Characteristics of the Histamine-sensitive Calcium Store in Vascular Smooth Muscle COMPARISON WITH NOREPINEPHRINE-OR CAFFEINE-SENSITIVE STORES*

Using the microfluorometry of an intracellularly trapped calcium indicator dye, quin2, characteristics of intracellular Ca2+ store sites sensitive to histamine, norepinephrine, or caffeine were investigated using rat vascular smooth muscle cells in primary culture at 25 degrees C. With similar time courses, both histamine- and the norepinephrine-sensitive Ca2+ store sites were readily depleted in Ca2(+)-free medium and almost completely replenished by loading the cells with 1.0 mM Ca2+ solution for 3 min, while the caffeine-sensitive Ca2+ store site was little affected. In the absence of extracellular Ca2+, transient elevations of cytosolic Ca2+ repeatedly appeared in response to repetitive applications of histamine, norepinephrine, or caffeine, with progressive reductions in peak levels. Histamine released Ca2+ from the norepinephrine-sensitive store site and norepinephrine released Ca2+ from the histamine-sensitive one. On the other hand, caffeine had little effect on the histamine- and/or the norepinephrine-sensitive Ca2+ store site in Ca2(+)-free medium, and vice versa. We propose that the location and mechanisms of release of Ca2+ of the histamine-sensitive Ca2+ store site are identical with events at the norepinephrine-sensitive site, and differ from the caffeine-sensitive one, in vascular smooth muscle cells in primary culture.

Ca2+ from the norepinephrine-sensitive store site and norepinephrine released Ca2+ from the histamine-sensitive one. On the other hand, caffeine had little effect on the histamine-and/or the norepinephrine-sensitive Ca2+ store site in Ca2+-free medium, and vice versa. We propose that the location and mechanisms of release of Ca2+ of the histamine-sensitive Ca2+ store site are identical with events at the norepinephrine-sensitive site, and differ from the caffeine-sensitive one, in vascular smooth muscle cells in primary culture.
It is now well established that cellular Ca2+ stores play an important role in the regulation of contractions of vascular smooth muscle (l-4).
However, the characteristics of this store and the mechanism of the Ca*+ release are not well understood.
Whether or not the norepinephrine-sensitive Ca'+ store is identical with the caffeine-sensitive one has remained debatable (4-6). When measuring tension development and Ca*+ fluxes in vascular strips, some workers found that overlap of the norepinephrine-and the caffeine-sensitive Ca2+ stores is not complete (1, 7-ll), while others thought that norepinephrine and caffeine share the same Ca2+ store (12-15).These contradictions mainly relate to the difficulty in directly measuring the levels of cytosolic Ca2+ concentration ([Ca"']J.' In particular, changes in [Ca"'], in the subthreshold levels required for muscle contraction were not directly monitored in these studies. Using quin2-microfluorometry (16-B), we found that the K+ depolarization-sensitive Ca*+ store site overlaps completely with the caffeinesensitive one (18) and that the norepinephrine-sensitive Ca*+ store site does not overlap with the caffeine-sensitive one, in rat aortic vascular smooth muscle cells (VSMCs) in primary culture (19). We also reported that in the absence of extracellular Ca'+, histamine (20) (as do norepinephrine (21) and caffeine (17)) induces a release of Ca" from cellular store sites in VSMCs in primary culture.
In the present study, we characterized the histamine-sensitive Ca2+ store site, compared it with the norepinephrineand the caffeine-sensitive one, and investigated the degree of overlap among these intracellular Ca2+ store sites in VSMCs from rat aorta in primary culture.  (23). It was reported that amounts of intracellular quin2, between 0.7-3 mM, did not alter the cytosolic calcium concentration, and a higher quin2 concentration did not lower the calcium concentration (27). It should be noted, however, that extremely rapid cytosolic calcium transients may be somewhat slowed and/or blunted with this extent of loading of quin2 (26 (27)(28)(29). The extent of the peak levels of [Ca'+], transient induced by histamine was concentrationdependent (p < 0.05 by analysis of variance), and the optimal dose of histamine was lo-" M (Fig. 1D). Ca2+ Release Induced by Norepinephrint-When VSMCs were exposed to norepinephrine in Ca*+-free PSS containing 2 mM EGTA, there was a transient elevation of [Ca2+li, which reached a peak level at 2 min with a duration of 6 min. The and 10e5 M norepinephrine induced a maximum response (Fig. 1, B and E). Ca2+ Release Induced by Caffeine-In Ca*+-free PSS containing 2 mM EGTA, caffeine induced a transient, which reached a peak level at 30 s with a duration of '2 min, and the concentration-dependent (p < 0.05 by analysis of variance) elevation of [Ca*+]i of VSMCs and the optimal dose of caffeine was lo-* M (Fig. 1, C and F Fig. 2 also shows the effect of reexposure to Ca'+-containing media on [Ca2+li of VSMCs after Ca2+ depletion. When VSMCs were incubated with 1.0 mM Ca2+ PSS for 3 min, and then re-exposed to Ca'+-free medium for 1 min prior to the application of histamine after 15 min or longer in Ca'+-free solution, the extent of peak elevation of [Ca"]i by histamine was as large as that observed in VSMCs not exposed to Ca'+-free solution (0 min), and independent of the duration of the first incubation with Ca'+-free medium, as is the case of norepinephrine (19). We suggest that the histamine-and the norepinephrine-sensitive Ca*+ store sites respond differently from the caffeine-sensitive one to Ca'+-free media and Ca2+ repletion.
