Fatty acid-mediated calcium sequestration within intracellular calcium pools.

Intracellular Ca2+ pools play an essential role in generating Ca2+ signals. The heterogeneity of intracellular Ca2+ pools reflects the complex and dynamic character of the endoplasmic reticulum within which they reside. Translocation of Ca2+ between distinct subcompartments of the endoplasmic reticulum is mediated by a sensitive and specific GTP-activated process involving formation of reversible communicating junctions (Rys-Sikora, K. E., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 31607-31613). In the presence of palmitate at 10 microM or above, this GTP-activated mechanism mediates substantial Ca2+ accumulation within a specific Ca2+-pumping pool. The fatty acid- and GTP-dependent accumulation of Ca2+ was highly chain length-specific; pentadecanoate (C15) and palmitate (C16) were equally effective, whereas fatty acids of shorter or longer chain length were either marginally effective or devoid of effect. Fatty acids with one or more unsaturated carbons were without effect, regardless of chain length. Palmitate-induced Ca2+ accumulation was immediately terminated with 2 microM palmitoyl-CoA, a blocker of the GTP-activated Ca2+-translocating mechanism. The anion transport inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid completely prevented both palmitate- and oxalate-mediated GTP-dependent Ca2+ accumulation, with EC50 approximately 30 microM. Ca2+ sequestered in the presence of palmitate and GTP could be immediately and completely released by A23187, whereas the sequestered Ca2+ was remarkably resistant to release induced by inositol 1,4,5-trisphosphate (InsP3). In contrast, oxalate-sequestered Ca2+ within the same pool could be effectively released by either ionophore or InsP3. The results indicate that fatty acids are specifically transported into the lumen of a subset of Ca2+ pools, wherein they mediate substantial sequestration of Ca2+ in a distinct membrane-associated substate that is not readily releasable by opened InsP3-sensitive Ca2+ channels.

Cytosolic Ca 2ϩ signals mediate control over a diverse array of cellular activities ranging from short-term responses such as contraction and secretion to longer term regulation of cell growth and proliferation (1)(2)(3)(4)(5). Intracellular pools of Ca 2ϩ are fundamental elements in the generation of Ca 2ϩ signals, yet little is known about the nature and distribution of the organelles that serve as Ca 2ϩ pools (4). It is widely held that the majority of Ca 2ϩ pools are contained within the endoplasmic reticulum (ER) 1 or subcompartments thereof (1,2,4,6,7). Most of the ER appears to pump and accumulate Ca 2ϩ . However, the ER represents an extensive and heterogeneous network of cisternae undergoing continuous dynamic changes in morphology through active membrane trafficking events. It is therefore not surprising that Ca 2ϩ pools are observed to be structurally and functionally heterogeneous in particular with respect to distribution of InsP 3 -sensitive Ca 2ϩ release channels (8 -11). In earlier studies, we identified and characterized distinct InsP 3sensitive and -insensitive intracellular Ca 2ϩ -pumping pools (8,10). In addition, we and others have characterized a sensitive and specific guanine nucleotide-activated process that appears to mediate translocation of Ca 2ϩ between organelles within cells (12)(13)(14)(15)(16) and that permits Ca 2ϩ movement between InsP 3sensitive and -insensitive Ca 2ϩ pools (8, 16 -19). This Ca 2ϩ translocation process is rapid, temperature-sensitive, and dependent on the hydrolysis of GTP and represents a movement of Ca 2ϩ quite distinct from that activated by InsP 3 (7,8,16). The process of Ca 2ϩ transfer may involve a close interaction between ER subcompartments and the cytoskeleton and may be particularly evident after disruption or fragmentation of the ER (20). Although no specific GTP-binding protein has been identified as mediating the Ca 2ϩ transfer, it is likely that transfer reflects the function of one or more of the class of small GTP-binding proteins that are known to control many of the membrane trafficking and translocation events that occur within the ER and other organelles of the secretory pathway (4,16,21).
