3.34 +/- 2.00 Wood units to 2.51 +/- 0.88 Wood units; p < 0.05). Comparisons of tissue samples taken at the time of implantation and at the time of transplantation showed a marked reduction in myocytolysis. Calcium uptake, calcium-binding rates, and lipid levels normalized in patients studied. Plasma norepinephrine levels decreased to near normal levels. CONCLUSION: Prospective studies are now indicated to determine whether device removal without transplantation may be beneficial in selected patients.

CT-guided percutaneous catheter cecostomy.

A patient with pseudomembranous colitis is described in whom a percutaneous cecostomy was performed using computed tomographic guidance. Several lines of evidence indicate the safety of this approach, and clinical circumstances are suggested in which the procedure may have potential therapeutic benefit.

Influence of dietary fish oil on mitochondrial function and response to ischemia.

Dietary supplementation with marine oils may reduce the incidence of thromboembolism and decrease cardiac arrhythmias during myocardial ischemia. However, function of subcellular organelles isolated from hearts of these animals is impaired. In contrast to studies where marine oil was the sole source of dietary lipid in rats, menhaden oil was used to supplement standard canine laboratory chow. In mitochondria isolated from hearts of dogs fed this diet for 60 wk, the phospholipid content of arachidonic acid was replaced by the n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Mitochondrial levels of linoleic and linolenic acids were not altered. The mitochondrial membrane phospholipid from the menhaden oil-fed dogs demonstrated increased cardiolipin. The respiratory function of heart mitochondria from the menhaden oil-supplemented dogs was not decreased from that of dogs on standard chow only. Increased succinate-supported respiration paralleled increased cytochrome oxidase in mitochondria from menhaden oil-fed dogs. The activity of the cardiolipin-dependent carnitine acylcarnitine translocase was unaffected. Myocardial ischemia decreased mitochondrial respiration in menhaden-fed dogs. Decreased palmitoylcarnitine-carnitine exchange following ischemia resulted from decreased matrix carnitine rather than decreased translocase activity. Normal levels of the essential fatty acids in the n-3-enriched mitochondrial membrane phospholipids appear to eliminate the mitochondrial dysfunction observed in essential fatty acid-deficient membranes.

Intracellular association between UDP-glucose:glycoprotein glucosyltransferase and an incompletely folded variant of alpha1-antitrypsin.

Genetic variants of human alpha1-antitrypsin unable to fold into the native structural conformation are poorly secreted from hepatocytes. The molecular chaperone calnexin coimmunoprecipitates with secretion-incompetent variant null(Hong Kong) retained in stably transfected mouse hepatoma cells (Le, A., Steiner, J. L., Ferrell, G. A., Shaker, J. F., and Sifers, R. N. (1994) J. Biol. Chem. 269, 7514-7519). Mobilization of intracellular Ca2+ stores with metabolic poisons diminished interaction with calnexin and coincided with coimmuoprecipitation of a 150-kDa protein (p150). Mobilization of endoplasmic reticulum lumenal Ca2+ with thapsigargin, an inhibitor of the microsomal Ca2+ATPase, gave a similar result. Coimmunoprecipitation of p150 was specifically disrupted in response to incubation of the cell lysate with exogenous CaCl2. Finally, in ECL Western blotting, p150 was recognized by polyclonal antiserum against UDP-glucose:glycoprotein glucosyltransferase that likely functions in glycoprotein folding and quality control (Sousa, M. C., Ferrero-Garcia, M. A., and Parodi, A. J. (1992) Biochemistry 31, 97-105). The data are consistent with a model in which perturbation of endoplasmic reticulum Ca2+ results in a stable physical association between unfolded human alpha1-antitrypsin and UDP-glucose:glycoprotein glucosyltransferase.

Membrane asymmetry in isolated canine cardiac sarcoplasmic reticulum: comparison with skeletal muscle sarcoplasmic reticulum.

