Dihydropyridine-sensitive Ca2+ channels in human glomerular mesangial cells

David A. Hall, Pamela K. Carmines, and Steven C. Sansom

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In mesangial cells (MC), the response of intracellular Ca2+ concentration ([Ca2+]i) to a contractile agonist is biphasic with a large, transient increase in [Ca2+]i followed by a smaller but sustained elevation as Ca2+ flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca2+ channels in the plasmalemmal membrane. However, a role for voltage-gated Ca2+ channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca2+ entry pathway responsible for the sustained response of [Ca2+]i in human MC. We found that ANG II at 10 nM, 100 nM, and 1 µM increased [Ca2+]i to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 µM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca2+. None of the peak responses (due to release of intracellular stores of Ca2+) were affected by removal of external Ca2+ or addition of diltiazem. Upon elevating the extracellular [K+] from 5 mM to 75 mM, [Ca2+]i reached a sustained level of 48% greater than baseline. This effect of high K+ was attenuated by either Ca2+ removal or addition of diltiazem. In the presence of 75 or 140 mM K+, the dihydropyridine agonist BAY K 8644 (1 µM and 10 µM) initiated sustained [Ca2+]i responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca2+ in the external solution, BAY K 8644 did not significantly affect [Ca2+]i. In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl2 and 10 µM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between -30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca2+ channels responsible for the voltage-regulated entry of Ca2+ into the cell during an agonist-induced contraction.

diltiazem; BAY K 8644; voltage-gated calcium channel; fura 2; patch clamp


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MESANGIAL CELLS (MC) are contractile cells that surround the glomerular capillaries and regulate rates of renal filtration by responding to vasoactive agonists such as angiotensin II (ANG II) and nitric oxide. The electrophysiology of MC, studied by several investigators (1, 17, 18, 23) and recently reviewed (24), has many similarities to that of vascular smooth muscle (VSM) and other smooth muscle cells. In VSM, contractile agonists stimulate the inositol trisphosphate-mediated release of intracellular Ca2+, which activates Cl- and nonselective cation channels leading to a depolarization of the membrane potential and activation of voltage-gated calcium channels (VGCC) in the plasmalemmal membrane (16, 27). Initially, release of Ca2+ from intracellular stores causes a large but transiently elevated intracellular Ca2+ concentration ([Ca2+]i) which then subsides toward baseline by internal buffering and the activity of Ca2+ pumps and exchangers (12). A smaller, but sustained increase in [Ca2+]i results from activating VGCC in the plasmalemmal membrane (6, 7). This influx of Ca2+ both sustains contraction and initiates the release of Ca2+ in "sparks" of activity from ryanodine-sensitive Ca2+ channels of the sarcoplasmic reticulum (15). The release of Ca2+ from ryanodine-sensitive stores activates large, Ca2+-activated K+ channels that hyperpolarize the membrane and inactivate VGCC in a negative feedback manner (4, 15).

Although VGCC are necessary for the maintenance of contractile tone in smooth muscle, it is not clear whether these calcium channels are operative in MCs. It was demonstrated with fluorescence microscopy that membrane depolarization by high KCl increases [Ca2+]i in rat MC (25, 31). Whole cell patch-clamp studies in rat MC revealed VGCC that were sensitive to the dihydropyridine antagonist nifedipine and agonist BAY K 8644 (21). However, in human MC, measurements of [Ca2+]i suggested that VGCC are not operative (19). Moreover, the single-channel properties of VGCC in human or rat MCs have not been described.

The present study was performed to determine whether human glomerular MC possess VGCC activated during an agonist-induced contraction and whether these VGCC possess pharmacological and single-channel properties similar to L-type Ca2+ channels of other excitable cells. Our observations regarding the pharmacological sensitivity of Ca2+ influx, together with single-channel analysis, indicate that ANG II leads to activation of L-type Ca2+ channels, allowing Ca2+ to enter the cell in a voltage-dependent manner after release of Ca2+ from intracellular stores.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MC cultures. Human MC, originally isolated by the laboratory of Hanna Abboud, were subcultured to no more than 10 generations by standard methods (13). Beyond 10 generations, MC lose the phenotypic shape that is characteristic of smooth muscle cells. Cells were cultured in DMEM media (pH 7.4) supplemented with 10 mM HEPES, 2.0 mM glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 µg/ml streptomycin, and 17% fetal bovine serum. For patch-clamp experiments, cells were grown on coverslips (22 × 22 mm; Fisher, Pittsburgh, PA) and maintained in a humidified tissue culture incubator at 37°C, 5% CO2 (IR Autoflow; Nuaire, Plymouth, MN).

