Cell type-specific modes of feedback regulation of capacitative calcium entry.

The Ca2+-ATPase inhibitor, thapsigargin, activated Ca2+ entry into pancreatic acinar cells, a process known as capacitative calcium entry. In cells loaded with the calcium chelator BAPTA, the transient Ca2+ release was blunted and the rise of [Ca2+]i on readdition of Ca2+ was slowed. However, the steady-state [Ca2+]i due to Ca2+ entry was substantially augmented compared with control cells. This indicates that [Ca2+]i exerts a negative feedback on Ca2+ entry from a compartment buffered by BAPTA and separated from the bulk of cytoplasmic Ca2+. This interaction probably occurs close to the calcium channel where [Ca2+] is higher than in the bulk of the cytoplasm. In support of this interpretation, the slower Ca2+ chelator, EGTA, also blunted the release of Ca2+ and slowed the rise of the sustained [Ca2+]i phase but failed to augment steady-state [Ca2+]i. In contrast, Ca2+ entry in NIH 3T3 cells was characterized by a transient rise of [Ca2+]i that decays to near prestimulus levels. This decay in Ca2+ entry also results from negative feedback by Ca2+ because the decrease in Ca2+ entry was reversed by incubation in a Ca2+-deficient medium. However, unlike its effects in acinar cells, BAPTA neither augmented steady-state [Ca2+]i nor prevented the inactivation of entry. Rather, in BAPTA-loaded cells, [Ca2+]i failed to increase substantially suggesting that negative regulation by Ca2+ may occur at a site distinct from the cytoplasmic compartment and inaccessible to cytoplasmic BAPTA. These two distinct types of feedback behavior may indicate subtypes of store-operated calcium channels expressed in different cells or a single type of channel which is differentially regulated in a cell type-specific manner.

gargin can activate Ca 2ϩ influx without changes in inositol phosphate levels (3,4). This entry of Ca 2ϩ is presumed to occur through Ca 2ϩ channels in the plasma membrane, termed "store-operated channels" (5,6), although the existence of such channels has not been proven unequivocally. Electrophysiological studies have described inward Ca 2ϩ currents (I CRAC ) that are activated by intracellular Ca 2ϩ pool depletion (7)(8)(9)(10) and which may reflect the activity of these store-operated channels.
The underlying mechanisms that link internal Ca 2ϩ pool depletion to the activation of Ca 2ϩ entry are not yet understood. However, recent data indicate that this mode of Ca 2ϩ entry can be regulated by Ca 2ϩ itself. Several studies have demonstrated that intracellular Ca 2ϩ can feed back to inhibit Ca 2ϩ influx current on two different time scales. Rapid inactivation of the inward current associated with capacitative calcium entry occurs on a millisecond time scale (8) and is believed to involve an action of cytoplasmic Ca 2ϩ close to the mouth of the Ca 2ϩ channel, 3-4 nm from the mouth of the pore (10). There is also a relatively slow inactivation process in Jurkat cells occurring over tens of seconds, but which also appears to be Ca 2ϩ -dependent (11).
While the majority of evidence for negative feedback by Ca 2ϩ has come from patch-clamp studies, a Ca 2ϩ -dependent regulation of Ca 2ϩ entry has also been proposed from steady-state measurements of [Ca 2ϩ ] i with fluorescent indicators. Missiaen et al. (12) demonstrated that, in HeLa cells, Ca 2ϩ entry inactivates with a time course of tens of seconds giving rise in some cells to slow [Ca 2ϩ ] i oscillations. The relationship of this slow inactivation of Ca 2ϩ entry to the [Ca 2ϩ ] i -dependent inactivation of Ca 2ϩ current is not known. In addition, it is not known whether these mechanisms are intrinsic to capacitative calcium entry in all instances or represent cell-specific modes of regulation. In this study, we have monitored thapsigargininduced capacitative Ca 2ϩ entry in intact cells with the fluorescent indicator, fura-2, and have perturbed intracellular Ca 2ϩ gradients by loading or injecting the cells with Ca 2ϩ chelators. Our findings confirm the existence of at least two important Ca 2ϩ -dependent negative feedback mechanisms, one which involves rapid effects of cytoplasmic Ca 2ϩ on Ca 2ϩ entry channels and a second slower mechanism which appears to depend on Ca 2ϩ acting at an extracellular site or at a site within the calcium channel. Our findings also indicate that these mechanisms do not represent an intrinsic property of capacitative calcium entry in all cell types but rather appear to be differentially expressed in a cell type-specific manner.