The mechanism by which GTP activates Ca 2ϩ transfer requires close contacts between membranes (7,16), but the underlying process mediating Ca 2ϩ transfer has not been elucidated. Work by Comerford and Dawson (22,23) has suggested that GTP-induced Ca 2ϩ movements may reflect the activation of fusion between membranes. Instead, we have interpreted our results to indicate that the rapid fluxes of Ca 2ϩ observed in response to GTP reflect a process that may precede a subsequently activated membrane fusion event (16,24). Indeed, studies have shown that acyl-CoA esters can not only block, but also reverse the action of GTP in inducing Ca 2ϩ transfer (24). Thus, we revealed that fatty acyl-CoA esters allosterically modify a component of the GTP-activated process, resulting in a rapid termination and reversal of Ca 2ϩ transfer. It is difficult to reconcile this rapid reversing action of acyl-CoA esters with * This work was supported by National Institutes of Health Grant HL55426, National Science Foundation Grant MCB 9307746, and a grant-in-aid from the American Heart Association, Maryland Affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  a mechanism by which GTP induces Ca 2ϩ movements via a membrane fusion event since, having taken place, membrane fusion is an essentially irreversible process. Instead, we concluded that GTP rapidly induces a communicating prefusion complex between membranes that allows Ca 2ϩ transfer and that, later, may lead to full fusion between membranes (4,24). To understand more about the progression of these events, we investigated the actions of agents that might modify the membrane and enhance the process of membrane fusion. Surprisingly, one class of agents used for such analysis, nonesterified fatty acids, induced a remarkable chain length-specific alteration in the accumulation of Ca 2ϩ observed after activation of the GTP-induced Ca 2ϩ transfer process. Our results indicate that specific fatty acids are transported into the lumen of a subset of Ca 2ϩ pools, wherein they mediate substantial sequestration of Ca 2ϩ in a distinct membrane-associated substate; although readily releasable by application of ionophore, the release of this fatty acid-complexed Ca 2ϩ through opened InsP 3 -sensitive Ca 2ϩ release channels is highly restricted.

EXPERIMENTAL PROCEDURES
Culture of Cells-Cells of the hamster smooth muscle cell line DDT 1 MF-2 were cultured in Dulbecco's modified Eagle's medium with 2.5% serum (Calf-Plus, Inovar Chemicals, Inc., Gaithersburg MD) as described previously (25)(26)(27). Cells were passaged every 7 days, and media changes were performed on days 3 and 5 after passaging. Cells used in experiments had been grown for 4 days after passaging.
Cell Permeabilization-The procedures for cell permeabilization were as described earlier (24). Briefly, suspensions of DDT 1 MF-2 cells (1 ϫ 10 6 cells/ml) were incubated with 0.0025% saponin in an intracellular-like medium (ICM; comprising 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl 2 , and 10 mM Hepes-KOH, pH 7.0) at 37°C until 95% permeabilization occurred (normally 2-3 min). After permeabilization, cells were washed twice in saponin-free ICM at 4°C and kept cold before use in experiments. To avoid problems of lipid dilution of added hydrophobic compounds, the final cell concentration in all experiments was kept at exactly 5 ϫ 10 5 cells/ml.
Calcium Flux Experiments-Ca 2ϩ flux measurements were conducted as described previously (24,28,29). The accumulation of Ca 2ϩ in intracellular organelles was measured using permeabilized DDT 1 MF-2 cells (5 ϫ 10 5 cells/ml) maintained with gentle stirring at 37°C in ICM containing 50 M CaCl 2 (with 150 Ci/mol 45 Ca 2ϩ ), EGTA (to buffer free Ca 2ϩ to exactly 0.1 M), 3% polyethylene glycol, and 5 M ruthenium red (to prevent mitochondrial Ca 2ϩ accumulation) in a total volume of 2 ml. Effectors indicated in the figures (GTP, InsP 3 , and A23187) were added at the times shown. Fatty acids or oxalate, with or without GTP or DIDS when present, were added immediately prior to the start of uptake or as shown in the figures. At required times, 200-l aliquots were removed from the stirred uptake medium, diluted immediately into 4 ml of ice-cold ICM containing 1 mM LaCl 3 , rapidly vacuumfiltered on glass-fiber filters (Schleicher and Schuell, type 31), washed, and counted. For the palmitate concentration curves, all additions of fatty acids with or without GTP were made prior to the start of the experiment. For the curves shown, Ca 2ϩ loading was for 6 min, at which time three successive aliquots were taken, and accumulation was determined. In other experiments (data not shown), Ca 2ϩ loading was monitored for 12 min as described. In fatty acid chain length specificity experiments, Ca 2ϩ accumulation was assessed only after 12 min of Ca 2ϩ loading. The figures show ATP-dependent Ca 2ϩ accumulation with that component of Ca 2ϩ retained by cells and filters in the absence of ATP subtracted (ϳ0.1% of total Ca 2ϩ ). All experiments shown are typical of at least three separate experiments (and in most cases, a considerably larger number). FIG. 2. Concentration dependence of palmitate-induced reversal of GTP-mediated Ca 2؉ movements. Ca 2ϩ accumulation was monitored with increasing concentrations of palmitate acid in the absence (E) or presence (q) of 20 M GTP. Palmitate at the concentrations shown and GTP were added just prior to the start of Ca 2ϩ uptake, which continued for 6 min, at which time three successive aliquots were rapidly taken, and radioactivity was determined as described under "Experimental Procedures." The measurements were carried out in duplicate, and results are the means Ϯ S.D. of six determinations and are representative of four similar experiments. 3 and A23187 were purchased from Calbiochem. ATP, GTP, EGTA, polyethylene glycol, saponin, ruthenium red, Hepes, DIDS, oxalate, and all fatty acids were purchased from Sigma. Fatty acids were each dissolved in methanol/ Me 2 SO (1:1), sonicated, and stored at Ϫ20°C in capped vials containing N 2 . Before addition to experiments, fatty acids were again sonicated and added to permeabilized cell suspensions in ICM in no more than 1% of the final 2-ml volume. All controls contained equivalent quantities of the solvent without fatty acid. Visible turbidity of solutions was only observed with palmitate at a final concentration of 300 M or higher. The DDT 1 MF-2 cell line was originally obtained from Drs. James Norris and Lawrence Cornett (University of Arkansas). Free Ca 2ϩ concentrations were controlled using EGTA, computing all complexes between EGTA, ATP, Ca 2ϩ , Mg 2ϩ , monovalent cations, and protons, as described previously (30).

