1Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China; and 2Department of Medicine, University of California, San Francisco, California 94143
Submitted 7 May 2002 ; accepted in final form 8 September 2003
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ABSTRACT |
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anion; inositol 1,4,5-trisphosphate; Ca2+ release
ANG II is a potent vasoconstrictor and growth factor in vascular smooth muscle (26). The renin-angiotensin system plays an important role in the regulation of blood pressure. Activity of the renin-angiotensin system is modulated by various factors, including Cl. Elevated extracellular Cl concentration ([Cl]e) may stimulate the renin-angiotensin system by acting directly on juxtaglomerular cells to stimulate renin release (10, 23). Cl also increases the activity of angiotensin-converting enzyme (21). In a Cl-sensitive hypertensive model described by Tanaka et al. (25), it has been found that supplemental dietary KCl exacerbated hypertension, and supplemental potassium citrate or bicarbonate attenuated hypertension in the stroke-prone spontaneously hypertensive rat (SHR). Interestingly, elevated plasma renin activity was associated with exacerbated elevation of blood pressure in this rat model with a high-Cl diet (25). Thus enhanced ANG II conversion may further increase peripheral resistance. In addition, ANG II-induced vasoconstriction of the resistant arterioles may be further enhanced by Cl (9) and contribute to the Cl-sensitive hypertension. Although ANG II has been considered as a paradigm of receptor signaling in the vasculature, it is not known how Cl enhances ANG II-induced vasoconstriction.
The mechanism by which receptor activation entrains contraction of vascular smooth muscle cells (VSMC) involves a transient increase in the intracellular concentration of Ca2+ ([Ca2+]i) (12). ANG II stimulates phospholipase C (PLC) which acts on phosphatidyl inositol to release inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Binding of Ins(1,4,5)P3 to a specific receptor on the sarcoplasmic reticulum releases stored Ca2+ into cytoplasm (12). The released Ca2+ may activate a Ca2+-sensitive Cl channel on the plasma membrane that sequentially entrains Cl efflux and membrane depolarization, which may be strong enough to cause Ca2+ influx through voltage-sensitive Ca2+ channels (11). Thus ANG II-stimulated Ca2+ transient results from both release of intracellular Ca2+ stores and influx of extracellular Ca2+ (12). However, it is not known whether [Cl]e affects ANG II-induced Ca2+ signaling in vascular smooth muscle. We report a positive test of the hypothesis that Cl enhances ANG II-induced Ca2+ transients in VSMC. The magnitude of ANG II-induced Ca2+ transients varies directly with [Cl]e over a physiological and lower range in both the absence and presence of extracellular Ca2+. To our knowledge, these results are the first to demonstrate in vascular smooth muscle that extracellular Cl can modulate intracellular Ca2+ release induced by receptor activation. This modulating effect of Cl on ANG II-induced Ca2+ transients may be mediated by a mechanism involving Ins(1,4,5)P3 metabolism via altering intracellular Cl concentration ([Cl]i). Some of the results have been presented previously as an abstract (16).
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MATERIALS AND METHODS |
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Cell culture. From abdominal aortas of male SHR (364 ± 14 g, at least 16 wk old, n = 14; Taconic Farms) anesthetized with halothane, primary cultures of VSMC were obtained by enzyme digestion. Cells from SHR were used because of our previous observation of Cl sensitivity in stroke-prone SHR (25). Slices of arteries were incubated with collagenase (402 U/ml), elastase (1.3 U/ml), and soybean trypsin inhibitor (1 mg/ml) for three 30-min intervals. Cell suspensions were pooled and cultured in minimum essential medium with fetal bovine serum (10%), tryptose phosphate broth (2%), glutamine (20 mM), penicillin (50 U/ml), and streptomycin (50 U/ml) in a humidified atmosphere of 5% CO2-95% air at 37°C for at least 4 days before experimentation to ensure establishment of appropriate cell-matrix interaction. Cells not used in primary culture were used for experiments within five passages if not otherwise mentioned. In addition, immortalized newborn human VSMC (HNB18E6E7, male) obtained from Dr. Karen Yee (University of Washington, Seattle, WA) were used at passage 7. These cells were maintained in Waymouth's medium containing 10% fetal bovine serum and were handled similarly to VSMC from rats. Identification of cells as smooth muscle was determined by immunofluorescence with a fluorescein isothiocyanate-conjugated anti--actin antibody.
