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Am J Physiol Heart Circ Physiol 289: H840-H844, 2005. First published March 18, 2005; doi:10.1152/ajpheart.00026.2005
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Sphingosine 1-phosphate prevents platelet-activating factor-induced increase in hydraulic conductivity in rat mesenteric venules: pertussis toxin sensitive

Fred L. Minnear, Longkun Zhu, and Pingnian He

Department of Physiology and Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia

Submitted 11 January 2005 ; accepted in final form 11 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sphingosine 1-phosphate (S1P) is a biologically active lipid. In vitro, S1P tightens the endothelial barrier, as assessed by a rapid increase in electrical resistance and a decrease in solute permeability. We hypothesized that this activity of S1P would also occur in vivo. Hydraulic conductivity (Lp), an assessment of endothelial barrier function, was measured in individually perfused venules in rat mesenteries. S1P (1 µM) decreased basal Lp by 63% when basal Lp was between 3.6 and 4.1 x 10–7 cm·s–1·cmH2O–1 but showed no effect when basal Lp was below 2 x 10–7 cm·s–1·cmH2O–1. Under either condition, S1P blocked the sixfold increase in Lp induced by platelet-activating factor (PAF, 10 nM). Perfusion of venules with pertussis toxin (0.1 µg/ml), a specific inhibitor of the inhibitory G protein, Gi, for 3 h did not affect basal Lp or the increased Lp induced by PAF. Pertussis toxin, however, significantly attenuated the inhibitory action of S1P on the PAF-induced increase in Lp, indicating the involvement of the Gi protein. Measurement of endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) in venules loaded with fura-2 AM showed that S1P alone transiently increased basal endothelial [Ca2+]i (from 89 nM to 193 nM) but had no effect on the magnitude and time course of the PAF-induced increase in endothelial [Ca2+]i. These results indicate that S1P functions in vivo to prevent the PAF-induced increase in microvessel permeability. The inhibitory action of S1P involves the pertussis toxin-sensitive Gi protein and is not mediated by prevention of the PAF-induced increase in endothelial [Ca2+]i.

permeability; endothelium; inhibitory G protein; calcium

SPHINGOSINE 1-PHOSPHATE (S1P) is a biologically active lipid that is found in low micromolar concentrations in blood and serum. S1P has been studied extensively using cultured cells and has been shown to induce a myriad of cellular events such as proliferation, migration, survival, chemotaxis, matrix assembly, tumor cell invasion, mitogenesis, and angiogenesis (6, 18, 19). S1P also enhances the barrier function of vascular endothelia, as demonstrated by an increase in electrical resistance and a decrease in albumin permeability across endothelial cell monolayers (5, 14). The apparent contrasting activities of cell migration, which results in part from the separation of attached cells, and increased barrier function, which implicates enhancement of cell-cell contacts, raises the question of how S1P would function in vivo in an intact vessel. One study has demonstrated in vivo that S1P reduces the endotoxin-induced extravasation of albumin in lung tissue of mice (17). Therefore, we hypothesized in the present study that S1P plays an important role in maintaining endothelial barrier function in intact microvessels.

To test this hypothesis, transvascular water movement or hydraulic conductivity (Lp) was measured in individually perfused venules in rat mesenteries. We investigated whether S1P affected basal Lp and the increased Lp induced by an inflammatory mediator, platelet-activating factor (PAF). In endothelial cell monolayers, S1P enhances endothelial barrier function via a G protein-coupled receptor pathway involving the inhibitory G protein, Gi, and the S1P-1 (Edg-1) receptor, which couples primarily via Gi, as well as the S1P-3 (Edg-3) receptor (5). To determine whether S1P also affects endothelial barrier function via the Gi protein in intact microvessels, the effect of S1P on the PAF-induced increase in Lp was investigated after pretreatment of venular microvessels with pertussis toxin, a specific inhibitor of the Gi protein. We also measured changes in endothelial cytoplasmic Ca2+ concentration ([Ca2+]i). S1P has been reported to increase [Ca2+]i in cell monolayers of human umbilical vein endothelium (13). Therefore, endothelial [Ca2+]i was measured in individually perfused microvessels to determine whether an effect of S1P on the PAF-induced increase in Lp was due to modification of the increase in endothelial [Ca2+]i induced by PAF (20).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Experiments were carried out in venular microvessels in rat mesenteries. The Animal Care and Use Committee at West Virginia University approved all procedures and animal use. Female Sprague-Dawley rats (2–3 mo old, 220–250 g; Hilltop Lab Animals, Scottdale, PA) were anesthetized subcutaneously with pentobarbital sodium. Female rats were used because the mesenteric vascular bed is more developed in female rats, making it easier to select a suitable venular microvessel for cannulation, thus requiring the use of fewer rats. The initial dose of pentobarbital sodium was 65 mg/kg body wt with an additional dose of 3 mg given as needed. The trachea was intubated, and a midline surgical incision (1.5–2 cm) was made in the abdominal wall. The rat was transferred to a tray and kept warm on a heating pad. The mesentery was gently removed from the abdominal cavity and spread over a pillar for the measurement of Lp or over a glass coverslip attached to an animal tray for the measurement of endothelial [Ca2+]i. The upper surface of the mesentery was continuously superfused with mammalian Ringer solution at 37°C during preparation and experimentation. All experiments were carried out in venular microvessels with diameters ranging between 40 and 50 µm. Each experiment used a single microvessel per animal.