Cardiac sarcoplasmic reticulum (CSR), isolated from dog hearts, was shown to be asymmetric in the distribution of phospholipids across the CSR bilayer. Phosphatidylethanolamine was mostly resident in the outer leaflet, phosphatidylcholine was equally distributed across both monolayers and phosphatidylserine was found primarily in the inner monolayer. This distribution of headgroups is similar to that found in fast skeletal muscle sarcoplasmic reticulum (SSR); however, the asymmetry in CSR is not as striking as that in SSR. Phospholipids retained by the CSR calcium pump protein (CaATPase) after detergent "stripping" were similar to those intimate to the SSR CaATPase, although the percentages of unsaturated phospholipids and plasmalogenic phospholipids are not as great as in the skeletal system. Lipids associated with the CSR CaATPase following DFDNB cross-linking showed a preference for retention of the aminophospholipids, again similar to the SSR CaATPase. Because the nonrandom distribution of membrane lipids modifies SSR function, it is likely these membrane lipids impact in situ the function of the CSR.

Nucleotide triphosphate utilization by cardiac and skeletal muscle sarcoplasmic reticulum. Further evidence for an alternative substrate hydrolysis cycle and the effect of calcium NTPase purification.

It was previously observed that the hydrolysis of GTP by cardiac sarcoplasmic reticulum (SR) (in contrast to skeletal muscle SR: (a) was identical in rate with total ATP hydrolysis; (b) gave a similar nonlinear substrate response; (c) was not Ca2+ dependent; and (d) stimulated Ca2+ accumulation but not Ca2+ translocation. Evidence was presented that both the ATPase and GTPase are effected by the same enzyme and represent different hydrolysis cycles (Van Winkle, W. B., Tate, C. A., Bick, R. J., and Entman, M. L. (1981) J. Biol. Chem. 256, 2268-2274). In the present paper, we report that purification of the NTPase from both muscle sources resulted in an alteration in the NTP concentration response compatible with a single high affinity binding site for ATP only in cardiac SR and for both substrates in skeletal muscle SR. As is the case with native skeletal muscle SR, purified skeletal muscle NTPase hydrolyzed GTP in a manner qualitatively similar to ATP (but with no Ca2+-independent NTPase) but with reduced velocity. In contrast, there was no GTPase activity or Ca2+-independent "basic" ATPase activity in the purified cardiac NTPase. Inclusion of oxalate or the ionophore, A23187, in assays with cardiac SR and ATP as the substrate increased the total ATPase activity but had no effect on GTPase activity. Furthermore, the nucleotide-dependent uptake of oxalate by cardiac SR was only apparent with ATP and not with GTP. In the presence of Ca2+, ATP was a potent inhibitor (noncompetitive, Ki of 2-5 microM) of GTPase activity, whereas it was a weaker competitive inhibitor in the absence of Ca2+. We suggest that GTPase and basic ATPase represent similar alternative enzyme cycles for the CaATPase enzyme that are inhibited by the presence of ATP plus Ca2+ but are rendered inactive during the purification of cardiac NTPase.

Nucleotide specificity of canine cardiac sarcoplasmic reticulum. Differential alteration of enzyme properties by detergent treatment.

We previously demonstrated that, in contrast to the hydrolysis of ATP, the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium. Based on a variety of qualitative and quantitative considerations (cf. Tate, C. A., Bick, R. J., Chu, A., Van Winkle, W. B., and Entman, M. L. (1985) J. Biol. Chem. 260, 9618-9623), we suggested that the hydrolysis of ATP and GTP appears to be effected by the same enzyme. In the present paper, we examined the sensitivity of both enzymatic activities to low concentrations of detergent. With nonsolubilizing concentrations of the nonionic detergent, octaethylene glycol monododecyl ether, the hydrolysis of GTP was rendered partially calcium-sensitive resulting from a slightly increased total (Ca2+ + Mg2+)-GTPase activity and a markedly inhibited calcium-independent (Mg2+-dependent) GTPase activity. Calcium-dependent ATPase activity was increased with octaethylene glycol monododecyl ether, mimicking the effect of the ionophore, A23187. Calcium-dependent ATPase activity and detergent-induced calcium-dependent GTPase activity were similar in (a) calcium sensitivity, (b) sensitivity to mersalyl, and (c) pressure inactivation through dilution and centrifugation, all of which differed from the untreated calcium-independent GTPase activity. Calcium-dependent ATPase activity differed from calcium-dependent GTPase activity with (a) a higher nucleotide affinity, (b) a lower vanadate sensitivity, and (c) a calcium sensitivity for phosphoenzyme formation. Thus, the detergent-induced perturbation of the GTPase resulted in an enzyme with many characteristics qualitatively and quantitatively similar to the calcium ATPase.

