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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Cardiac Membrane Research Lab, Simon Fraser University, Burnaby and 2Cardiovascular Sciences, Child and Family Research Institute, Vancouver, British Columbia, Canada; and 3Laboratorio de Fisiología Celular, Cardiología, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Submitted 7 June 2007 ; accepted in final form 3 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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sarco(endo)plasmic reticulum Ca2+ pump; sarcoplasmic reticulum Ca2+ loading; cytosolic Ca2+ concentration; L-type Ca2+ channel; Na+/Ca2+ exchanger; neonate cardiomyocytes; store-operated Ca2+ channel
70% and 30% of the total Ca2+ during cell contraction, respectively (8, 17). Accordingly, 28% of the Ca2+ removed from the cytosol during cell relaxation is extruded by the Na+/Ca2+ exchanger (NCX), and the sarco(endo)plasmic reticulum Ca2+ (SERCA)2a pump transports 70% of total Ca2+ back into the SR (3). The sarcolemmal Ca2+-ATPase (plasma membrane Ca2+-ATPase, PMCA) and mitochondrial Ca2+ uniporter, collectively referred to as the slow Ca2+ removal system, remove the remaining 2% (2).
Ultrastructural data from the neonatal heart have led to the suggestion that the SR is relatively sparse at birth and develops gradually from neonate to adult through the first few weeks of life (11, 25). Several studies have also indicated that SERCA mRNA and protein expression levels are 50% of adult levels at birth and gradually increase to adult levels by postpartum day 15 (9, 11), leading further support to the notion that SERCA2a function is less important in neonates than in adults (5, 10). Consequently, it has been assumed that the SR in neonatal ventricular myocytes is unable to store amounts of Ca2+ comparable to those of adult myocytes. However, recent studies have challenged this assumption. Observations such as robust and spatially homogeneous caffeine (Caf)-induced Ca2+ transients and contractures have been reported in neonate hearts (1, 10, 16). Moreover, integration of the Caf-induced inward NCX current (INCX) in neonatal rabbit ventricular myocytes revealed that SR Ca2+ loading (loadSR) at rest was significantly larger in early neonatal than in late neonatal and adult ventricular myocytes (14), suggesting that store-operated Ca2+ entry (SOCE) may contribute significantly to loadSR in the neonate rabbit heart. The aim of the present study was therefore to elucidate the cellular mechanisms contributing to refilling of the SR on a beat-to-beat basis during postnatal development.
To achieve this, we used a whole cell perforated patch-clamp technique and Ca2+ transient measurements combined with pharmacological manipulation of the L-type Ca2+ channel and the NCX, the main sarcolemmal Ca2+ sources in the adult mammalian ventricular myocytes (3, 12, 14). Our results show that the SR Ca2+ pump is capable of efficiently loading the SR with Ca2+ even at the earliest neonatal stage, but that the principal Ca2+ sources contributing to loadSR change during ontogeny.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Whole cell perforated-patch voltage clamp. Whole cell amphotericin-perforated voltage-clamp technique was used at room temperature as described previously (12, 13, 17). The internal pipette solution contained (in mM) 110 CsCl, 5 MgATP, 1 MgCl2, 20 tetraethylammonium, 5 Na2 phosphocreatine, and 10 HEPES, pH 7.1 (adjusted with CsOH). The standard external solution contained (in mM) 130 NaCl, 5 CsCl, 1 MgCl2, 2.0 CaCl2, 5 Na-pyruvate, 10 glucose, and 10 HEPES, pH 7.4 (adjusted with NaOH). All drugs were purchased from Sigma (St. Louis, MO). Because nifedipine (Nif) is very sensitive to light, particular precautions were taken. A fresh working solution of 15 µM Nif was made by diluting a fresh 15 mM stock solution (dissolved in DMSO), resulting in a final DMSO concentration of 0.1%. The entire Nif delivery pathway including the micromanifold was light tight.
Measurement of Ca2+ fluorescence.