MATERIALS AND METHODS
Preparation of Rat Pancreatic Cells-Pancreatic acinar cells were prepared essentially as described previously (13). Briefly, excised pancreata from 75-100-g rats were distended by injection of a HEPESbuffered physiological saline solution (HPSS; composition, in mM: 140 NaCl, 5 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES, 10 glucose, 0.1% bovine serum albumin, pH 7.4), supplemented with 10 mM pyruvate and 0.02% soybean trypsin inhibitor (Sigma). After mincing the tissue, the acinar * 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 U.S.C. Section 1734 solely to indicate this fact. cells were liberated by incubation with collagenase (3 mg/10 ml; Sigma) for 10 min at 37°C. Following collagenase digestion, the pancreatic cells were washed and suspended in Dulbecco's modified Eagle's medium (DMEM), 2 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The isolated cells were then allowed to attach to glass coverslips coated with Matrigel (Collaborative Biomedical Products).
Preparation of Mouse Lacrimal Cells-Lacrimal acinar cells were prepared essentially as described previously (14). Briefly, lacrimal glands, excised from 6 mice (30 -40 g), were finely minced, suspended in DMEM, and then incubated with trypsin (0.25 mg/10 ml; Sigma) for 1 min in 37°C. The trypsin was removed by centrifugation, followed by a 5-min incubation with soybean trypsin inhibitor (2 mg/10 ml; Sigma), in the presence of 2.5 mM EGTA at 37°C. Finally, acinar cells were isolated following incubation with collagenase (4 mg/10 ml; Boehringer Mannheim) for 10 min at 37°C. The digested tissue was then washed and suspended in DMEM, supplemented with 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Subsequently, the isolated cells were allowed to attach to glass coverslips coated with Matrigel (Collaborative Biomedical Products).
NIH 3T3 Cell Culture-NIH 3T3 cells were maintained at 37°C and 5% CO 2 in DMEM containing 10% fetal bovine serum, 5 mM glutamine, 50 units/ml penicillin, and 50 units/ml streptomycin. After 3 days culture, the cells were passed at a dilution of 1:10. In preparation for experiments, cells were plated on glass coverslips 2 days before use.
Cellular Loading of the Fluorescent Ca 2ϩ Indicator fura-2-In all cases, cell-attached coverslips were mounted in a Teflon microscope chamber (Bionique) before dye loading. NIH 3T3 cells were incubated in DMEM containing 1 M fura-2/AM (Molecular Probes) for 15 min at 37°C, whereas pancreatic and lacrimal cells were incubated in DMEM containing 1.6 M fura-2/AM for 30 min at room temperature. After dye loading, cells were washed and bathed in HPSS and incubated at room temperature for an additional 20 min before Ca 2ϩ measurements were made to ensure complete hydrolysis of cellular fura-2/AM. In some experiments (indicated below), following the fura-2 loading procedure, cells were further incubated with either BAPTA/AM (50 -100 M) and/or EGTA/AM (500 M) for 20 min at room temperature.