Temporal effects of cytokines on neonatal cardiac myocyte Ca2+ transients and adenylate cyclase activity.

This study investigates the hypothesis that inflammatory cytokines, interleukin (IL)-1alpha IL-1beta, and tumor necrosis factor (TNF), influence cardiac function by affecting calcium homeostasis and that this effect is mediated by the beta-adrenergic-adenylate cyclase system. After 4 days in culture, neonatal rat ventricular myocytes were treated with cytokines (10 ng/ml) for short (2 h) or longer (18 h) times. Myocyte calcium, contractility, and adenylate cyclase were measured under each condition. Anticipated stepwise increases in adenylate cyclase and intracellular calcium were found in controls (non-cytokine-treated) with 10(-7) M isoproterenol, 10(-7) M isoproterenol + 0.1 mM guanosine triphosphate, and 10(-9) M forskolin. Cells in the presence of cytokine for 2 h show increased basal calcium levels but no changes in adenylate cyclase activities, and isoproterenol fails to elevate adenylate cyclase levels or affect contractile shortening. After long-term treatment with IL-1beta or TNF, but not IL-1alpha, the significantly elevated levels of basal systolic calcium remain, and isoproterenol increases adenylate cyclase activity, unlike after short exposure. Forskolin maximally activates adenylate cyclase following both short- and long-term incubation, but the stepwise increase in activity is blunted following prolonged exposure. Thus short-term cytokine treatment blocks the adrenergic receptor-mediated increases in adenosine 3',5'-cyclic monophosphate, dissociating adenylate cyclase activation from cytokine-mediated increases in cell calcium, whereas longer treatment apparently produces direct affects on adenylate cyclase. Time-dependent differences in contractile response were found with IL-1alpha at 2 h and TNF at 18 h, implying that myofibrillar responsiveness to increased cytoplasmic calcium is dependent on both cytokine species and exposure time.

Sclerosing cholangitis: cholescintigraphy with Tc-99m-labeled DISIDA.

Four patients with primary sclerosing cholangitis (PSC) were examined with the hepatobiliary agent Tc-99m-labeled DISIDA (diisopropylphenylcarbamoyl iminodiacetic acid), and the results correlated with those of invasive cholangiography. Three of the four patients exhibited a typical pattern of multiple, persistent focal "hot spots" in the duct system, representing stasis within the segmental ductal dilatations (beading), also seen on cholangiography. Cholescintigraphy is superior to cholangiography in cases of suspected PSC where there is nonfilling of biliary radicals due to high-grade stenosis. The finding of delayed hepatic parenchymal clearance can allow estimation of the degree of obstruction of the various branches of the major bile ducts. Cholescintigraphy offers a noninvasive method of investigating patients with suspected sclerosing cholangitis, leading to earlier diagnosis. Confirmation with invasive cholangiographic procedures is recommended.

Evidence for membrane microheterogeneity in the sarcoplasmic reticulum of fast twitch skeletal muscle.

Sarcoplasmic reticulum (SR) from rabbit back muscles can be readily subfractionated into two morphologically and compositionally different vesicular populations, SRH (heavy) and SRL (light) derived from terminal cisternae and longitudinal SR, respectively. Polyacrylamide gels indicate that SRH contains most of the calsequestrin. Quantitation of freeze-fractured isolated preparations reveals that, while differences in vesicular dimensions are seen in SRH and SRL, the intramembrane particle (Ca2+ ATPase) density is identical. Phospholipid headgroup composition is the same in SRH and SRL, but fatty acyl moieties show significant differences in the ratio of saturated to unsaturated phospholipids in the two fractions. The vesicular dimensions of the purified Ca2+-ATPases, SRHP and SRLP, from the two fractions are identical, but the freeze-fracture particle density is higher in the SRLP fraction. The phospholipid composition remains similar after purification, but the differences in phospholipid fatty acyl composition of the preparations are maintained. SRH and SRHP contain almost twice as much of the unsaturated species as compared to SRL and SRLP. Differences in intramembrane particle density in purified fractions, thermotropic segregation of particles in freeze-fractured purified fractions, as well as differences in turnover of the acyl phosphate, appear to reflect the differences in fatty acyl chain composition of the two SR fractions and provide evidence of microheterogeneity in lipid-protein environment of the SR.