Composition of solutions. All salt solutions (pH 7.2) used in the experiments contained NaCl (111 mM), NaHCO3 or Na-gluconate (29 mM), KCl (5 mM), MgSO4 (1 mM), NaHPO4 (1 mM), CaCl2 (0 or 2 mM), glucose (25 mM), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (25 mM), and bovine serum albumin (0.05%). Where [Cl] was varied, NaCl was replaced isosmotically with NaHCO3, if not otherwise mentioned. The amount of Cl in other ingredients was taken into consideration for calculation of the final [Cl] reported in the study. The pH of solutions was titrated to 7.2 with NaOH or acetic acid. The concentration of ionized, free Ca2+ in various salt solutions was measured using a Ca2+/pH analyzer (Ciba Corning).
[Ca2+]i measurement. [Ca2+]i was determined by using a Nikon inverted epifluorescence microscope with the UMANS analytic software (Bio-Rad) or an SLM 8000 spectrofluorimeter (Urbana, IL) at 37°C. Cells plated on a coverslip were incubated with a mixture containing fura 2-AM (4.2 µM), Pluronic F127 (0.03%), and bovine serum albumin (0.4%) in salt solutions with various [Cl] (as described above) at room temperature for 30 min. The ratio of fluorescence intensity at excitation wavelengths of 340 and 380 nm was used to determine [Ca2+]i, as previously described (4). Each cell preparation was exposed to ANG II or PDGF once only. In some experiments, VSMC was incubated with 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a Cl channel inhibitor, for 40 min before the addition of ANG II and throughout the experiment.
[Cl]i measurement. Relative [Cl]i changes were assessed by fluorescence dye MQAE, as described previously (13). Briefly, VSMC were incubated with 5 mM MQAE at room temperature with 120 or 20 meq/l Cl for 30 min. After removal of extracellular MQAE and equilibration at 37°C for 10 min, solutions were changed to alter [Cl]e for 12 min before the fluorescence intensity was recorded with a spectrofluorimeter (Hitachi F4500) with an excitation wavelength of 350 nm. The intensity of fluorescence emitted at a wavelength of 460 nm during a period of 30 s was averaged for each preparation.
Ins(1,4,5)P3 measurement. VSMC were equilibrated with salt solutions containing 120 vs. 20 meq/l Cl for 30 min before the addition of ANG II. LiCl (10 mM) was added 10 min before the addition of ANG II. The response was terminated by 0.2 volume of 50% ice-cold perchloric acid at the indicated time. After 20-min incubation on ice, the cells and medium were collected and the mixture was centrifuged at 2,000 g for 15 min at 4°C. Supernatants were titrated to pH 78.5 with 10 N KOH and kept on ice. After centrifugation, the total mass of Ins(1,4,5)P3 was assayed by using an assay kit from Amersham (TRK 1000). The levels of Ins(1,4,5)P3 with both samples and standards were determined in duplicates.
Statistical analysis. Data are presented as means ± SE. Significant differences were established using Student's t-test or two-way analysis of variance followed by Duncan's post hoc test.
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RESULTS |
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When bicarbonate replaced Cl, the response to ANG II at a [Cl]e of 80 and 20 meq/l was 42 ± 12% (n = 11, from 4 rats) and 25 ± 16% (n = 9, from 3 rats), respectively, of that at a [Cl]e of 120 meq/l (n = 15, from 4 rats). When gluconate replaced Cl, the response to ANG II (2 µM) at a [Cl]e of 80 and 20 meq/l was 36 ± 14% (n = 7, from 5 rats) and 15 ± 5% (n = 9, from 6 rats), respectively, of that at a [Cl]e of 120 meq/l (n = 11, from 6 rats) (Fig. 1C). These results suggest that the effect of Cl replacement was independent on the anion used for replacement. Given that the resolution for measuring [Ca2+]i above 1 µM is reduced (4), most of the following experiments measuring ANG II-induced Ca2+ transients were conducted with a smaller concentration of ANG II, i.e., 100 nM, to induce a smaller amplitude of responses.