RESULTS AND DISCUSSION
Palmitate Reverses GTP-activated Ca 2ϩ Translocation, Resulting in Substantial Ca 2ϩ Accumulation-The role of distinct Ca 2ϩ pools in accumulating, releasing, and transferring Ca 2ϩ has been studied extensively in several permeabilized cell systems (16,29), including cells from the DDT 1 MF-2 smooth muscle line. An important feature of the Ca 2ϩ movements observed has been the role of a sensitive and specific guanine nucleotide-induced Ca 2ϩ translocation process (4,7,8,16). As shown in Fig. 1A, Ca 2ϩ accumulated within internal pools of permeabilized DDT 1 MF-2 cells via SERCA pump activity in the presence of 1 mM ATP was rapidly released by addition of 20 M GTP; more than half of the accumulated Ca 2ϩ was released within 30 s. Thus, GTP caused release from only a proportion of the pools able to accumulate Ca 2ϩ as compared with the action of the Ca 2ϩ ionophore A23187, which released Ca 2ϩ from all of the Ca 2ϩ -pumping pools. The action of GTP on Ca 2ϩ pools has been studied extensively and is believed to result from a GTPinduced transfer of Ca 2ϩ between distinct subcompartments (8,13,(17)(18)(19)31). This process requires close contact between membranes (17,18) and the hydrolysis of GTP (7,8,13,32) and can be fully reversed by fatty acyl-CoA esters (24). Ca 2ϩ transfer is believed to result from the activation of junctional complexes between distinct Ca 2ϩ -pumping compartments of the endoplasmic reticulum (16,24). Such junctions allow the passage of Ca 2ϩ ions and perhaps other small molecules, but, since they can be reversed (24), appear not to represent fusion between membranes. Our studies indicate that the GTP-activated process may represent a rapid prefusion event (16, 24) and that, in a substantially longer period following the GTPinduced interaction, fusion between different vesicular components of the ER may take place (7,16,22,23). Since, in permeabilized cells, a small component of the ER appears to exist as non-intact vesicular membranes, the rapid transfer process induced by GTP results in the fast release of a substantial proportion of the accumulated Ca 2ϩ (7,16), as observed in Fig.  1A.
To determine more about the mechanism of the GTP-induced Ca 2ϩ transfer process, we investigated the actions of fatty acids. As stated earlier, the rationale for examining fatty acids was to determine whether such lipid molecules might modify the effectiveness of GTP by promoting membrane interactions and enhancing the rate of subsequent membrane fusion. The introduction of up to 100 M palmitate into the Ca 2ϩ uptake medium induced only a slight augmenting action on Ca 2ϩ pump-mediated Ca 2ϩ accumulation within the permeabilized DDT 1 MF-2 cells (Fig. 1B). However, in stark contrast to the results shown in Fig. 1A, addition of GTP in the presence of palmitate resulted in a rapid and dramatic increase in the accumulation of Ca 2ϩ (Fig. 1B). Under these conditions, the action of GTP was biphasic, inducing initially a slight release of Ca 2ϩ before this changed to a large and continuous increase in Ca 2ϩ accumulation. The methyl ester of palmitate had no effect on Ca 2ϩ accumulation (data not shown), indicating that the carboxyl group was important for the stimulation of Ca 2ϩ uptake with GTP. Without GTP addition, the Ca 2ϩ accumulated in the presence of palmitate could still be fully released with the Ca 2ϩ ionophore A23187 (Fig. 1B), indicating no obvious difference in the state of accumulated Ca 2ϩ induced by palmitate. Likewise, after loading pools in the presence of palmitate, passive Ca 2ϩ release induced by Ca 2ϩ pump inhibition with thapsigargin was unchanged (data not shown), suggesting no obvious alteration in Ca 2ϩ release. As shown below (see Fig. 7), palmitate did not alter the function of Ca 2ϩ release through InsP 3 receptors. The large accumulation of Ca 2ϩ induced by GTP in the presence of palmitate was dependent on Ca 2ϩ pumping, with no change in Ca 2ϩ accumulation being observed without ATP also being present. Under the standard conditions for Ca 2ϩ accumulation, the low concentration of free Ca 2ϩ (0.1 M) and the presence of ruthenium red ensured that no mitochondrial Ca 2ϩ uptake was occurring (7,10). At a higher free Ca 2ϩ concentration (10 M), in the absence of ruthenium red, the accumulation of Ca 2ϩ was predominantly mitochondrial (7), yet under this condition, palmitate and/or GTP again induced no alteration of the mitochondrial component of Ca 2ϩ accumulation (data not shown).