Anion effects on in vitro sarcoplasmic reticulum function. Co-transport of anions with calcium.

In isolated sarcoplasmic reticulum vesicles, calcium-chelating but non-calcium-precipitating dicarboxylates, such as maleate and succinate, stimulated ATP-dependent Ca2+ accumulation and its ensuring spontaneous Ca2+ accumulation and its ensuring spontaneous Ca2+ release, and Ca2+-dependent ATPase activity (Chu, A., Tate, C. A., Bick, R. J., Van Winkle, W. B., and Entman, M. L. (1983) J. Biol. Chem. 258, 1656-1664). We further examined the effect of dicarboxylates on enzyme turnover. The anionic buffer maleate enhanced the rate of rapid acyl phosphoenzyme hydrolysis compared to that in the zwitterionic buffer piperazine-N,N'-bis(2-ethanesulfonic acid) but had no effect on the phosphoenzyme formation. The presence of a calcium-precipitating anion, oxalate, or a Ca2+ ionophore, A23187, eliminated the differences observed in the phosphoenzyme decay between the two buffers, but accelerated the rate of decay. Furthermore, the catalytic activity of the purified Ca2+-dependent ATPase was not affected by maleate, whether oxalate was present or not. [14C]Succinate was transported into the sarcoplasmic reticulum in a manner which was dependent on Ca2+ transport, and occurred over a similar time course as Ca2+ accumulation/release. The net succinate uptake was equivalent to the amount of succinate-stimulated Ca2+ accumulation. Rapid efflux of both [14C]succinate and 45Ca2+ was induced by A23187, whereas the efflux induced by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid was slower and less compared to A23187. Succinate accumulation exhibited saturation kinetics with positive cooperativity (Km congruent to 20 mM; Hill coefficient = 1.70). When maleate and succinate were both present, they were equipotent, and had an additive stimulatory effect on peak 45Ca2+ accumulation at low concentrations. Maleate was a competitive inhibitor of succinate accumulation (Ki approximately equal to 17 mM; Hill coefficient = 1.75). KCl in the presence or absence of valinomycin did not influence succinate accumulation or release. The data suggest that succinate accumulation is Ca2+-dependent, but occurs at a saturable, divalent, anion-specific site. While this carrier or channel requires Ca2+ transport, it may be controlled by additional factors as well.

Anion effects on in vitro sarcoplasmic reticulum function. The relationship between anions and calcium flux.

Isolated sarcoplasmic reticulum vesicles exhibited different functional characteristics in the presence of zwitterionic as compared to anionic buffers. In the absence of oxalate, dicarboxylic anions (e.g. maleate, succinate) in a dose-dependent manner enhanced ATP-supported Ca2+ accumulation, the ensuing spontaneous Ca2+ release, and Ca2+-dependent ATPase activity compared to zwitterionic buffers (e.g. piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Hepes). This was not attributed to ionic strength and osmotic effects. The additional anion-dependent Ca2+ accumulation was linked to augmented Ca2+-dependent ATPase activity, and both could be induced by the addition of anion at any time during Ca2+ accumulation as long as ATP was present. Since the initial Ca2+ accumulation rates and acyl phosphoenzyme formation were the same between the two buffer classes, and the presence of either oxalate (a Ca2+-precipitating anion) or A23187 (a Ca2+ ionophore) abolished differences in Ca2+-dependent ATPase activity between the two buffer classes, it is likely that conditions favoring high intravesicular Ca2+ concentration allow the expression of the observed effect of the anions. Initial spontaneous Ca2+ release in the presence of maleate was not caused by ATP depletion, and it was virtually absent in Pipes buffer. The rate of spontaneous release was also stimulated in a dose-dependent manner by the dicarboxylic anions, with the time of release being related to the time of anion addition and not ATP addition. A later, more rapid release phase in either maleate or Pipes buffer corresponded to ATP depletion, and could be duplicated at any time in the Ca2+ accumulation/release cycle by the addition of an ATP trap. With an ATP-regenerating system present or with very high ATP concentrations, the maximal peak Ca2+ accumulation in Pipes buffer could approach that in maleate buffer. The data suggest that dicarboxylic anions stimulate the filling of a Ca2+ compartment from which spontaneous Ca2+ release occurs.

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.