Patch-clamp procedure. Patch-clamp experiments were performed with the pipette attached to the membrane (cell attached). The bath solution contained (in mM) 140 KCl, 10 HEPES, 2 MgCl2, and 1 CaCl2, pH 7.4. The pipette solution contained 90 mM BaCl2 and 10 mM HEPES, pH 7.4. The free Ca2+ concentration of the bath was adjusted to 1.0 µM by buffering with 1.08 mM EGTA, according to the Ca2+ concentration program by MTK Software.

Single-channel analysis was made at 23°C using standard patch-clamp techniques as previously described (10, 23). The patch pipette was inserted into a polycarbonate holder connected to the head stage of the patch amplifier (patch clamp model 501A; Warner Instrument, Hamden, CT) by an Ag-AgCl wire. After lowering the pipette onto the cell membrane, a high-resistance (>5 GOmega ) seal was obtained by applying suction. The unitary current (i) was determined as the mean of the best-fit Gaussian distribution of the amplitude histograms and was defined as zero for the closed state (C). Channels were considered to be in an open state (S) when the total current (I) was >(n-1/2)i and <(n + 1/2)i, where n is the maximum number of current levels observed. The open probability (Po) was defined as the time spent in a conducting state (S) divided by the total time of the recording. When multiple channels occupied a patch, the channel activity was calculated as NPo = Sigma nPn, where Pn is the probability of finding n channels open. Currents were recorded and analyzed using the Axoscope acquisition program and pClamp program set 6.02 (Axon Instruments, Foster City, CA).

Fura 2 measurements of [Ca2+]i. Measurements of [Ca2+]i in human MC using fura 2 was performed as previously described (5, 9). Dual-excitation wavelength fluorescence microscopy was used to monitor [Ca2+]i of individual MC. The perfusion chamber (Warner RC-2OH) was mounted on the stage of an inverted microscope (Nikon Diaphot 300). Cells attached to the coverslip floor of the chamber were illuminated alternately with light at 340- and 380-nm wavelengths (3-nm bandwidths) provided by a Deltascan dual monochromator system (Photon Technology International, Monmouth Junction, NJ). An adjustable optical sampling window was positioned to allow emission fluorescence (510 nm, 20-nm band pass) from a single cell to be detected by a photon-counting photomultiplier. Background-corrected data were collected at 5 points/s, stored, and processed using the FELIX software package (Photon Technologies). Calibration of the fura 2 signal was performed according to established methods (9), as described in detail previously (5). Cells were fura 2 loaded with 60-min incubation (37°C) in Waymouth culture media containing 7 µM fura 2-AM, 0.09 g/dl DMSO, and 0.018 g/dl Pluronic F-127 (Molecular Probes, Eugene, OR). The bathing solution contained (in mM) 135 NaCl, 5 KCl, 10 HEPES, and 1 CaCl2. In some experiments, bath [Ca2+] was reduced to less than 10 nM by addition of EGTA, and [K+] was increased to 75 or 140 mM by substituting for Na+.

Diltiazem and BAY K 8644 were purchased from Sigma Chemical (St. Louis, MO). Differences among groups of data were determined using the one-way ANOVA plus Student-Newman-Keuls test. P < 0.05 was considered statistically significant. Data are reported as means ± SE; n = number of cells.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Measurements of [Ca2+]i. Figure 1 illustrates the impact of VGCC blockade and external [Ca2+]i on the responses of human MC to ANG II. Figure 1A depicts representative [Ca2+]i responses to 1 µM ANG II and the effects of VGCC blockade and removal of bath Ca2+ on this response. In the presence of 1 mM Ca2+ (top, Fig. 1A), ANG II evoked a transient increase in [Ca2+]i from 39 nM (baseline) to 730 nM (peak), subsequently subsiding to a sustained value (68 nM) that was ~75% greater than baseline in this example. Neither diltiazem nor the nominally Ca2+-free bath significantly influenced baseline [Ca2+]i; however, the pattern of the response to ANG II was modified by these treatments. In the nominally Ca2+-free bath (middle, Fig. 1A), [Ca2+]i transiently increased from 41 to 950 nM in response to ANG II; however, [Ca2+]i subsequently declined to a value less than baseline despite continued exposure to ANG II. During treatment with 5 µM diltiazem in the presence of 1 mM Ca2+ (bottom, Fig. 1A), ANG II still elicited a large transient increase in [Ca2+]i (from 53 to 1,239 nM in this example), but the sustained response was reduced to a value approximating baseline. There was no significant difference in the peak response to ANG II in the presence (Delta  = 420 ± 73 nM, n = 7) or absence of Ca2+ (Delta  = 609 ± 99 nM, n = 6) or with addition of diltiazem to the Ca2+-containing bath (Delta  = 723 ± 225 nM, n = 6). As shown in the summary bar graph of Fig. 1B, ANG II increased the sustained [Ca2+]i response in a dose-dependent manner at peptide concentrations of 10 nM, 100 nM, and 1 µM in the presence of external Ca2+. The sustained [Ca2+]i response evoked by 1 µM ANG II was significantly greater than the response to 10 nM ANG II. The sustained [Ca2+]i response to 1 µM ANG II achieved a value significantly below baseline in the absence of external Ca2+ and was abolished by addition of diltiazem to the Ca2+-containing bathing solution.