The concentration dependence of the effect of palmitate on reversing GTP-induced Ca 2ϩ fluxes in permeabilized cells is shown in Fig. 2. In this experiment, Ca 2ϩ accumulation was measured after 6 min with the simultaneous addition of ATP and palmitate, with or without 20 M GTP, all at the start of Ca 2ϩ uptake. Under these conditions, concentrations of palmitate above 3 M caused significant Ca 2ϩ entry; the action of palmitate was half-maximal at ϳ25 M and maximal at 100 M. In the absence of GTP, little change in Ca 2ϩ accumulation occurred with palmitate concentrations as high as 300 M. If a longer time interval for Ca 2ϩ accumulation was used, the total amount of Ca 2ϩ that could be sequestered in the presence of GTP and palmitate continued to increase; after 12 min, total Ca 2ϩ accumulation could reach as high as 8 nmol/10 6 cells (data not shown). Measured with a 12-min incubation, the sensitivity to palmitate was slightly increased, with half-maximal activation closer to 10 M. Although 300 M palmitate had no greater effect than 100 M, introduction of concentrations beyond 300 M induced a sharply concentration-sensitive de-crease in Ca 2ϩ accumulation in the presence or absence of GTP (data not shown). This reduction in Ca 2ϩ accumulation at high palmitate levels likely reflects a generalized deleterious action of palmitate on the integrity of the membrane through detergent-like effects of the fatty acid. Indeed, at or above 300 M, some turbidity following addition of palmitate indicated the formation of larger fatty acid complexes. The critical micelle concentration for fatty acids is highly dependent on ionic conditions and, although not known in the presence of the intracellular-like conditions used, was likely to have been exceeded at 300 M palmitate. Physiological levels of free nonesterified fatty acids inside cells are difficult to assess. In serum, the level of total nonesterified fatty acid can be in the millimolar range, palmitate being one of the most abundant of fatty acids; however, a large proportion of this total fatty acid is bound to albumin and other serum proteins. Fatty acids inside cells are likely to be similarly bound to proteins and/or partitioned within membranes. Experiments with sarcoplasmic reticulum vesicles have revealed that palmitate in the mid-micromolar range is efficiently incorporated into the vesicle bilayer (33). These studies revealed that the monounsaturated C 18 fatty acid oleate at these concentrations caused considerable disruption and fusion of SR vesicles, whereas, in contrast, palmitate induced no fusion of the vesicles and no morphological alteration in the membrane except for an increase in the lateral volume of the membranes (33).
Structural Specificity of Fatty Acid-mediated Stimulation of GTP-dependent Ca 2ϩ Sequestration-Important to assess was any structural requirement among fatty acids for stimulating GTP-dependent Ca 2ϩ accumulation, particularly with respect to chain length and degree of saturation. Indeed, the results reveal a striking structural specificity for fatty acids. A comparison of the effects of saturated fatty acids of different chain length is shown in Fig. 3. In this experiment, each fatty acid was added at 100 M at the beginning of Ca 2ϩ uptake either with or without GTP, and uptake continued for a total of 12 min. The control condition (i.e. without fatty acid) revealed the ϳ50% inhibition of Ca 2ϩ accumulation due to GTP, as a result of GTP-induced Ca 2ϩ release. Both pentadecanoate (C 15 ) and Experiments also investigated the actions of unsaturated fatty acids. It was noted that the unsaturated fatty acids had significant inhibitory effects on Ca 2ϩ accumulation at the higher levels (100 M). Such effects may relate to the inhibitory effects of unsaturated fatty acids on Ca 2ϩ pump activity as observed using SR vesicles (34 -36) and, as described above, may be indicative of the greater effectiveness of the unsaturated fatty acids in disrupting the lipid bilayer, resulting in leak of Ca 2ϩ (33). The experiment shown in Fig. 4 compared the actions of a range of different unsaturated fatty acids with that of palmitate, each added at a lower concentration (10 M). With the longer incubation period of 12 min, the action of palmitate in inducing Ca 2ϩ accumulation in the presence of GTP was clearly apparent even at 10 M. In contrast, neither of the monounsaturated C16:1-⌬ 9 fatty acids (either the cis form, palmitoleate, or the trans form, palmitoleidate) had any effect on Ca 2ϩ accumulation. Moreover, heptadecenoate (C17:1-cis-⌬ 10 ), oleate (C18:1-cis-⌬ 9 ), linoleate (C18:2-cis-⌬ 9,12 ), and arachidonate (C20:4-cis-⌬ 5,8,11,14 ) also did not induce any reversal of the action of GTP. As discussed below, the specificity of action of fatty acids in inducing Ca 2ϩ accumulation, particularly with respect to chain length of saturated fatty acids, may relate to differences in the ability of fatty acids to be transported into the ER and/or to different intrinsic abilities of fatty acids to bind to and sequester Ca 2ϩ .