The effects of [Cl]e described above were not limited to cells obtained from SHR; VSMC from Sprague-Dawley (SD) rats of a different genetic origin exhibited similar results. With VSMC from SD rats, the ANG II (100 nM)-induced Ca2+ transient with a [Cl]e of 120 meq/l (283 ± 29 nM; n = 10) was significantly higher than that with a [Cl]e of 20 meq/l (176 ± 29 nM; n = 10; P < 0.05). Corresponding values were not different from those induced by 100 nM ANG II in VSMC from SHR (data not shown, n = 18).
Assessment of effect of Cl on Ca2+ measurement. When Cl was replaced with bicarbonate, extracellular free Ca2+ measured 1.3, 1.1, and 0.9 mM in solutions containing 120, 80, and 20 meq/l Cl, respectively. For gluconate replacement, these values were 1.0, 0.6, and 0.3 mM. Thus free [Ca2+] did vary significantly with anion composition. However, when additional CaCl2 (3.5 mM) was added to increase free [Ca2+] to that measured with a [Cl] of 120 meq/l, the amplitude of the ANG II (2 µM)-induced Ca2+ transient remained greatly reduced in solutions containing 20 meq/l Cl, i.e., 16 ± 6% (n = 3) and 23 ± 11% (n = 3) of that in a [Cl]e of 120 meq/l. Addition of CaCl2 to control for ionized [Ca2+] in solution did not reverse the effect of low [Cl]e on ANG II-induced Ca2+ transients. Thus the results with different [Cl]e in Fig. 1 cannot be explained by differences in extracellular ionized [Ca2+] in the solutions.
To assess whether cellular handling of fura 2-AM may be modulated by Cl, the maximal-to-minimal fluorescence ratio (Rmax/Rmin) was determined with different [Cl]e. Rmax/Rmin in 120 meq/l Cl was 7.2 ± 0.7 (n = 22) for bicarbonate replacement and 7.2 ± 1.1 (n = 12) for gluconate replacement. This ratio was unaffected by changes in [Cl]e. (see MATERIALS AND METHODS; data not shown). Finally, Cl did not affect the Ca2+ transient-induced by the Ca2+ ionophore A-23187 (1 µM) with either bicarbonate or gluconate replacing Cl (n = 511, data not shown). Therefore, it is unlikely that the effects of Cl on ANG II-induced Ca2+ transient were due to an effect on fluorescent characteristics of fura 2.
Effects of [Cl]e on Ca2+ release and entry. To determine how [Cl]e modulates ANG II-induced Ca2+ transients, ANG II (2 µM) was first added in the absence of extracellular Ca2+ to induce intracellular Ca2+ release; after [Ca2+]i recovered to near basal level (28 min), CaCl2 (2 mM) was added to the medium to examine Ca2+ entry in a primary culture of VSMC from SHR. ANG II-induced intracellular Ca2+ release and the subsequent Ca2+ entry were both attenuated when [Cl]e was reduced to 20 meq/l, as shown in Fig. 2A. This Cl-dependent Ca2+ release was observed with a wide range of ANG II concentrations from 108 to 3 x 106 M in subsequently cultured VSMC (passages 810; P < 0.05; n = 1330, 2 rats), as shown in Fig. 3. Our results demonstrate that Cl replacement dramatically reduced the efficacy of ANG II to induce Ca2+ release from intracellular stores in VSMC from SHR. Although the amplitude of ANG II-induced Ca2+ release might be blunted with time in culture, the effect of Cl persisted to at least passage 10 of cultured VSMC. Similar results were observed with PDGF (25 ng/ml) (Fig. 2C). Ca2+ release and entry induced by PDGF (25 ng/ml) in a [Cl]e of 80 meq/l were 15 ± 7 and 50 ± 19% (n = 4, 2 rats) of those in a [Cl]e of 120 meq/l (n = 5, 3 rats).