Effects of Ca2+ Loading on Ca" Store Sites after Depletion by Repetitive Applications of Histamine, Norepinephrine, and Caffeine in Ca2+-free Medium- Fig.  3 shows the effect of Ca" loading on the histamine-, norepinephrine-, and caffeinesensitive Ca*+ store sites after depletion by repetitive applications of these agonists in the absence of extracellular Ca*+. When VSMCs were repeatedly exposed to 10e5 M histamine, low5 M norepinephrine, or lo-' M caffeine in Ca2+-free PSS containing 2 mM EGTA, there were transient [Ca'+]i elevations in response to each application, but the peak levels of the elevations of [Ca'+]i were reduced progressively by each exposure, and the third application of histamine, third application of norepinephrine, and fifth application of caffeine produced little or no response. This suggested that the stored Ca2+ sensitive to each stimuli was completely depleted. When the cells were reincubated with 1.0 mM Ca2+ PSS for 3 min after the depletion of histamine-sensitive stored Ca'+, and then incubated in Ca'+-free PSS for 10 min, histamine induced a near-maximal [Ca'+], elevation (Fig. 3A), an event indicating an almost complete replenishment of Ca'+ at the histamine-sensitive store site. Also in the case of norepinephrine, the norepinephrine-sensitive Ca*+ store site was almost completely replenished (Fig. 3B) (Fig. 3C). Overlap of the Histamine-and the Norepinephrine-sensitive Co'+ Store Sites-At 10 min in Ca'+-free PSS containing 2 mM EGTA, the extent of the elevation of [Ca'+]; induced by the first application of 10m5 M norepinephrine (Fig. 4Aa) was almost equal to that induced by the first application of 10e5 M histamine (Fig. 4Ab) When VSMCs were exposed to 10m5 M norepinephrine after the first application of 10m5 M histamine in Ca'+-free PSS (Fig. 4Ab), the peak level of [Ca'+]: transient was almost equal to that observed when VSMCs were exposed to lo-" M histamine after the first application A, prior to the application of histamine, cells were not exposed to norepinephrine (a), cells were exposed to norepinephrine once (b), twice (c), and three times ( There is a positive slope of 0.90 with a correlation coefficient of 0.99. of 10m5 M histamine (Fig. 4Ac). After the second or further applications of 10m5 M histamine, 10e5 M norepinephrine induced little or no Ca2+ release ( Fig. 4Ac and d), although 10e5 M norepinephrine did produce an elevation of [Ca'+]: at the same incubation time in Ca2+-free solution, without the pretreatment of histamine (Fig. 2).