What Is the Process by Which Fatty Acids Mediate GTP-dependent Ca 2ϩ Accumulation?-The actions of fatty acids in promoting the substantial GTP-dependent accumulation of Ca 2ϩ could be explained by several possible mechanisms. First, palmitate could be augmenting the activity of the Ca 2ϩ pump. Second, fatty acids could be promoting the action of GTP to induce fusion between membrane components of the ER, resulting in larger Ca 2ϩ accumulation. Third, fatty acids might be entering the ER and mediating sequestration of Ca 2ϩ within the lumen. Several lines of evidence support the latter conclusion. Direct effects of palmitate on the initial rate of Ca 2ϩ pumping were not observed (Fig. 1, A and B). Others have reported that palmitate at 100 M has no effect on intracellular Ca 2ϩ pumping in permeabilized pancreatic cells (37), whereas saturated fatty acids including palmitate, stearate, and arachidate are reported to actually have an inhibitory effect on SERCA pumping in SR vesicles (38). The chain length specificity of the action of fatty acids did not appear consistent with their effect on promoting membrane fusion; indeed, experiments described below appear to more directly rule out membrane fusion as a mechanism mediating GTP-and palmitateinduced Ca 2ϩ accumulation. Instead, the action of palmitate in promoting Ca 2ϩ accumulation in the presence of GTP is strongly analogous with the action of the dicarboxylic acid oxalate (7,16). We previously revealed that concentrations of oxalate in the millimolar range were also able to reverse the action of GTP, resulting in significant Ca 2ϩ accumulation; the conclusions from a number of studies were that two distinct Ca 2ϩ -pumping pools exist, one of which is InsP 3 -releasable and oxalate-permeable, the other being InsP 3 -nonreleasable and impermeable to oxalate (reviewed in Ref. 16). As described above, Ca 2ϩ transfer between these pools is mediated through interorganelle junctions activated by GTP hydrolysis, representing a rapid and reversible "prefusion" event (24). With oxalate present, Ca 2ϩ can be sequestered within the oxalatepermeable pool as an oxalate precipitate; GTP then permits the additional Ca 2ϩ pumped into the oxalate-impermeable pool to have access to oxalate, resulting in greatly increased Ca 2ϩ accumulation. Even though the total pumping activity does not increase, oxalate acts as a sink for Ca 2ϩ to provide a large increase in Ca 2ϩ accumulation (19). As mentioned earlier, the GTP-induced release of Ca 2ϩ observed without oxalate results from junctional activation between a small proportion of nonintact membrane vesicles and the larger ER continuum (7,16,32). Since oxalate-complexed Ca 2ϩ cannot be transferred through the GTP-activated Ca 2ϩ translocation process, this small leak does not prevent buildup of oxalate-complexed Ca 2ϩ inside the ER (37).
Whereas the analogy between the actions of palmitate and oxalate was suggestive of a similar Ca 2ϩ -sequestering action of palmitate, there was also a major difference in the actions of the two agents, namely, the much greater sensitivity of the response to palmitate. Thus, measured under optimal conditions, the effect of palmitate was ϳ100-fold more potent than that of oxalate. In view of this, it was important to more definitively investigate a possible alternative effect of palmitate through enhancement of fusion between membranes of distinct pools. We recently revealed that fatty acyl-CoA esters can reverse the GTP-activated Ca 2ϩ translocation process (24). As shown in Fig. 5, maximally activated uptake of Ca 2ϩ promoted by GTP in the presence of palmitate could be immediately terminated by addition of 2 M palmitoyl-CoA. If palmitate were stimulating Ca 2ϩ accumulation by inducing GTPdependent fusion between the Ca 2ϩ -pumping organelles, then such an effect would not be reversible; in other words, membrane fusion, once having occurred, is an irreversible event. We therefore conclude that the action of palmitate is to promote sequestration of Ca 2ϩ . There is ample precedent for believing this to occur. Thus, details of the binding of Ca 2ϩ to palmitate have been measured (39). In the 10 -80 M palmitate range, Ca 2ϩ binding was shown to be half-maximal at 30 M Ca 2ϩ and to occur with a stoichiometry of 0.4 mol of Ca 2ϩ /mol of palmitate. The rate of association was highly Ca 2ϩ -dependent and maximal at ϳ1 mM Ca 2ϩ . Considering that the luminal Ca 2ϩ concentration is in the high micromolar to millimolar range (40 -42), then entry of palmitate into the ER lumen could give rise to substantial Ca 2ϩ sequestration within the lumen. The question of whether and how palmitate might cross the ER membrane was therefore addressed.