Fluorescence Measurements-The fluorescence of fura-2-loaded cells was monitored with a photomultiplier-based detection system, mounted on a Nikon Diaphot inverted microscope equipped with a Nikon 40x (1.3 numeric aperture) Neofluor objective. The fluorescence light source was provided by a Deltascan D101 (Photon Technology International Ltd.), equipped with a light path chopper and dual excitation monochromators. The light path chopper enabled rapid interchange between two excitation wavelengths (340 nm and 380 nm), and a photomultiplier tube monitored the emission fluorescence at 510 nm, selected by a barrier filter (Omega Optical). All experiments were carried out at room temperature. Calibration and calculation of [Ca 2ϩ ] i were carried out as described previously (15).
Cell Microinjection-Pancreatic, lacrimal, and NIH 3T3 cells were microinjected with 2 mM fura-2 solution (in H 2 O) via a glass micropipette attached to WPI PV830 Picopump (World Precision Instruments, New Haven, CT). In some experiments, the injection solution also contained 250 mM BAPTA or 250 mM EGTA.
Statistical Analysis-Statistical significance was determined by analysis of variance or by Student's t test where appropriate. The level of significance (p) was 0.05.

RESULTS
Receptor-activated Ca 2ϩ mobilization involves two phases: Ca 2ϩ release from intracellular stores and extracellular Ca 2ϩ entry (16). In the presence of 1.8 mM extracellular Ca 2ϩ , the addition of 2 M thapsigargin to pancreatic acinar cells produced an elevation in [Ca 2ϩ ] i that was sustained or slowly falling and which remained above baseline for a period of at least 30 min (Fig. 1A). Addition of thapsigargin to fura-2-loaded pancreatic acinar cells incubated in a Ca 2ϩ -free medium caused a transient increase of [Ca 2ϩ ] i which was followed on restoration of external Ca 2ϩ by an elevated and sustained [Ca 2ϩ ] i signal (Fig. 1A). This biphasic [Ca 2ϩ ] i signal induced by the Ca 2ϩ -ATPase inhibitor, thapsigargin, is diagnostic of cal-cium entry linked to the depletion of intracellular calcium stores or capacitative calcium entry (4,17).
In previous electrophysiological studies (8 -10), a hallmark of the regulation by [Ca 2ϩ ] i of Ca 2ϩ entry was the ability of intracellular Ca 2ϩ chelators to augment inward Ca 2ϩ current. In this study, we investigated the effects of intracellular BAPTA and EGTA on the thapsigargin-induced Ca 2ϩ entry in pancreatic cells. In most experiments, the Ca 2ϩ chelators were loaded into cells by incubation with their acetoxymethyl ester derivatives as described under "Materials and Methods." In pancreatic acinar cells loaded with BAPTA (50 M BAPTA/AM for 20 min), the transient release of Ca 2ϩ (observed in control cells after the addition of thapsigargin in a calcium-free medium) was blunted (Fig. 1B). On restoring extracellular Ca 2ϩ , [Ca 2ϩ ] i rose at a rate of rise substantially slower than in control cells. The slower rate of rise in the presence of BAPTA is expected, since additional Ca 2ϩ entry is required to overcome the additional Ca 2ϩ buffering capacity of the cell before a steady-state elevation of [Ca 2ϩ ] i is observed. Unexpectedly, however, the resulting steady-state [Ca 2ϩ ] i due to Ca 2ϩ entry was significantly higher in BAPTA-loaded cells compared to control cells (Fig. 1B).
This apparent augmentation of Ca 2ϩ entry was found to depend on the species of intracellular Ca 2ϩ buffer. Pancreatic acinar cells were loaded with even greater concentrations of the Ca 2ϩ chelator, EGTA (500 M EGTA/AM, 20 min), and treated with thapsigargin; however, steady-state [Ca 2ϩ ] i was not augmented (Fig. 1C). As observed with BAPTA-loaded cells, EGTA loading blunted the transient release of stored Ca 2ϩ in the absence of external Ca 2ϩ , and, following the restoration of external Ca 2ϩ , the initial rate of Ca 2ϩ entry was slower than in control cells (0.045 Ϯ 0.015 versus 0.81 Ϯ 0.11 nM/s, respectively). To ensure that the difference in effects of these two chelators was not a result of nonspecific inhibition of Ca 2ϩ entry by EGTA, pancreatic cells were loaded with both BAPTA and EGTA (50 M BAPTA/AM and 500 M EGTA/AM, 20 min); these cells responded to thapsigargin with an augmented Ca 2ϩ entry as if they had been loaded with BAPTA alone (Fig. 1C).