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Fig. 1.   Effects of extracellular [Ca2+] and diltiazem on mesangial cell (MC) intracellular Ca2+ concentration ([Ca2+]i) (plotted on log scale) responses to ANG II. A: representative responses to 1 µM ANG II observed in presence of 1 mM Ca2+ (top), in the nominally Ca2+-free bath (middle), and upon addition of 5 µM diltiazem to the Ca2+-containing bath (bottom). B: average sustained [Ca2+]i responses to ANG II, expressed as percentage change from baseline. Bath concentrations of ANG II, diltiazem (Dltzm), and Ca2+ are specified for each experiment. *P < 0.05 vs. baseline; dagger P < 0.05 vs. 1 µM ANG II + 1 mM Ca2+ (0 Dltzm); n = 5-7 cells.

To determine whether a voltage-dependent mechanism can drive Ca2+ entry into these cells, some cells were subjected to membrane depolarization achieved by increasing bath [K+] from 5 to 75 mM. Typical responses are illustrated in Fig. 2A. The top tracing in Fig. 2A illustrates that K+-induced membrane depolarization increased [Ca2+]i from 31 nM to a peak value of 571 nM in the presence of 1 mM Ca2+, subsequently achieving a sustained plateau at ~41 nM in this example. In the nominally Ca2+-free bath (middle, Fig. 2A) or in the presence of both 1 mM Ca2+ and 5 µM diltiazem (bottom, Fig. 2A), K+-induced membrane depolarization did not evoke any change in [Ca2+]i. Results from these experiments are summarized in Fig. 2B, indicating that the significant increase in [Ca2+]i evoked by 75 mM K+ (in the presence of 1 mM Ca2+) was abolished by diltiazem treatment or removal of bath Ca2+.



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Fig. 2.   Effects of extracellular [Ca2+] and diltiazem on MC [Ca2+]i responses to membrane depolarization, achieved by increasing bath [K+] from 5 to 75 mM. A: representative responses to 75 mM K+ observed in presence of 1 mM Ca2+ (top), in the nominally Ca2+-free bath (middle), and upon addition of 5 µM diltiazem to the Ca2+-containing bath (bottom). B: average sustained [Ca2+]i responses to 75 mM K+, expressed as percentage change from baseline. Bath concentrations of Ca2+ and diltiazem (Dltzm) are specified for each experiment. *P < 0.05 vs. baseline; dagger P < 0.05 vs. 1 mM Ca2+ (0 Dltzm); n = 7-9 cells.

At depolarizing potentials, the dihydropyridine agonist BAY K 8644 promotes Ca2+ influx through VGCC in several cell types. Accordingly, further experiments assessed the impact of BAY K 8644 on [Ca2+]i in human MC studied during K+-induced membrane depolarization. Figure 3A presents representative tracings illustrating the critical involvement of Ca2+ influx in evoking the [Ca2+]i response to BAY K 8644 in solutions containing 140 mM K+. Exposure to 10 µM BAY K 8644 (top, Fig. 3A) increased [Ca2+]i from 48 nM to a maximal value of 565 nM, with a subsequent sustained plateau at 65 nM (35% above baseline in this example). In contrast, exposure of depolarized MCs to 10 µM BAY K 8644 under nominally Ca2+-free conditions failed to elicit any change in [Ca2+]i (bottom, Fig. 3A). The impact of BAY K 8644 on mesangial [Ca2+]i is summarized in Fig. 3B. In the presence of 75 mM K+, 1 and 10 µM BAY K 8644 evoked sustained increases in [Ca2+]i that averaged 18 and 25% above baseline, respectively. Sustained responses to 10 µM BAY K 8644 were not significantly enhanced by further membrane depolarization (140 mM K+) but were abolished in the absence of extracellular Ca2+. These observations document the presence of a dihydropyridine-sensitive Ca2+ influx mechanism in human MC studied under depolarizing conditions.