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With a [Cl]e of 120 meq/l, thapsigargin (2 µM) almost completely prevented Ca2+ release induced by ANG II (2 µM) (n = 3, data not shown). Although intracellular Ca2+ stores were believed to be heterogeneous in nature (3, 12), the results suggest that thapsigargin-sensitive stores were responsible for most, if not all, of the Ca2+ released by these agents in our preparation. Figure 2B shows that, in a primary culture of VSMC, the magnitude of the Ca2+ release induced by thapsigargin (2 µM) was similar with a [Cl]e of both 120 and 20 meq/l. After passages in culture, VSMC also exhibited similar Ca2+ release to thapsigargin in high and low [Cl]e (total n = 13, from 6 rats). In addition, we found no difference in the subsequent Ca2+ entry with a [Cl]e of 120 vs. 20 meq/l when extracellular Ca2+ was restored (Fig. 2B). Cl did not affect Ca2+ release induced by a submaximal concentration (1 µM) of ionophore A-23187 (n = 6, data not shown).
To determine how Cl channel may participate in the Cl-sensitive Ca2+ signaling, a Cl channel inhibitor, NPPB (10 µM), was added 40 min before the addition of ANG II, and the treatment was sustained throughout the experiment. As shown in Fig. 4, NPPB increased ANG II-induced Ca2+ release and the subsequent Ca2+ entry at low [Cl]e (P < 0.05). The results indicate that only with low [Cl]e did the NPPB-sensitive Cl channel have a role in modulating ANG II-induced Ca2+ signaling.
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Mechanism of [Cl]e effect on intracellular Ca2+ release. First, to examine how [Cl]e modulates the amplitude of ANG II-induced Ca2+ release, [Cl]e was acutely altered before the addition of ANG II. VSMC were preincubated with solutions containing 120 or 20 meq/l Cl for 40 min before [Cl]e was changed. ANG II (100 nM) was added to cells 1 min after extracellular Ca2+ was removed and [Cl]e was changed. As shown in Table 1, ANG II-induced Ca2+ release in VSMC preincubated with 120 meq/l Cl (group A) was >1.6-fold that in VSMC preincubated with 20 meq/l Cl (group B). Acutely increasing [Cl]e from 20 to 120 meq/l 1 min before the addition of ANG II did not restore the peak of released Ca2+ (group D); acutely decreasing [Cl]e from 120 to 20 meq/l 1 min before the addition of ANG II did not attenuate the peak of released Ca2+ (group C). However, addition of ANG II 30 min after [Cl]e was increased from 20 to 120 meq/l restored ANG II-induced Ca2+ release (116 ± 19 nM; n = 6). ANG II-induced Ca2+ release correlated well with MQAE fluorescence intensity under these situations. When the MQAE fluorescence intensity was low, such as in groups A and C, indicating relative high intracellular [Cl], the amplitude of Ca2+ release was significantly larger; and vice versa. These results demonstrated that the capacity of Cl to modulate the ANG II-induced increase in [Ca2+]i was associated with the intracellular level of [Cl].
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To determine whether ANG II-induced Ca2+ entry as well as release was affected by acute changes of [Cl]e, Ca2+ was maintained at 2 mM throughout the experiment and [Cl]e was acutely altered 1 min before ANG II was added. After incubation with a [Cl]e of 20 meq/l for 40 min, the amplitude of ANG II (100 nM)-induced Ca2+ transients, 198 ± 23 nM (n = 18), was 67% of that with a [Cl]e of 120 meq/l (n = 18, P < 0.05). After 40-min preincubation with a [Cl]e of 20 meq/l, an acute increase of [Cl]e to 120 meq/l 1 min before the addition of ANG II did not amplify the ANG II-induced Ca2+ transients, which remained unchanged (198 ± 27 nM, n = 18). These results suggest that ANG II-induced release and entry of Ca2+ might be modulated by intracellular [Cl].