We assume that the loading of EGTA into the cytoplasm of the cells was at least comparable to BAPTA or likely even greater since EGTA was applied at a higher concentration than BAPTA. However, as this cannot be know for certain, we also carried out a series of experiments in which the pancreatic cells were microinjected with the free acid forms of these compounds (pipette concentration of BAPTA or EGTA was 250 mM, final cytoplasmic concentration of chelators is estimated as 2.5-5 mM (18)). As shown in Fig. 1D, the effects of the microinjected chelators were essentially the same as those observed using the acetoxymethyl ester loading. The summarized results from all experiments carried out with the protocols shown in Fig. 1 are given in Table I.
The different actions of BAPTA and EGTA are not likely due to differences in their affinities for Ca 2ϩ , since the K D for BAPTA (192 nM) is actually somewhat greater than for EGTA (67 nM). Rather, the differential effects of BAPTA and EGTA on Ca 2ϩ entry probably result from the faster calcium binding kinetics of BAPTA (6 ϫ 10 8 M Ϫ1 s Ϫ1 versus 1.5 ϫ 10 6 M Ϫ1 s Ϫ1 for EGTA). The ability of BAPTA to enhance the steady-state Ca 2ϩ entry would seem to reflect the rapid negative feedback on Ca 2ϩ entry seen in earlier patch-clamp experiments (8,10). Because this occurs in the face of an elevated level of Ca 2ϩ in the cytoplasm, it must result from an action of BAPTA in a compartment separated to some degree from the bulk of cytoplasm. In all likelihood, this interaction would occur at a site close to the mouth of the calcium channel where the [Ca 2ϩ ] is normally maintained higher than the bulk of the cytoplasm by rapid entry through the channel. Consistent with this interpretation, the slower Ca 2ϩ chelator, EGTA, mimicked the effect of BAPTA on the transient release of Ca 2ϩ and on the rate of rise of the sustained [Ca 2ϩ ] i phase, but EGTA failed to augment the steady-state [Ca 2ϩ ] i level (Fig. 1, C and D). That is, the [Ca 2ϩ ] gradient which is responsible for the negative feedback is maintained by diffusion of Ca 2ϩ through the channel at a rate too rapid to be buffered by the slower chelator, EGTA.
We next examined the effects of BAPTA loading on two other cell types to determine if a similar mode of negative feedback could be detected. We chose mouse lacrimal acinar cells and NIH 3T3 fibroblast cells, because the former display large, sustained elevations in [Ca 2ϩ ] i due to capacitative calcium entry ((15) and Fig. 2), while the latter exhibit a [Ca 2ϩ ] i entry signal which approaches base-line with prolonged activation (Fig. 3). As seen in pancreatic acinar cells, in lacrimal acinar cells loaded with BAPTA (100 M BAPTA/AM for 20 min), the thapsigargin-induced transient [Ca 2ϩ ] i release was abolished, and the time to steady-state [Ca 2ϩ ] i on addition of extracellular Ca 2ϩ was also prolonged compared with controls. Also similar to pancreatic cells, BAPTA-loaded lacrimal cells had a significantly greater steady-state [Ca 2ϩ ] i level, compared with control cells (Fig. 2 and Table II)   [Ca 2ϩ ] i but did not augment the sustained Ca 2ϩ -entry phase compared with control cells (Fig. 2). Microinjection of the calcium chelators gave results similar to AM-loading. Summarized data from the lacrimal acinar cell experiments are presented in Table II. The similarity between pancreatic and lacrimal cells in the effects of BAPTA and EGTA on Ca 2ϩ entry suggests the presence of similar Ca 2ϩ -dependent feedback mechanisms in these two cell types.