Does the Ability of Fatty Acids to Induce GTP-dependent Ca 2ϩ Accumulation Represent Function of an ER Anion Transporter?-In previous work (7,16), we had speculated that the action of oxalate might be related to the function of anion channels present in the ER membrane. Thus, evidence has shown that relatively nonselective anion channels exist in the SR membrane of muscle cells and the ER membrane of nonmuscle cells, the function of which is considered to help equilibrate the charge buildup that would otherwise accompany rapid release of a large quantity of Ca 2ϩ ions from the SR or ER (7,16,(43)(44)(45). Plasma membrane anion channels have been characterized in detail, and the stilbene-disulfonic acid derivative DIDS has been widely used as an inhibitor of such channels (46). Anion channels in both the SR (45, 47-49) and ER (44, 50) have also been well described. In rabbit SR, there appear to be two different anion channels that are blocked by 8 and 80 M DIDS, respectively (51), and in rat brain ER, anion channels are blocked by DIDS in the 15-100 M range (52). We therefore investigated whether DIDS would modify the action of either palmitate or oxalate in promoting Ca 2ϩ accumulation in the presence of GTP. As shown in Fig. 6, in both cases, 100 M DIDS completely prevented the anion-mediated accumulation of Ca 2ϩ . The experiment reveals that the actions of 100 M palmitate (Fig. 6A) and 2.65 mM oxalate (Fig. 6B) added at the start of Ca 2ϩ uptake were very similar. In the presence of palmitate when 20 M GTP was also present, Ca 2ϩ accumulation was initially retarded (due to increased release, as described above) and then, within a few minutes, became greatly augmented. This biphasic effect is similar to that shown in Fig.  1A, but is significantly slower. The biphasic action most likely reflects the rate of palmitate entry. When added before GTP (Fig. 1A), there is a very rapid transition since palmitate has presumably equilibrated within the ER lumen; in Fig. 6A, entry of sufficient palmitate must take place before significant Ca 2ϩ sequestration can occur, hence a lag of ϳ3 min. The effect of DIDS was to block any action of GTP and palmitate in promoting Ca 2ϩ uptake. This action of DIDS was half-maximal between 10 and 30 M in preventing both palmitate-and oxalatedependent Ca 2ϩ accumulation in the presence of GTP (data not shown), clearly within the sensitivity range described above for anion channels of the SR and ER (51,52). DIDS had little inhibitory effect on the initial rate of ATP-dependent Ca 2ϩ pumping in the absence of palmitate or oxalate; at longer time intervals, a slight reduction in Ca 2ϩ accumulation may reflect the permissive role of anion channels in mediating charge equilibration and allowing an increase in the equilibrium level of Ca 2ϩ to be attained within the ER.
These results provide evidence to indicate that the actions of palmitate and oxalate are indeed reflections of a similar mechanism involving mediated anion entry. One question that arises is whether the movement of oxalate or palmitate to support the large accumulation of Ca 2ϩ is directly mediated by a possible anion channel or whether the large sequestration of Ca 2ϩ that occurs is facilitated as a result of anion channels permitting movement of other smaller ions (for example, Cl Ϫ ), which themselves dissipate charge. Although we have not undertaken studies on the effects of anion replacement, it is interesting to note that there is considerable precedent for DIDS-sensitive long chain fatty acid transport. Thus, much evidence points to the movement of fatty acids across plasma membranes being mediated by specific transport proteins. Interestingly, the function of these transporters has been shown to be blocked by DIDS in the 40 -200 M range (53, 54); indeed, labeled DIDS has been revealed to specifically bind one such plasma membrane fatty acid transport protein, FAT, which has been isolated, sequenced, and expressed (55,56). Controversy surrounds how important such fatty acid transport proteins really are for the movement of fatty acids across membranes. Whereas evidence suggests that such proteins exist (55,57), some believe that the flip-flop of fatty acids across membranes is sufficiently fast that transport proteins are not necessary (58). Our data may throw light on this area. Thus, it is most unlikely that the charged hydrophilic dicarboxylic acid oxalate traverses membrane bilayers, yet both palmitate and oxalate mediate similar effects on GTP-induced Ca 2ϩ movements, and the actions of both are prevented by DIDS. Thus, it may be reasonable to conclude that a DIDS-sensitive transporter for mono-or dicarboxylic acids at least facilitates the entry of the two Ca 2ϩ -complexing anions. A further very significant inference from these data is that fatty acid transport appears to be facilitated across intracellular membranes as well as the plasma membrane. Last, the question of how fatty acid-induced sequestration of Ca 2ϩ within the ER has such narrow chain length specificity is interesting. It is unlikely that the Ca 2ϩbinding properties of fatty acids are so stringently related to chain length; therefore, it appears more likely that the specificity of the effect we observed is derived from specificity of the transporter itself.