To estimate the amount of Ca2+ released with each treatment, the area bound by the fluorescence trace and the steadystate base line in Ca'+-free media was measured using a computerized manipulator (Houston Instrument). The calculated area was expressed as "fluorescence (F) X minute (min)," indicating relative amounts of Ca'+ transiently re- leased from intracellular store sites (18). As shown in Fig. 4B, the relative changes in [Ca'+]i, estimated from "fluorescence x minutes," induced by norepinephrine after (n -1) time application (1 < n < 3) of histamine was almost equal to that observed during n time application of histamine. On the other hand, when VSMCs were repeatedly exposed to 10e5 M norepinephrine in Ca'+-free PSS containing 2 mM EGTA, the extent of the subsequent release of Ca2+ induced by histamine was progressively reduced with the increasing number of previous exposures to norepinephrine (Fig. 5,Aad). As shown in Fig. 5B, the relative changes in [Ca2+lL, induced by histamine after (n -1) time exposure (1 < n < 3) to norepinephrine was almost equal to that observed during n time application of norepinephrine. Thus, the histaminesensitive Ca*+ store site almost completely overlaps the norepinephrine-sensitive one. Histamine-and Norepinephrine-sensitive Ca2+ Store Sites Differ from Caffeine-sensitive Ones-When VSMCs were repeatedly exposed to 10m5 M histamine in Ca*+-free PSS containing 2 mM EGTA, the third application produced little or no Ca2+ release, thereby indicating that histamine-sensitive stored Ca2+ was almost completely depleted. However, the subsequent application of 10m5 M caffeine induced a transient [Ca'+]; elevation and the peak level of the [Ca'+]i transient was almost equal to that induced by 10e5 M caffeine with the same incubation time (40 min, 30 s) in Ca*+-free solution and without the pretreatment of histamine (Fig. 6A). Conversely, when VSMCs were repeatedly exposed to lo-' M caffeine in Ca'+-free PSS containing 2 mM EGTA, the fifth application produced little or no response, thereby indicating the complete depletion of caffeine-sensitive stored Ca*+ (Fig. 6B). The subsequent application of 1Om5 M histamine produced an elevation of [Ca'+],, and the extent of [Ca2+li was almost equal that observed during the first application of 10m5 M histamine for the same duration of incubation (31 min) in Ca*+-free PSS, without the pretreatment of caffeine. Thus, it is suggested that the histamine-sensitive Ca2+ store site differs from the caffeine-sensitive one. In the case of norepinephrine, we obtained results similar to those seen with histamine, in comparison with caffeine and as reported (19). This would suggest that the norepinephrinesensitive Ca2+ store site also differs from the caffeine-sensitive one.

DISCUSSION
In the present study, using the quin2-microfluorometry of intact VSMCs of rat aortic media in primary culture, we obtained evidence that Ca2+ in the histamine-and the norepinephrine-sensitive store sites is readily depleted in Ca'+free solution and is readily replenished by loading with 1.0 mM Ca2+ PSS, whereas Ca2+ in the caffeine-sensitive store site is little affected. Histamine induced a release of all the stored Ca2+ in the norepinephrine-sensitive store site, and norepinephrine released Ca2+ from the histamine-sensitive store. However, histamine and norepinephrine could not release Ca2+ from the caffeine-sensitive store site, and caffeine could not release Ca2+ from the histamine-and norepinephrine-sensitive Ca2+ store. We reported that there is a complete overlap of the caffeine-and K' depolarization-sensitive intracellular Ca*+ store sites (18) and that the norepinephrinesensitive intracellular Ca2+ store differs from the caffeinesensitive one in VSMCs of rat aorta in primary culture (19). The present findings clearly show that the histamine-sensitive Ca2+ store site completely overlaps with the norepinephrine-sensitive one and differs from the caffeine-sensitive one in VSMCs in primary culture.
The present study shows the advantages of the quin2microfluorometry of [Ca2+li over the estimation of [Ca"], changes by tension measurement, when attempting to determine the characteristics of the intracellular Ca*+ store site in VSMCs. For example, the peak levels of [Ca2+li transients induced by the second or more applications of histamine or norepinephrine and also those induced by histamine or norepinephrine after five repetitive pretreatments of caffeine were lower than the [Ca*+]i level in cells in normal PSS containing 5 mM K+ and 1 mM Ca'+. Thus, using quin2microfluorometry, changes in [Ca'+], below the resting cell levels can be recorded, namely, changes in [Ca*+]; in the subthreshold levels required for the contractile response. Repetitive applications of histamine and norepinephrine induced a progressive reduction in the amount of Ca2+ released from the intracellular store site in VSMCs in Ca'+-free PSS. The possibility that desensitization to histamine and norepinephrine occurs during repetitive treatments with these agonists can be excluded by the finding that histamine and norepinephrine induced a near-maximal release of Ca*+ after repletion of Ca'+, by replacement of Ca'+-free medium with 1.0 mM Ca'+-PSS at 3 min after repetitive treatments with agonists in Ca'+-free solution.
With regard to the source of Ca2+ released by norepinephrine, there is no general agreement. Grover et al. (9) suggested that the norepinephrine-sensitive stored Ca2+ is a component of bound Ca*+ on the plasma membrane of rabbit aortic smooth muscle. Daniel (5) proposed that there is a norepinephrine-sensitive Ca*+ store in the sarcoplasmic reticulum near the plasma membrane or at the inner aspect of the plasma membrane. Our findings that Ca2+ in the histamine/ norepinephrine-sensitive store is readily depleted and replenished may indicate that the site is located in close proximity to the cell surface. It is also possible that this site may have communication with extracellular milieu, and may easily lose and accumulate Ca*+ during depletion and repletion, respectively. Conversely, Ca*+ in the caffeine-sensitive store was hardly depleted and replenished during depletion and repletion of extracellular Ca*+, respectively suggesting that the caffeine-sensitive store might be located in the central part of the cell and/or communication with extracellular milieu may be inadequate.