In comparison to the two acinar cell types described thus far, NIH 3T3 cells exhibit a strikingly different pattern of Ca 2ϩ entry in response to thapsigargin. As shown in Fig. 3, the response of NIH 3T3 cells to thapsigargin in the presence of extracellular Ca 2ϩ is characterized by a transient rise in [Ca 2ϩ ] i that decays over a period of minutes to a level close to the prestimulus level. A small entry of Ca 2ϩ is maintained, however, because when external Ca 2ϩ is removed, [Ca 2ϩ ] i decreases slightly ( Fig. 3; but not in untreated cells, not shown). In contrast, when the cells are first activated by thapsigargin in a Ca 2ϩ -deficient medium, and then Ca 2ϩ is restored extracellularly, a substantial Ca 2ϩ entry response is revealed. Unlike the case for the acinar cells, this entry is transient and decays over a period of minutes returning close to the prestimulus levels. This inactivation is dependent on the presence of Ca 2ϩ , as shown by the results in Fig. 4. Following the decay of the Ca 2ϩ entry response, incubation of the 3T3 cells in a Ca 2ϩdeficient medium can partially reverse this inactivation, and the extent of this reversal depends on the duration of incubation in Ca 2ϩ -deficient medium. The response recovers with a half-time of about 1 min, a rate similar to the rate of decline of the [Ca 2ϩ ] i signal in the presence of Ca 2ϩ . Note that approximately 25% of the response cannot be restored by this procedure. This is not simply due to prolonged incubation in Ca 2ϩfree medium, or nonspecific "run-down" of the preparation, because responses to Ca 2ϩ addition at 2000 s are not significantly different from those at 1000 s, unless an intervening exposure to Ca 2ϩ occurs at 1000 s. These data are shown in Fig.  5 and indicate that this essentially irreversible component of inactivation also appears to be dependent on previous exposure to Ca 2ϩ .
The reversible and Ca 2ϩ -dependent decline in steady-state [Ca 2ϩ ] i could result from a Ca 2ϩ -dependent modulation of either Ca 2ϩ entry or Ca 2ϩ efflux. To distinguish between these possibilities, we examined Ca 2ϩ -dependent changes in the entry of Ba 2ϩ as a surrogate for Ca 2ϩ . Ba 2ϩ is known to permeate the Ca 2ϩ channels involved in capacitative Ca 2ϩ entry (19,20), but is a poor substrate for Ca 2ϩ -ATPases (19 -22). Fig. 6 depicts the experimental protocol: after the Ca 2ϩ stores of NIH 3T3 cells were depleted by 2 M thapsigargin, they were bathed in a medium containing 10 mM Ba 2ϩ for 100 s and subsequently returned to a nominally Ca 2ϩ -free medium; a second addition of 10 mM Ba 2ϩ followed 400 s later. These additions of Ba 2ϩ resulted in a rising fura-2 ratio indicative of Ba 2ϩ entry, and, as shown in the inset to Fig. 6, in control cells the rate of Ba 2ϩ entry during the second exposure was similar to that during the first. However, if during the period between the two test exposures to Ba 2ϩ the cells were incubated in a Ca 2ϩ -containing medium, then the rate of Ba 2ϩ entry at the second exposure to Ba 2ϩ was significantly reduced. This indicates that the exposure to Ca 2ϩ inactivates divalent cation entry, and that the decline in steady-state [Ca 2ϩ ] i in 3T3 cells results at least in part from inactivation of Ca 2ϩ influx.
Although apparently much slower, such an inactivation could reflect the same, or a similar, mechanism to the one seen with pancreatic and lacrimal cells. As with pancreatic and lacrimal cells, loading of 3T3 cells with BAPTA blunted the transient rise of [Ca 2ϩ ] i . However, in contrast to pancreatic and   (Fig. 7, Table III). Microinjection of BAPTA (pipette concentration, 250 mM) into NIH 3T3 cells produced the same result as loading with BAPTA/AM (Table III).