In our preparation of VSMC, ANG II-induced Ca2+ release appears to be stimulated primarily by an increase in Ins(1,4,5)P3 that is produced by PLC activation, given that exogenous ryanodine (10 µM), an Ins(1,4,5)P3-independent Ca2+-releasing agent, induced no Ca2+ release (data not shown). Table 2 shows the effects of a PLC inhibitor, U-73122, on ANG II-induced Ca2+ release at 120 vs. 20 meq/l Cl. U-73122 nearly completely blocked ANG II-induced Ca2+ release with either [Cl]e, whereas U-73343, an inactive analog of U-73122, did not.
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In our preparation, ANG II induced a peak increase in Ins(1,4,5)P3 level 15 s after its initiation (Fig. 5A). Total Ins(1,4,5)P3 level in cells was significantly higher with high [Cl] 15 and 30 min after the addition of ANG II (P < 0.05). The peak increase that occurred with 120 meq/l Cl was approximately sixfold that with 20 meq/l Cl. Similar results were observed with gluconate replacement of Cl (n = 5, P < 0.05, data not shown). Furthermore, in the absence of extracellular Ca2+, the ANG II-induced peak increase in Ins(1,4,5)P3 level with 120 meq/l Cl was approximately sixfold that with 20 meq/l Cl (Fig. 5B). In addition, PDGF (25 ng/ml for 30 s)-induced peak increase in Ins(1,4,5)P3 (2.7 ± 0.2 pmol/well, n = 3) with 120 meq/l Cl was completely blocked with 80 meq/l Cl (P < 0.05, data not shown), suggesting that both the ANG II- and PDGF-induced increase in Ins(1,4,5)P3 was Cl dependent.
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DISCUSSION |
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In our preparation, Ins(1,4,5)P3 production appears to be a major immediate upstream event of intracellular Ca2+ release, because inhibition of PLC by U-73122 completely blocked Ca2+ release in both 120 and 20 meq/l Cl (Table 2). An effect of Cl on the levels of Ins(1,4,5)P3 after ANG II stimulation may be contributed to the effect of Cl on ANG II-induced Ca2+ release. Total Ins(1,4,5)P3 at [Cl]e of 20 meq/l was less than that at 120 meq/l both 15 and 30 min after addition of ANG II. This result is consistent with the previous finding that Cl may modulate Ins(1,4,5)P3 level in rat mesangial cells (18). Cl may increase production of Ins(1,4,5)P3 by modulating G protein-mediated PLC activation. It has been shown that Cl increased affinity of Go with GTP to decrease subsequent hydrolysis of the nucleotide, which increases the population of activated G proteins (6). However, the amplitude of Ins(1,4,5)P3 increase and Ca2+ release induced by PDGF also depends on [Cl]. It is unlikely that the site of action is primarily or solely on G proteins. Alternatively, Cl may increase the level of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to facilitate conversion of Ins(1,4,5)P3; however, a previous study (2) has demonstrated that 1.1 M NaCl does not alter the level of PtdIns(4,5)P2, which makes it unlikely that a concentration of 120 meq/l Cl may alter PtdIns(4,5)P2 in the present study. Nevertheless, the current results do not rule out the possibility that Cl might either increase the total Ins(1,4,5)P3 level by reducing Ins(1,4,5)P3 catabolism or modulate the capacity of Ins(1,4,5)P3 generated in response to ANG II to induce the release of Ca2+ from intracellular stores.
Alternatively, increased [Cl]e might increase the size of intracellular Ca2+ stores, which could contribute to enhancement of ANG II-induced Ca2+ release (12). Despite the heterogeneous nature of intracellular Ca2+ stores (3), Ca2+ released by ANG II was primarily from thapsigargin-sensitive stores in our preparation; however, [Cl]e did not affect thapsigargin-induced Ca2+ release in the absence of extracellular Ca2+ (Fig. 2B), suggesting that the size of this ANG II- and thapsigargin-sensitive Ca2+ stores may not be altered by Cl. Furthermore, Ca2+ entry following thapsigargin-induced Ca2+ release was also unaffected by [Cl]e (Fig. 2B), suggesting that Cl does not alter the capacitative Ca2+ entry induced by thapsigargin.