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Fig. 3.   Mesangial [Ca2+]i responses to BAY K 8644 observed under depolarizing conditions (75 mM external K+) A: representative responses to BAY K 8644 (10 µM) with 1 mM Ca in bath (top) and under nominally Ca2+-free conditions (bottom). B: average sustained [Ca2+]i responses to BAY K 8644, expressed as percentage change from baseline. Bath concentrations of K+, BAY K 8644, and Ca2+ are specified for each experiment. *P < 0.05 vs. baseline (n = 6-9 cells).

Table 1 shows a summary of the absolute baseline and sustained response values for [Ca2+]i evoked by the various experimental conditions of this study. In groups which contained 1 mM external Ca2+, basal levels of [Ca2+]i varied from 32.9 ± 3.0 to 46.7 ± 9.0 nM. In groups in which external [Ca2+]i was reduced to less than 10 nM with EGTA, baseline values ranged from 27.3 ± 3.8 to 30.2 ± 3.5 nM. None of these baseline values was significantly different from the other values when using ANOVA plus Student-Newman-Keuls test. However, the baseline [Ca2+]i for the combined groups with <10 nM external calcium (27.0 ± 1.9, n = 24) was significantly less (P < 0.001) than the combined groups with 1 mM external calcium (39.4 ± 2.1, n = 63) when compared using the unpaired t-test.

                              
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Table 1.   Baseline and sustained response values of [Ca2+]i

Patch-clamp experiments. Single-channel current analysis (Fig. 4) using the patch-clamp technique provided direct evidence for the existence of L-type Ca2+ channels in MC. Figure 4A shows typical tracings of single-channel currents in the cell-attached configuration with 90 mM BaCl2 in the presence and absence of 10 µM BAY K 8644 in the pipette. In the absence of BAY K 8644, inward currents of this amplitude were never observed. In the presence of 10 µM BAY K 8644, single-channel currents were observed at potentials as low as -30 mV. As shown in Fig. 4B, the open probability increased in an exponential manner from 0.03 at -30 mV and saturated at 0 mV at a Po of 0.87. Figure 4C shows a summary current-voltage plot of five separate Ca2+ channels. The single-channel conductance was 11.2 pS, and the extrapolated reversal potential was 33 mV. Assuming [K+]i = 120 mM, we estimated the selectivity for Ba2+/K+ at 4.2. 




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Fig. 4.   Patch-clamp analysis of single-channel inward currents in cell-attached configuration. Pipette solution contained 90 mM BaCl2 and 10 µM BAY K 8644. Bathing solution contained 140 mM KCl. A: tracings of single-channel inward barium currents at various command potentials (-Vp = pipette potential). Voltage-gated calcium channels (VGCC) were observed only in presence of 10 µM BAY K 8644 in pipette. B: summary current-voltage (I-V) plot of 5 separate Ca2+ channels found in cell-attached patches. Single-channel conductance is 11.2 pS, and extrapolated reversal potential is 33 mV. C: plot of -Vp vs. Po of single Ca2+ channel in cell-attached patch.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study provide evidence for a role for a VGCC in the sustained entry of Ca2+ into MC during an agonist-induced contraction. The ANG II-induced sustained increase in [Ca2+]i is mimicked by depolarizing the cell with 75 mM K+, indicating that the Ca2+ entry pathway is voltage dependent. That this VGCC is of the L-type variety is supported by the effects of diltiazem, which attenuated the sustained [Ca2+]i responses to ANG II and K+-induced depolarization, and of BAY K 8644, which evoked an increase in sustained [Ca2+]i. This contention is further supported by the ability of BAY K 8644 to enhance single-channel barium currents in a voltage range consistent with the L-type VGCC.

Analysis of the sustained increase in [Ca2+]i. The results of this study are consistent with earlier studies that demonstrated an elevation of [Ca2+]i in rat GMC upon depolarizing the membrane potential with high-K+ solution (25, 31). However, other studies indicated that human MC do not possess VGCC (19). This latter study showed that an increase in [Ca2+]i, resulting from raising external Ca2+, was not potentiated by membrane depolarization with 50 mM K+. This result may have been partly due to analyzing the average [Ca2+]i of several cells in a monolayer rather than focusing on individual cells. Because some cells have a delayed or cyclical response to elevating external [Ca2+], the average change in [Ca2+]i would appear as a large sustained elevation with extreme variability, which would make it difficult to discern a significant difference in a sustained response. Moreover, it is well known that MC in culture are phenotypically heterogeneous, with some cells not having the ability to contract. It is has been observed by this laboratory that these cells, which are "star"-shaped or "diamond"-shaped do not contract in culture and do not have an ANG II-evoked sustained [Ca2+] response. In the present study, only spindle-shaped cells, known to contract in vitro, are used in the measurements of [Ca2+]i. The response of [Ca2+]i to depolarizing potentials would be blunted if these cells were included in the analysis.