Intracellular Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fluo-3 AM as described previously (12, 13, 17). The [Ca2+]i was calculated from the formula [Ca2+]i = Kd·(F – Fmin)/(Fmax – F), where Fmin is the background fluorescence determined from a cell-free area and Fmax is the fluorescence acquired after the cell was depolarized to +150 mV for 10–20 s to maximize [Ca2+]i and kill the cell at the end of each experiment. F0 was taken as the difference between the background fluorescence determined in the absence and presence of a cell in the area of measurement. 
F is the incremental fluorescence measured from baseline or the background fluorescence in the presence of a cell. Kd is the fluo-3 Ca2+ dissociation constant, and a value of 400 nM was used for all age groups (20).
General protocol.
Figure 1A shows the SR Ca2+ loading experimental protocol applied in Fig. 1, B and C, as well as 
Figs. 3 and 4. The SR Ca2+ was first cleared by a brief Caf application. A train of 20 repetitive depolarizations at 0.2 Hz (first depolarized to –40 mV for 50 ms in order to inactivate Na+ channels and T-type Ca2+ channels, and then depolarized to +10 mV for 400 ms, at a holding potential of –80 mV) was then initiated 5 s after Caf removal. Immediately after the 20th depolarization, Caf was applied again to evaluate the SR Ca2+ content.
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Figure 6A shows the experimental protocol used to measure the SR Ca2+ uptake rate (VSR) and [Ca2+]i. The first Caf application was used to clear the SR Ca2+ content, and the cell was then depolarized to various voltages (from –30 mV to +50 mV, in increments of 20 mV) for 3 s to load the SR. Long depolarizations were used to induce a relative spatial and temporal homogeneity of [Ca2+]i. Pulses longer than 3 s were also tried, but these frequently resulted in cell death, particularly in the younger age groups. More positive depolarizations were also tried, but these often resulted in saturation of loadSR and as a consequence frequently generated spontaneous release. Therefore, these data were not included in the analysis. The time integral of the INCX (
INCX) induced by the second Caf application was used as a measure of loadSR during the preceding 3-s depolarization (12). The average VSR (amol·s–1·pF–1) was obtained by dividing loadSR (INCX, amol/pF) by the duration of the loading period (3 s).
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0.05 was taken to be significant. |
RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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tail INCX) and Caf INCX (Caf INCX) are shown in Fig. 1E. Peak ICa in the 3d group did not show a significant decrease in magnitude with depolarization number, although this phenomenon was observed in older age groups (data not shown). Both the Ca2+ transient and tail INCX showed a depolarization-dependent increase that reached a steady state near the 10th depolarization. Assuming that sarcolemmal Ca2+ entry and extrusion are equal in magnitude at steady state, subtraction of the average value of the last 5 tail INCX from each individual tail INCX should give the net Ca2+ entry during each depolarization. Figure 1F shows the net Ca2+ entry for each depolarization, and the cumulative net Ca2+ entry during the 20 depolarizations is shown in Fig. 1G. Age-dependent differences in sarcolemmal Ca2+ entry, Ca2+ transients, and SR Ca2+ loading. Figure 2A shows that the cumulative net Ca2+ entry significantly decreased with age while the number of depolarizations required to achieve steady state was comparable in all age groups. Figure 2B shows the maximum amplitude of [Ca2+]i as a function of number of depolarizations. The data were well fit with a Boltzmann function (R2 0.98). The peak [Ca2+]i increased with subsequent depolarizations and reached steady state near the 10th depolarization. The response was comparable for all age groups, which was neither proportional to cumulative Ca2+ entry nor to loadSR. Figure 2C compares the cumulative net Ca2+ entry and the loadSR (loadSR-Con) as a function of age. Both decreased significantly with age, but loadSR was larger than cumulative Ca2+ entry in all age groups, suggesting that an additional source of loadSR exists.
Age-dependent difference in nifedipine-insensitive Ca2+ transients and sarcolemmal Ca2+ entry.