In addition to Ca2+ release, Ca2+ entry may contribute to the effects of Cl on ANG II- and PDGF-induced Ca2+ transients (Fig. 2, A and C). It has been proposed that receptor activation-induced Ca2+ release may trigger Cl efflux via a Cl channel, resulting in depolarization and subsequent Ca2+ entry through a voltage-sensitive Ca2+ channel in smooth muscles (15); ANG II-induced Ca2+ entry may thus be affected by [Cl]e. However, an inhibitor of Cl-channel, NPPB, exhibited no effect on ANG II-induced Ca2+ release or entry with a [Cl]e of 120 meq/l (Fig. 4), suggesting that Cl efflux may not play a significant role in Ca2+ movement in our preparation with a [Cl]e of 120 meq/l. In contrast, NPPB increased Ca2+ transients at a [Cl]e of 20 meq/l, suggesting Cl efflux may have a role in the reduced Ca2+ transients with low [Cl]e, probably due to a Cl gradient between intracellular and extracellular space that may serve as a driving force to induce Cl efflux with low [Cl]e. Furthermore, the magnitude of Ca2+ entry may depend on the amount of Ca2+ released from the intracellular store, and hence the extent to which it is depleted of Ca2+ (24). It is possible that the reduced Ca2+ entry with lower [Cl]e is secondary to the effect of lower [Cl]e on Ca2+ release. On the other hand, Ca2+ entry may occur independently of Ca2+ store depletion (8, 17), and [Cl]e could have an effect on Ca2+ entry independent of intracellular Ca2+ release. Previous studies have demonstrated that in isolated perfused afferent arterioles of rabbit, Cl was essential for vasoconstrictor responses to K+ (5), which increases intracellular Ca2+ solely by an entry mechanism. Nevertheless, our results are consistent with previous findings that [Cl]e is essential for the Ca2+ influx induced in mesangial cells by vasopressin and endothelin (14), and for that induced in endothelial cells by histamine and ATP (7).
Finally, both intracellular alkalinization and acidification have been found to acutely increase basal [Ca2+]i (1, 22). Possibly, replacing Cl with other anions might alter ANG II-mediated Ca2+ transients by altering intracellular pH (pHi). However, previous studies with VSMC have shown that complete replacement of Cl in the medium with either gluconate or aspartate induces only a minimal reduction in pHi of 0.05 pH units (20). In addition, replacement of extracellular Cl with gluconate, acetate, or methanesulfonate has no significant effect on the basal pHi of mesangial cells (14). Thus, in the present study, altering pHi is unlikely to mediate the effects of partial replacement of Cl on ANG II- or PDGF-induced Ca2+ transients.
In conclusion, our results provide evidence for a modulable mechanism for ANG II-induced Ca2+ signaling by Cl in VSMC. Cl may play a role in modulating both intracellular Ca2+ release and Ca2+ entry induced by ANG II and, perhaps, other vasoconstrictors. The possible mechanism(s) for the effect of Cl on Ca2+ release includes modulation of ANG II-induced Ins(1,4,5)P3 increase. Our results provide a potential mechanism for Cl-dependent responses to vasoconstrictors observed in isolated arteries (9) and for Cl-sensitive hypertension described in stroke-prone SHR with high plasma renin activity (25).
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ACKNOWLEDGMENTS |
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This work was supported by National Science Council (Taiwan) Grant NSC89-2320-B-182-078; Chang Gung Memorial Hospital Grant CMRP870; National Institutes of Health Grants 5M01-RR-000079, R01-HL-47943, and R01-HL-41210; the Research Evaluation and Allocation Committee of the University of California, San Francisco, and gifts from the Church and Dwight Company and the Emil Mosbacher, Jr., Foundation.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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