Measurement of hydraulic conductivity. All measurements of Lp were based on the modified Landis technique, which measures the volume flux of water across the microvessel wall (2). The assumptions and limitations of the original method and its application in mammalian microvessels have been evaluated in detail elsewhere (2, 11). Briefly, a single venular microvessel was cannulated with a glass micropipette and perfused with albumin-Ringer solution containing 1% (vol/vol) rat red blood cells as markers. A hydrostatic pressure (range 50–80 cmH2O), controlled by a water manometer, was applied through the micropipette to the microvessel lumen. The initial water filtration per unit area of the microvessel wall [(Jv/S)0, where Jv is water flux and S is unit area of the microvessel wall] was calculated from the velocity of the marker cell after the vessel was occluded, the vessel radius, and the length between the marker cell and the occlusion site. Microvessel Lp was calculated from the Starling equation, Lp = (Jv/S)/{Delta}P, where {Delta}P is the effective difference in hydrostatic and oncotic pressures across the microvessel wall. Based on experimental data and theoretical estimations, the tissue hydrostatic and oncotic pressures are assumed negligible (10, 11). {Delta}P represents the pressure difference between the hydrostatic pressure applied to the microvessel and the effective oncotic pressure generated from the albumin in the perfusate. In each experiment, baseline Lp and the stimulus-induced Lp were measured in the same venule, which allows for comparisons to be made in the same microvessel. If Lp was relatively constant throughout the time course, the mean value of Lp for each perfusate was calculated from all of the occlusions during that perfusion period. If a transient increase in Lp was observed, then Lp is reported as the means of peak and sustained values. Theoretically, the velocity of the red blood cell might overestimate Jv/S and the absolute value for Lp. In our Lp calculations, we did not correct for this potential error. However, the changes in Lp in the present study are presented as ratios of the test Lp to control Lp, which would cancel out this potential error, if it existed. The baseline Lp values were used as a reference for Lp levels. This potential error will not affect the results and conclusions.

Measurement of endothelial [Ca2+]i. Endothelial [Ca2+]i was measured in individually perfused microvessels using the fluorescent calcium indicator fura-2 AM. Experiments were conducted on a Nikon Diaphot 300 microscope equipped with a Nikon photometry system, computer-controlled shutter, and filter changer. Details have been described (8, 9), but briefly, a venular microvessel in rat mesentery was cannulated and perfused first with albumin-Ringer solution containing fura-2 AM (10 µM) for 45 min. The venule was then recannulated and perfused with albumin-Ringer solution for 10 min to remove fura-2 AM from the venular lumen. Fluorescent intensity (FI) was collected by a Nikon Fluor lens (x20, NA 0.75) from a measuring window (150 x 50 µm) positioned about 100 µm downstream from the cannulation site of the microvessel. The excitation wavelengths for fura-2 were selected by two narrow-band filters (Oriel, 340 ± 5 and 380 ± 5 nm), and the emission was separated with a dichroic mirror (DM400) and a wide-band filter (Oriel, 500 ± 35 nm). Values of FI340 and FI380 were collected with a 0.25-s exposure at each wavelength. At the end of the experiment, the microvessel was superfused with a modified Ringer solution (5 mM Mn2+ without Ca2+) while being perfused with the same solution containing ionomycin (10 µM) to bleach the Ca2+-sensitive form of fura-2. The background FIs due to unconverted fura-2 AM and other Ca2+-insensitive forms of fura-2 were subtracted from FI340 and FI380. The ratios of the two FIs were converted to Ca2+ concentrations with an in vitro calibration curve (8, 9).

Solutions and reagents. Mammalian Ringer solution was used for dissecting mesenteries, superfusing tissue, and preparing the perfusion solutions. The composition of the mammalian Ringer solution was (in mM) 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5 NaHCO3, and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and Na-HEPES. The pH of the Ringer solution was maintained at 7.40 to 7.45 by adjusting the ratio of Na-HEPES to HEPES. All perfusates used for control and test perfusion contained BSA (10 mg/ml).