Figure 3, A and B, are representative traces from a 3d myocyte and a 56d myocyte, respectively, using the same protocol as shown in Fig. 1A but in the presence of 15 µM Nif, a selective inhibitor of L-type Ca2+ channels that completely blocked ICa in all age groups (data not shown). [Ca2+]i, inward INCX (tail INCX and Caf INCX), and their time integrals (INCX and Caf INCX) are shown from top to bottom, respectively, in Fig. 3, A and B. The depolarization-dependent Ca2+ transient and the tail INCX were abolished by the addition of Nif in 56d but not in 3d myocytes. Both Caf [Ca2+]i and Caf INCX were still observed in 56d myocytes, although tail INCX and Ca2+ transients were abolished. Figure 3, C and D, show the cumulative net Ca2+ entry and the [Ca2+]i amplitude in the presence of Nif for the different age groups. Note that Nif abolished sarcolemmal Ca2+ entry and Ca2+ transients in older but not in younger age groups.
Age-dependent difference in nifedipine and KB-R7943-insensitive SR Ca2+ loading.
Figure 4, A and B, show representative traces from a 3d myocyte and a 56d myocyte in the presence of both 15 µM Nif and 10 µM KB-R7943 (KB-R). [Ca2+]i, inward INCX (tail INCX and Caf INCX), and their time integrals (INCX and Caf INCX) are shown from top to bottom, respectively, in Fig. 4, A and B. The depolarization-dependent Ca2+ transient and tail INCX observed in 3d myocytes with Nif were abolished by the subsequent addition of KB-R. However, a significant Caf-induced Ca2+ transient and a considerable amount of Caf INCX were still observed in both 3d and 56d myocytes, although the values were significantly greater for 3d than for 56d myocytes, confirming that an additional loadSR source exists and is insensitive to Nif and KB-R. Figure 4C shows that Nif- and KB-R-insensitive loadSR was significantly smaller in older compared with younger age groups.
Age-dependent changes in relative contributions of different sarcolemmal Ca2+ sources.
Figure 5A shows the cumulative Ca2+ entry in control and Nif conditions for the different age groups (there is no cumulative Ca2+ entry in the presence of NIF+KB-R). The relative contributions of Nif-sensitive and KB-R-sensitive (but Nif insensitive) Ca2+ entry are shown in Fig. 5B. KB-R-sensitive but Nif-insensitive Ca2+ entry was dominant at the earliest developmental stage and significantly decreased with age; in contrast, Nif-sensitive Ca2+ entry significantly increased with age and became dominant at the latest developmental stage examined. Figure 5C shows the corresponding control, Nif, and Nif+KB-R or Nif+KB-R-insensitive loadSR as shown in Fig. 4C. The relative contributions (Con = 1) of Nif-sensitive, KB-R-sensitive, and Nif+KB-R-insensitive loadSR are shown in Fig. 5D for the different age groups. The calculation of the contribution of Nif+KB-R-insensitive loadSR is predicated on two observations: 1) steady state in control solution was achieved at 10th depolarization (50 s) and 2) Nif+KB-R-insensitive loadSR is linear with time for up to 100 s (13). Therefore the contribution of Nif+KB-R-insensitive loadSR relative to Con is derived from the assumption that its loading is the half-value of Nif+KB-R-insensitive loadSR (100 s). In accordance with Fig. 5B, Nif-sensitive loadSR significantly increased with age, while KB-R-sensitive loadSR and Nif+K-BR-insensitive loadSR significantly decreased with age.
Age-dependent changes in k0.5 of SR Ca2+ uptake.