The effects of diltiazem on the sustained increase in [Ca2+]i evoked by ANG II are consistent with the view that Ca2+ enters the cell through an L-type VGCC (26). Diltiazem abolished the sustained [Ca2+]i increase but did not reduce it to the level obtained with removal of external Ca2+, which was 25% less than the original baseline. These results suggest the presence of an additional Ca2+ influx pathway, distinct from and in parallel with the VGCC that is activated during agonist-induced contraction. It is possible that this channel is a capacitative calcium entry pathway as described for lymphocytes and other nonexcitable cells (3, 14). Mené et al. (19) previously provided evidence for the existence of store-operated Ca2+ channels in human MC in culture (19). The existence of a Ca2+ influx pathway normally identified in lymphocytes is interesting since MC can acquire an immunocyte phenotype in culture and in vivo.

L-type VGCC are prototypically identified by sensitivity to dihydropyridines such as BAY K 8644 (20, 22). At 1 µM, BAY K 8644 caused a significant sustained elevation in [Ca2+]i that was eliminated when Ca2+ was removed from the bathing solution. This observation provides firm evidence for an L-type VGCC that is notably activated by BAY K 8644 in the presence of depolarizing potentials (2, 11). However, it is not understood why further depolarization of the membrane potential with 140 mM K+ instead of 75 mM K+ failed to enhance the effect of BAY K 8644. A possible explanation is that membrane depolarization by 75 mM K+ maximizes the combination of Po and electrochemical driving force for Ca2+ entry, whereas further depolarization to ~0 mV by 140 mM K+ may not enhance the Po sufficiently to overcome the reduced electrical driving force.

Single-channel properties of the mesangial VGCC. Activation of a Ca2+ influx pathway by high K+ and BAY K 8644 strongly suggests the presence of VGCC of the L-type variety in human MC in culture. Therefore, patch-clamp experiments were performed to determine the biophysical properties of these channels. Although several types of VGCC have been described in excitable cells (22), the pharmacological and biophysical properties determined with single-channel analysis in this study are most consistent with those defined for L-type channels. The voltage-dependent activation range of the mesangial VGCC was from -30 mV to a maximum of 0 mV. L-type channels in a variety of cell types are activated in the range beginning from -40 to -20 mV to a maximal Po at potentials from 0 to 20 mV (2, 8, 29, 30). Although an in depth kinetic analysis was not performed on these channels, the bursting pattern of channel gating is consistent with two closed and one open state previously described for L-type channels in chick sensory neurones (8), cardiac cells (11), and cerebral arteries (29).

Elevating the bath [K+] to 75 mM would be expected to depolarize the cell membrane potential to approximately -10 to -30 mV, an increase high enough to evoke a sustained increase in [Ca2+]i. It is not understood why L-type Ca2+ channels were not evident in cell-attached patches in the absence of BAY K 8644 in the pipette. However, in the fura 2 experiments, conditions were set to observe a response of [Ca2+]i immediately after changing the solution to high K+. In the patch-clamp experiments, the cells were depolarized in high-K+ solution for at least 30 min before observing the channel. It is possible that during this time of sustained depolarization the L-type channel in the patch downregulated via the effects of an inactivating enzyme such as a phosphatase.

The single-channel conductance of 11.2 pS is smaller than most L-type Ca2+ channels, which are normally 20-25 pS. However, similar channels of ~9-12 pS with L-type properties have been described in VSM cells from cerebral (29) and mesenteric arteries (28). Moreover, a 10-pS L-type channel has been described in renal proximal tubule cells (32). These smaller L-type channels are often found in the same cell preparations that exhibit larger conductance L-type channels (29). It is not known whether the small channel is a splice variant of the larger channel or whether it originates from a distinct gene.

In summary, the results of this study are consistent with the notion that membrane depolarization activates L-type VGCC, causing Ca2+ influx into human MC. Functional expression of this electrophysiological mechanism involving L-type Ca2+ channels for maintaining tone is further confirmation that the contractile phenotype of MC is very similar, if not identical, to that of VSM cells.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Hanna Abboud of the University of Texas Health Science Center at San Antonio for providing us with cultures of human mesangial cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49561 (to S. C. Sansom).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Physiology and Biophysics, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: ssansom{at}unmc.edu).

Received 12 April 1999; accepted in final form 31 August 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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