DISCUSSION
The addition of a high concentration of a fast calcium buffer to the cytoplasm of acinar cells resulted in an elevated steadystate [Ca 2ϩ ] i in response to thapsigargin. It is likely that this effect is due to the dissipation of a standing [Ca 2ϩ ] i gradient in the cell that inhibits the activity of Ca 2ϩ influx channels; the most likely locus for such a gradient would be the mouth of the channel where rapid inward flow of Ca 2ϩ ions can maintain the concentration of Ca 2ϩ higher than in the bulk of the cytoplasm. It is also likely that this is a manifestation of the same phenomenon described in patch-clamp studies of mast cells by Hoth and Penner (8) and Jurkat cells by Zweifach and Lewis (10). These investigators reported a rapid inactivation of inward calcium current in calcium store-depleted cells which was reversed by BAPTA but not by the slower chelator, EGTA. In principle, it would be possible for the results in the current study to be ascribed to a diminution of Ca 2ϩ extrusion at the plasma membrane; however, a steady-state gradient due to active extrusion would produce a localized [Ca 2ϩ ] lower than the bulk of cytoplasm. Thus, the ability of BAPTA to dissipate standing Ca 2ϩ gradients would result, if anything, in an increase in the concentration of Ca 2ϩ in the vicinity of Ca 2ϩ extrusion sites, and this would be expected to increase rather than reduce the rate of Ca 2ϩ efflux.
Zweifach and Lewis (11) have also described a slow inactivation of depletion-activated current in Jurkat cells. Because of the experimental protocol used by Zweifach and Lewis (11), the slow inactivation was due in part, but not wholly, to reuptake of Ca 2ϩ into intracellular stores. Zweifach and Lewis (11) also reported that the slower calcium chelator, EGTA, diminished this inactivation leading them to conclude that the site of this feedback was not close to the mouth of calcium channel but was located somewhere within the major cytoplasmic compartment. In the present study, in NIH 3T3 cells, we observed a similarly slow inactivation of calcium entry that was dependent of the presence of Ca 2ϩ in the incubation medium. However, in the presence or absence of excessive intracellular Ca 2ϩ buffering, thapsigargin induced an elevated steady-state [Ca 2ϩ ] i that was only about 20 nM greater than the base-line, prestimulus [Ca 2ϩ ] i level. While one could envision microdomains of [Ca 2ϩ ] i much higher than this level causing inactivation of calcium entry in control cells, this would not be expected to occur in the cells loaded with high concentrations of BAPTA. It is thus difficult to understand how such a minor elevation in [Ca 2ϩ ] i could maintain almost complete inhibition of Ca 2ϩ entry in the 3T3 cells. Rather, we conclude that the control by Ca 2ϩ must occur at a site readily accessible to extracellular Ca 2ϩ , but inaccessible to cytoplasmic BAPTA. This could be either a site on the extracellular domain of the Ca 2ϩ channel which is inhibited by extracellular Ca 2ϩ when it is in the activated state or possibly a site within the channel pore inaccessible to BAPTA in the cytoplasm. Our results do not readily distinguish between these possibilities.