Since age-dependent changes in loadSR may be due to changes in the SERCA2a Ca2+ affinity, we estimated this parameter at the different postnatal stages by measuring the SR Ca2+ uptake rate as a function of bulk phase [Ca2+]i. Figure 6B shows representative traces of the Ca2+ transient and membrane current elicited by the protocol shown in Fig. 6A (on depolarization to +30 mV) in a 3d cell. The magnitude of the Ca2+ transient after it reached a plateau state ([Ca2+]PS) was used as the [Ca2+]i corresponding to its average VSR. Figure 6C shows the average Ca2+ transient traces and its corresponding second Caf-induced INCX from five each of 3d and 56d cells at depolarization steps from –30 mV to +50 mV. It is clear that the second Caf-induced INCX is greater in 3d than 56d cells despite the fact that the amplitudes of the Ca2+ transients were comparable in the two groups. The representative average VSR as a function of [Ca2+]PS derived from Fig. 6C (including data point of VSR at –80 mV) is shown in Fig. 6D. Data points were fit with the Hill equation: VSR = Vmax·[Ca2+]PS
/(K+ [Ca2+]) (R2 0.98) to obtain the asymptotic value of VSR (Vpeak), k0.5 (the value of [Ca2+]PS at half of Vpeak) and Hill coefficient nH (the slope of the sigmoid curve). From Fig. 6D, it is clear that k0.5 is smaller in 3d than 56d cells; k0.5 increased significantly after 10 days of age. In contrast, there were no significant differences in either Vpeak or nH between groups (Table 1).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Age-dependent changes in relative contributions of ICa and reverse-mode NCX. The present study shows that 10 consecutive depolarizations are sufficient to fully reload the SR after transient exposure to 10 mM Caf. This was true for all rabbit ventricular myocytes examined independent of the age stage. In the older age groups, the calcium reloading was primarily due to an enhanced ICa and a decreased tail INCX during the first 10 depolarizations. This is in accordance with previous reports on adult rat and ferret hearts (28) and is also consistent with a previous study from our laboratory (14). It is generally agreed that the ICa is the main pathway for sarcolemmal Ca2+ entry in the adult rabbit heart (8, 17), and in agreement with this notion, we find that loadSR as well as the Ca2+ transient were sensitive to nifedipine in older age groups (Fig. 3, C and D, and Fig. 5). In contrast, in the early developmental stages, loadSR as well as the Ca2+ transient were insensitive to Nif but were sensitive to subsequent addition of KB-R (Fig. 4A and Fig. 5). The findings indicate that reverse-mode NCX plays an important role in both excitation-contraction (E-C) coupling and SR Ca2+ refilling at early neonatal stages. Moreover, total sarcolemmal Ca2+ efflux estimated via tail INCX at steady state in 3d hearts was about twice the magnitude of that in 56d hearts (0.4 vs. 0.2 pC/pF), and the first [Ca2+]i transient in younger age groups was larger than that in 56d hearts and sensitive to KB-R (Fig. 2B and Fig. 3D), confirming previous findings of a more prominent role of reverse-mode NCX in neonatal E-C coupling (14, 30).
It should be noted that the slow Ca2+ removal system has been suggested to play a greater role in neonatal compared with adult heart (3, 16, 29). In rat ventricular myocytes, the slow Ca2+ removal system has been reported to be approximately two times greater in immature than in adult cardiac myocytes (3). Therefore, it is possible that the contributions of sarcolemmal Ca2+ efflux and SR Ca2+ content determined by the integral of INCX are underestimated in younger age groups. However, the contribution of the slow Ca2+ removal system in the early developmental stage is still too small (5% of total Ca2+) to be considered physiologically significant (3).