It is also possible that prompt Ca*' depletion and repletion of histamine/norepinephrine-sensitive Ca2+ store may be related to their close proximity of location to cell surface rather than to their primary characteristics. By measuring the tension development of vascular smooth muscle in Ca*+-free PSS containing EGTA, Guan et al. (30) found that "EGTA in Ca*+-free medium may remove the superficially bound Ca*+ and subsequently reduce the intracellular Ca*+ pool via extraction of the intracellular Ca'+ at the cell membrane surface. This would lead to destabilization of the cell membrane and to an increased permeability to the subsequently added Ca*+ to refill the receptor-released intracellular Ca2+ pool." Saida and van Breemen (11) noted in tension studies of the rabbit mesenteric artery that norepinephrine releases Ca*+ from a store near the receptor, which diffuses to the caffeinesensitive store to release Ca*+ through the Ca*+-induced Ca2+ release mechanism. This occurs when [Ca*+]i exceeds 2 x 10m6 M (31). Using rat aortic VSMCs in primary culture, Yamamoto and van Breemen (32) noted that there was no significant decrease in the caffeine-releasable content in the range 3 X 10e6 to 1 x 10m5 M free Ca*+ concentrations and that there was a decrease in Ca2+ content of the sarcoplasmic reticulum at 1 X 10d4 M Ca2+. Thus, the threshold for the (norepinephrine/histamine-released) Ca*+-induced Ca'+ release from caffeine-sensitive Ca2+ store is very much higher than the Ca2+ concentration obtained by the application of 1O-5 M histamine in the absence of extracellular Ca'+, as it was in the present study. It may be that the Ca"-induced Ca*+ release is not actively involved in the mechanism of Ca*+ release from the sarcoplasmic reticulum in VSMCs because the threshold Ca2+ concentration would be too high for physiological conditions. However, since primary cultured smooth muscle cells, albeit retaining most of their original characteristics, may be modified to some extent during cultivation, no conclusions can be drawn concerning the importance of this mechanism in intact VSMCs, under physiological conditions. On the other hand, Bond et al. (12) and Kowarski et al. (15) reported that using electron probe x-ray microanalysis, norepinephrine releases Ca*+ from both the junctional (near the plasma membrane) and internal (central) sarcoplasmic reticulum. With regard to the mechanism of Ca*+ release from the internal (central) sarcoplasmic reticulum, Somlyo (4) proposed several possible mechanisms, including diffusion of Ca*+ from the internal to the junctional sarcoplasmic reticulum through luminal communications, the release of Ca2+ mediated by a chemical transmitter such as inositol 1,4,5trisphosphate and change in Ca*+ permeability propagated from the junctional to the internal sarcoplasmic reticulum. In the present study, there were differences not only in the rates of Ca2+ depletion and replenishment but also in the mechanism of Ca2+ release between the histamine/norepinephrinesensitive Ca2+ store site and the caffeine-sensitive Ca2+ store site. Histamine and norepinephrine had little effect on the caffeine-sensitive Ca*+ store site, and caffeine had little effect on the histamine/norepinephrine-sensitive Ca*+ store site, in Ca*+-free medium. We reported that a GTP-binding protein, a pertussis toxin substrate, couples the cu,-adrenoceptor and the H1 histamine receptor to mediate the release of intracellular Ca2+ and that this protein is not involved in the caffeineinduced Ca*+ release in VSMCs (33). These results are compatible with the idea that the mechanism of Ca*+ release from the histamine/norepinephrine-sensitive Ca2+ store site differs from that of the caffeine-sensitive one. Thus, there may be no overlap between the histamine/norepinephrine-sensitive Ca2+ store site and the caffeine-sensitive one. However, it has to be noted that these results were obtained in the absence of extracellular Ca'+ and that norepinephrine or histamine may release Ca*+ from the caffeine-sensitive store in an indirect manner when extracellular Ca"+ is present. In conclusion, our observations suggest that the histamineand the norepinephrine-sensitive Ca" store sites completely overlap, and the histamine/norepinephrine-sensitive Ca2+ store site does not overlap with the caffeine-sensitive Ca2+ store site. These two Ca*+ store sites also have different properties for Ca*+ leakage and accumulation during Ca*+ depletion and repletion, respectively.