State of Intraluminal Ca 2ϩ Sequestered in the Presence of Fatty Acids: Implications for the Location and Releasability of Fatty Acid-Ca 2ϩ Complexes-The effects of palmitate and oxalate on Ca 2ϩ accumulation provide some important parallels. Their similarity of action and blockade by DIDS suggest that a similar anion transport mechanism facilitates both processes. And the promotion by GTP of the actions of both anions suggests that the same two differentially anion-permeable Ca 2ϩ pools are functioning. Yet, despite these parallels, some significant differences in the state of the sequestered Ca 2ϩ were observed for the two anions. The data in Fig. 7 compare the actions of InsP 3 and A23187 on Ca 2ϩ sequestered in the presence of GTP together with correspondingly effective concentra-tions of either palmitate or oxalate. With palmitate (Fig. 7A), after attainment of maximal Ca 2ϩ sequestration, addition of a maximally effective InsP 3 concentration (10 M) induced the slow onset of a reduced rate of Ca 2ϩ accumulation. With oxalate (Fig. 7B), introduction of InsP 3 prevented any further Ca 2ϩ accumulation and induced a modest release of Ca 2ϩ . A more striking difference in effect was observed upon addition of A23187. With palmitate (Fig. 7A), a remarkably rapid release of most of the accumulated Ca 2ϩ was observed within a few seconds, whereas with oxalate (Fig. 7B), the action of A23187 was very much slower. In the latter case, there was almost no effect of A23187 after 30 s, and even after 4 min, scarcely more than half the Ca 2ϩ had been released. These effects point to a significant difference in the state and/or location of Ca 2ϩ sequestered with the two anions. Similar effects are revealed from the data shown in Fig. 8. In this case, InsP 3 or ionophore was added from the beginning of Ca 2ϩ accumulation. In the presence of palmitate (Fig. 8A), InsP 3 without GTP prevented accumulation of Ca 2ϩ within the InsP 3 -sensitive pool, indicating that the action of InsP 3 was not inhibited by palmitate. With GTP present, substantial sequestration of Ca 2ϩ was only reduced but not prevented by InsP 3 . In the presence of oxalate (Fig. 8B), InsP 3 completely prevented accumulation of Ca 2ϩ within the InsP 3 -sensitive pool whether or not GTP was present.
These data indicate that, whereas the operational Ca 2ϩ pools are likely the same, the state of Ca 2ϩ sequestered with the two anions is different. Oxalate is known to form easily observable insoluble Ca 2ϩ precipitates within the lumen of muscle SR vesicles (59,60) or the ER of permeabilized liver cells (61). Such precipitates can redissolve, albeit slowly, if free luminal Ca 2ϩ decreases, as reflected by the actions of InsP 3 and A23187 (Fig.  7B). If the free luminal Ca 2ϩ is prevented from reaching the threshold for precipitation as a result of the action of InsP 3 or A23187, the Ca 2ϩ -oxalate complex does not form (Fig. 8B). With palmitate, the Ca 2ϩ complex is relatively resistant to InsP 3 -induced release of Ca 2ϩ , but is remarkably sensitive to the action of A23187 (Fig. 7A). We conclude from this result that the Ca 2ϩ -palmitate complex is associated intimately with the membrane and as such is able to efficiently exchange Ca 2ϩ with the ionophore, which itself is partitioned within the membrane. In contrast, the far slower release of oxalate-complexed Ca 2ϩ by A23817 (Fig. 7B) reflects no particular advantage of the ionophore and the release of Ca 2ϩ at a rate that reflects dissociation of Ca 2ϩ from oxalate precipitated in the ER lumen. In this experiment, the more complete effect of A23187 as compared with InsP 3 likely reflects ionophore-mediated release from both the InsP 3 -sensitive and -insensitive pools. Whereas the membrane-bound Ca 2ϩ -palmitate complex is highly acces-sible to ionophore, in contrast, this complex appears not to be easily releasable by InsP 3 . Thus, passage of Ca 2ϩ through the InsP 3 receptor channel is likely restricted to free uncomplexed luminal Ca 2ϩ . The slow rate of Ca 2ϩ release by InsP 3 may reflect the slower dissociation of Ca 2ϩ from the Ca 2ϩ -palmitate complex due to a relatively higher affinity of palmitate for Ca 2ϩ as compared with oxalate (the actual affinity of palmitate in the membrane may be enhanced by the presence of other negatively charged membrane lipids). Similarly, as shown in Fig.  8, InsP 3 receptors are relatively less efficient in competing with palmitate for Ca 2ϩ being pumped into the lumen of the pool in the presence of GTP (Fig. 8A) as opposed to oxalate (Fig. 8B). Interestingly, the rate of dissociation of Ca 2ϩ from the Ca 2ϩpalmitate complex may be even slower than that indicated in Fig. 7A. Thus, as shown in Fig. 9, in an extension of the experiment shown in Fig. 5, the action of InsP 3 was compared on permeabilized cells either maximally accumulating Ca 2ϩ in the presence of palmitate and GTP or after this maximal uptake had been blocked by palmitoyl-CoA. Whereas a small reduction in the rate of Ca 2ϩ accumulation was observed in the former case (consistent with Fig. 7A), after fatty acyl-CoA addition, there was no release with InsP 3 . In this case, the accumulation of Ca 2ϩ as a result of GTP-activated Ca 2ϩ transfer has been terminated, and the palmitate-complexed Ca 2ϩ is not released by InsP 3 . In other words, addition of palmitoyl-CoA has effectively isolated the InsP 3 -sensitive pool, which, although containing palmitate-complexed Ca 2ϩ , is not in a state releasable through InsP 3 receptors. In contrast, the effect of InsP 3 in the absence of fatty acyl-CoA likely reflects the ability of Ca 2ϩ being pumped and transferred into the pool to be competed for and released by InsP 3 receptors before it is sequestered with palmitate.