Age-dependent changes in Nif+KB-R-insensitive SR Ca2+ loading. Interestingly, a considerable amount of loadSR was observed at all age stages despite the fact that the Ca2+ transient was abolished by the addition of Nif and KB-R (Fig. 4). This is in accordance with observations of Trafford et al. (28) in quiescent adult ferret and rat myocytes and a previous study from our laboratory (13) showing that this phenomenon can likely be ascribed to SOCE in rabbit cardiac myocytes. In agreement with this study, we observed that the contribution of SOCE to total SR Ca2 refilling significantly decreased with age. The average rate of sarcolemmal Ca2+ influx induced by depolarization was 1.1 and 0.5 pC·pF–1·s–1 for 3d and 56d myocytes, respectively, which is 15- to 50-fold greater than our previous estimate of the average Ca2+ influx rate via SOCE during the first 10 s after SR Ca2+ depletion (13). Thus, although SOCE may contribute to SR refilling after Ca2+ depletion, this mechanism is unlikely to make a significant contribution to E-C coupling on a beat-to-beat basis even in the neonate myocyte. The remarkable loadSR via SOCE (40%) after SR Ca2+ depletion does, however, suggest that it might contribute to regulate loadSR at the earliest developmental stages. Our previous results indicated that SOCE loadSR was intimately modulated by NCX (13), which we suggested resulted from the SOCE influx pathway and NCX being in the same microdomain. More recent evidence from two different laboratories has strongly supported the concept that TRP-C3 channels and NCX colocalize in heart tissue (7, 8). Although not the focus of this study, it is tempting to speculate that TRP-C3 channels may contribute to the observed SOCE.
The cellular mechanisms underlying loadSR provide important insight into the intact working heart. With an increase in the number of depolarizations, [Ca2+]i and loadSR dramatically increase, providing the bases for the phenomenon of the positive force-frequency relationship observed in rabbit ventricular muscle strip and intact heart (19, 31) preparations. Although the intact heart might be less dependent on Ca2+ influx via depolarization compared with isolated muscle (31), the developmental changes in the sources of Ca2+ for SR refilling in an intact heart are likely to be similar to those observed for the single myocyte, and these observations are clinically significant. For example, a prolonged whole heart arrest during open-heart surgery in the newborn will likely be more prone to cause SR Ca2+ overload and arrhythmogenesis as a result of a greater SOCE; therefore, a different approach for arresting the newborn heart during cardiopulmonary bypass may be beneficial.
SR Ca2+ pump activity during development. Our findings that 10 consecutive depolarizations were sufficient to reload the SR independently of the age stage although loadSR (normalized by membrane capacitance) was more than threefold greater in 3d than in 56d myocytes (Fig. 2C) suggest that at the early developmental stages there is an increase in 1) SERCA2a Vmax, 2) affinity of SERCA2a for calcium, and/or 3) [Ca2+] in the microdomain in which SERCA2a resides. Our estimates of Vpeak (12.8 amol Ca2+·pF–1·s–1 or 82 µM·liter cytosol–1·s–1) and k0.5 (0.38 µM) in the 56d group agree well with values reported by Bassani et al. (2) in intact myocytes from adult rabbits. The nH value observed from our data is close to the theoretical maximum of 2, but smaller than that observed by other groups (2) using intact cardiomyocytes, which might be explained by differences in the measurement and analysis techniques. The data shown in Table 1 indicate that Vpeak, as measured in our study, is not different between the age groups. Studies of SR vesicles have shown that the maximum SR Ca2+ uptake rates increase during development in a variety of species (23, 24) and are consistent with previous observations of sparse SR and a lower density of SERCA2a in neonates (25, 27). The discrepancies between the results of the present study and those presented above are probably related to differences in technique. The advantages of in vitro preparations such as microsomal vesicles include control over "cytosolic" [Ca2+], measurement of initial rates for a more accurate determination of Vmax and affinity, as well as regulation of intravesicular [Ca2+] with oxalate or similar compounds. However, the major disadvantage is that the SERCA2a has been removed from its native environment with the attendant potential loss of regulatory cofactors [e.g., Gi proteins, β-receptors, SR-associated phosphatases, and phospholamban (PLB)] and destruction of the microdomain in which SERCA2a resides. Despite these differences, however, it is useful to compare our data in the 56d intact myocyte with data from SR vesicles from the adult rabbit heart. In one such a study by Xu and Narayanan (33) in which uptake was determined at 37°C, they found a Vmax of 489 nmol Ca2+·mg protein–1·min–1, which is comparable to that determined by others in different species (10, 15). In addition, they found a k0.5 value of 0.6 µM and an nH of 1.4, which compare favorably to the k0.5 of 0.4 µM and nH of 2.2 determined in the present study. To convert the microsomal Vmax (in nmol Ca2+·mg protein–1·min–1) to units comparable to the Vpeak (in amol Ca2+·pF–1·s–1) observed in the present study, we used the following assumptions: 10-fold microsomal purification factor, 120 mg homogenate protein/g wet wt, 2.43 g wet wt/ml cytosol, 4.58 pF/pl cytosol (4, 26), and a Q10 of 2 to derive a value of 12.1 amol Ca2+·pF–1·s–1 in vesicles vs. 12.8 amol Ca2+·pF–1·s–1 determined in intact myocytes in the present study. Because we cannot unequivocally state that our VSR reflects initial rates because of the complexity of the preparation, we chose to refer to the maximal VSR in our study as Vpeak instead of Vmax. However, the fact that the Vpeak values are comparable to the Vmax determined under the simpler and more controlled in vitro conditions serves to allay many of these concerns.