Regardless of the underlying mechanism, the lack of an effect of BAPTA on the steady-state [Ca 2ϩ ] i and the transient nature of the capacitative Ca 2ϩ entry in NIH 3T3 cells suggest that a mechanism of negative regulation, distinct from that . NIH 3T3 cells were treated with thapsigargin and calcium entry was assessed by addition of 1.8 mM Ca 2ϩ at t ϭ 1,000 s (black bar) and, at t ϭ 1500 s, the extracellular medium was changed to a Ca 2ϩ -deficient medium in which the cells remained for 500 s. A second challenge with 1.8 mM Ca 2ϩ at t ϭ 2,000 s promoted an influx which was inhibited. In a second series of experiments, cells were treated with thapsigargin in the same manner but remained in the Ca 2ϩ -deficient medium for 2,000 s (i.e. the first addition of Ca 2ϩ at 1,000 s was omitted). Addition of 1.8 mM Ca 2ϩ resulted in an influx which was greater than the response at 2,000 s in cells which had been pretreated with Ca 2ϩ at 1,000 s and was not significantly different from the Ca 2ϩ entry measured at 1,000 s. observed in pancreatic and lacrimal cells, accounts for the reduced [Ca 2ϩ ] i level. Thus, there appear to be at least two different mechanisms for regulation of Ca 2ϩ entry by Ca 2ϩ , and, if one considers the Ca 2ϩ -dependent irreversible inactivation shown by the results in Fig. 4D and Fig. 5, there may be three. It is the effect of BAPTA on Ca 2ϩ entry that clearly distinguishes the action of Ca 2ϩ in 3T3 cells from that in acinar cells. In the case of 3T3 cells, we considered that the slow rate of inactivation and recovery might indicate a process of Ca 2ϩdependent covalent modification, perhaps by phosphorylation, of the channel or a protein regulating it. However, we have been unable to obtain pharmacological evidence for such regulation; pretreatment (15 min at room temperature) with the following modifiers of kinase or phosphatase activity failed to prevent the slow inactivation of Ca 2ϩ entry in 3T3 cells (data The appearance of these distinct modes of Ca 2ϩ entry regulation may indicate that the different cell types express distinct subtypes of capacitative calcium entry channels. Alternatively, the channels may be regulated differently because the cell types express different kinases or other Ca 2ϩ -dependent regulatory proteins. Until the capacitative Ca 2ϩ channel is isolated, cloned, and sequenced, these possibilities cannot be tested. Ca 2ϩ -dependent inactivation of Ca 2ϩ channels occurs in a variety of cells and is likely of physiological importance; this negative feedback loop will limit the rise of [Ca 2ϩ ] i in activated cells and thus provide a mechanism to limit the amplitude of Ca 2ϩ signals. Furthermore, it is possible that the distinct modes of regulation occurring in different cell types could be exploited to develop pharmacological agents with cell typespecific actions. FIG. 7. Effect of BAPTA on thapsigargin-induced Ca 2؉ entry in single NIH 3T3 cells. Using a procedure similar to that described for pancreatic cells in Fig. 1, the effects of BAPTA (BAPTA/AM, 100 M) on thapsigargin-induced entry in NIH 3T3 cells was examined. In the trace labeled Control, the addition of extracellular Ca 2ϩ (1.8 mM) resulted in an elevated [Ca 2ϩ ] i which decreases with time toward prestimulus levels (n ϭ 8). In the trace labeled BAPTA, the thapsigargininduced Ca 2ϩ entry is not augmented by BAPTA loading, but almost completely blocked, and the slight elevation in steady-state [Ca 2ϩ ] i was not affected by BAPTA when compared with control cells (n ϭ 8).  6. Inhibitory effect of external Ca 2؉ on Ba 2؉ influx. NIH 3T3 cells were treated with 2 M thapsigargin in nominally Ca 2ϩ -free medium. Ca 2ϩ influx was assessed by adding 10 mM Ba 2ϩ for 100 s as indicated to obtain a measure of Ba 2ϩ influx. The extracellular medium was then either restored to a Ca 2ϩ -deficient medium (control, dashed line) or changed to a medium containing 1.8 mM Ca 2ϩ (solid line). Approximately 400 s later, the solution was changed to a Ca 2ϩ -deficient medium containing 10 mM Ba 2ϩ . The second rate of Ba 2ϩ influx was measured and expressed as a percentage of the first rate of Ba 2ϩ influx in each tracing. The data averaged from 5-7 such experiments are summarized in the inset.