Concluding Remarks-A scheme depicting the carboxylic acid-induced sequestration of Ca 2ϩ and the function of the two pools is presented in Fig. 10. The entry of either oxalate or palmitate is shown through a single transport system, the FIG. 10. Scheme depicting the localization and actions of palmitate and oxalate in mediating Ca 2؉ sequestration within intracellular Ca 2؉ pools. A DIDS-sensitive anion channel is depicted as permitting entry of oxalate or palmitate into the InsP 3 -sensitive Ca 2ϩ pool. Ca 2ϩ is pumped into this pool and the InsP 3 -insensitive pool via the action of thapsigargin (TG)-sensitive SERCA Ca 2ϩ pumps. Palmitate is shown exclusively attached to the inner membrane surface within the Ca 2ϩ pool, where two molecules of palmitate are associated with one Ca 2ϩ ion (39). In contrast, oxalate (shown as a dumbbell structure) reversibly complexes Ca 2ϩ and remains within the lumen of the pool. Although the state and location of Ca 2ϩ complexes are distinct for palmitate and oxalate, both can effectively create a Ca 2ϩ sink, the filling of which is greatly augmented by the GTP-activated translocation process depicted as a junctional complex, allowing transfer of Ca 2ϩ between the InsP 3 -sensitive and -insensitive pools (7,16). Details of the scheme are provided under "Results and Discussion." function of which is restricted to the InsP 3 -sensitive Ca 2ϩ pool. GTP-induced transfer of Ca 2ϩ occurs between this pool and a distinct Ca 2ϩ -pumping pool that does not contain either InsP 3 receptors or the anion transporter. Palmitate is shown as being present only in the membrane, an inference highly likely considering the avid partitioning of fatty acids into membranes. The binding of two molecules of palmitate to one Ca 2ϩ ion is consistent with the binding stoichiometry mentioned earlier (39). In contrast to palmitate, oxalate is shown as being within the lumen. An important message from this scheme is that, whereas separate pools of Ca 2ϩ do appear to exist in cells, the Ca 2ϩ within a single pool can exist in substates that are very different with respect to releasability by channels. Thus, pools are frequently defined on the relative effectiveness of a given release channel versus the action of an ionophore. On this basis, the palmitate-complexed Ca 2ϩ would be defined as existing in a separate pool, yet every indication is that it is within the same pool as that complexed by oxalate and the same pool as that containing InsP 3 receptors. Thus, fatty acids may provide an important sink of membrane-associated Ca 2ϩ distinct from the bulk phase of Ca 2ϩ . Such a substate of Ca 2ϩ can be compared with that bound to low affinity, high capacity, calcium-sequestering proteins within the SR or ER lumen (such as calsequestrin and calreticulin) that provide additional buffering capacity at high intraluminal Ca 2ϩ levels; however, with these proteins, the bound Ca 2ϩ is readily releasable through Ca 2ϩ release channels (62). Whether fatty acids sequester significant Ca 2ϩ within pools under physiological conditions is yet to be proven. However, considering that (a) significant quantities of nonesterified fatty acids are present inside cells, (b) fatty acids appear to be selectively transported into specific subcompartments of the ER, and (c) fatty acids are highly partitioned in the lipid bilayer, where they can bind substantial quantities of Ca 2ϩ , we conclude that fatty acid-mediated Ca 2ϩ sequestration may be significant in the function of Ca 2ϩ pools. Their role may be to provide a slowly exchangeable sink of Ca 2ϩ in specific subcompartments of the ER. It is also intriguing that the binding of Ca 2ϩ to fatty acids within such membranes may itself confer a signaling role, for example, by modifying the activity of ER membrane channel or pump proteins; thus, the transport of fatty acids into the ER described here may be a means for regulating such control.