Consistent with the notion that affinity of SERCA2a for Ca2+ was altered, the k0.5 for SR loading significantly increased with age (Table 1). However, studies using electrical field stimulation of immature ventricular myocytes have suggested a diminished contribution of the SR Ca2+ pump to cytosolic Ca2+ removal (1, 3, 34), which would appear to contradict our findings. On the other hand, another study using electrical field stimulation recently demonstrated that a functional SR is present long before birth in a linear heart tube (22), and the different results are likely due to the specific experimental conditions and/or species differences.
Indeed, one possible explanation for a higher SR Ca2+ affinity without a change of Vmax in neonate rabbit ventricular myocytes may be a lower PLB expression relative to that of SERCA2a (9, 18). In agreement with this notion, it has been demonstrated that PLB-deficient myocytes exhibit a higher SR Ca2+ load (6), and it was recently demonstrated that the degree of PLB phosphorylation per SERCA2a was greater in the fetus and newborn compared with adult (32). It has been reported that the SR Ca2+ depletion prompts the phosphorylation of PLB to stimulate store refilling (5). An alternative explanation to the apparently higher Ca2+ affinity of the SR Ca2+ pump in neonate myocytes is that there are age-dependent differences in the subsarcolemmal microdomain (12, 13) resulting in a higher [Ca2+] in the immediate vicinity of the SERCA2a in the neonate heart for a given level of bulk phase [Ca2+]. Indeed, we recently showed that a narrow cleft (20 nm) between the sarcolemma and SR observed in both 3d and 56d myocytes was delimited by a threefold longer SR in 3d than 56d myocytes (300 vs. 100 nm), resulting in a much more restricted microdomain in the early developmental stages. It is therefore possible that an apparent greater Ca2+ affinity of the SR Ca2+ pump observed in neonate myocytes may at least partly result from measurement of VSR as a function of the average bulk [Ca2+]i.
Conclusions. In conclusion, there is a switch in the sarcolemmal calcium fluxes contributing to SR Ca2+ refilling from a predominance of NCX and SOCE at the earliest stage to a predominance of ICa at later stages. Moreover, the number of depolarizations required to achieve steady state did not vary during development, although steady-state loadSR was threefold larger in the neonatal heart. This may be explained by either a higher Ca2+ affinity of the SERCA2a in neonatal myocytes or a higher local [Ca2+] around SERCA2a for a given level of [Ca2+] in the bulk phase. Together, this suggests that age-dependent downregulation of SR calcium sequestration and increased dependence on calcium entry through L-type calcium channels during the first 20 days postpartum in rabbit ventricular myocytes is due to a selective downregulation of NCX-dependent SR Ca2+ refilling.
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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.
* L. Hove-Madsen and G. F. Tibbits are co-senior authors of this article. 
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P < 0.05 for 3d vs. either 20d or 56d in Nif+KB-R insensitive). n = 12.