D-Erythro-sphingosine-1-phosphate (S1P) was purchased from Avanti Polar Lipids, pertussis toxin was purchased from List Biological Laboratories, and 1-O-alky1–2-acetyl-sn-glycero-3-phosphocholine (PAF) was purchased from Sigma. Fura-2 AM was purchased from Molecular Probes. The stock solution of fura-2 AM (10 mM) was prepared with 100% dimethyl sulfoxide. The final concentration of fura-2 AM (10 µM) was achieved by 1:1,000 dilution of the stock with albumin-Ringer solution. PAF was initially dissolved in 95% ethyl alcohol (5 mM) and further diluted to a final concentration of 10 nM with albumin-Ringer solution. S1P was first dissolved with 95% methanol at a concentration of 1 mM and then further diluted to 1 µM with albumin-Ringer solution for experiments. Pertussis toxin (0.1 µg/ml) was prepared with albumin-Ringer solution. All perfusates containing the test agent were freshly prepared before each cannulation.

Data analyses and statistics. All values in the text are means ± SE, except where noted otherwise. The mean values (control and test) measured in the same microvessel were used as paired data. The significance of the differences within or between groups, respectively, was evaluated by a paired t-test or analysis of variance, where P < 0.05 was considered statistically significant. Changes in Lp, measured in the same microvessel, were expressed as the ratio of the test Lp versus the control Lp. In summary Figs. 1B, 2B, 3B, and 4B, asterisks indicate a significant increase from the negative control and daggers indicate a significant decrease from the positive control.



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  		Fig. 1. Sphingosine 1-phosphate (S1P) prevents platelet-activating factor (PAF)-induced increase in hydraulic conductivity (Lp). A: paired measurements of Lp in a single venular microvessel were obtained in response to PAF (10 nM) in the presence and absence of S1P (1 µM). S1P abolished the PAF-induced increase in Lp. After S1P and PAF were washed from the microvessel lumen, the Lp response to PAF was recovered to a peak value of 6 times the control value. B: summary results compare the effect of S1P on basal Lp and PAF-induced increase in Lp. When basal Lp was higher (3.9 ± 0.1 x 10–7 cm·s–1·cmH2O–1), S1P caused a significant reduction in Lp (2nd group, n = 4), whereas no significant reduction occurred in microvessels with a lower basal Lp (1.5 ± 0.1 x 10–7 cm·s–1·cmH2O–1; 1st group, n = 6). In both groups, S1P significantly suppressed the PAF-induced increase in Lp ({dagger}P < 0.05). The third group shows a normal PAF response in the absence of S1P (*P < 0.05 vs. control, n = 7).

 


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  		Fig. 2. S1P increases endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) but does not effect PAF-induced increase in endothelial [Ca2+]i. A: an individual experiment shows magnitude and time course of changes in endothelial [Ca2+]i induced by S1P alone and in response to PAF in the presence of S1P. S1P alone caused a smaller transient increase in endothelial [Ca2+]i but did not modify the PAF-induced increase in [Ca2+]i. B: summary data compare the magnitude of PAF-induced increase in endothelial [Ca2+]i in the presence and absence of S1P. *P < 0.05 vs. control.

 


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  		Fig. 3. Pertussis toxin (PTx) has no effect on basal Lp and on PAF-induced increase in Lp. A: paired measurements of Lp in a single microvessel were obtained in response to PAF (10 nM) in the absence and presence of PTx (0.1 µg/ml). Perfusion with PTx for 3 h did not modify either basal Lp or PAF-induced increase in Lp. B: summary results from 4 microvessels. *P < 0.05 vs. control.

 


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  		Fig. 4. PTx attenuates the inhibitory effect of S1P on PAF-induced increase in Lp. A: an individual experiment shows the time course of changes in Lp in response to PAF in the presence of S1P in a vessel that was pretreated with PTx (0.1 µg/ml) for 3 h. PTx had no effect on basal Lp but did significantly attenuate the inhibitory effect of S1P on the PAF-induced increase in Lp. B: summary results from 4 microvessels. *P < 0.05 vs. control.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
S1P prevents PAF-induced increase in Lp. Perfusion of PAF in seven venular microvessels increased Lp by 6.0 ± 0.9 times the control value. The effect of S1P (1 µM) on the PAF (10 nM)-induced increase in microvessel Lp was examined in 10 microvessels from 10 different animals. After the measurement of baseline Lp, S1P was applied for 20 min to the perfusate before exposure to PAF. In 4 of the 10 microvessels, baseline Lp was between 3.6 and 4.1 x 10–7 cm·s–1·cmH2O–1, with a mean value of 3.9 ± 0.1 x 10–7 cm·s–1·cmH2O–1. In these four microvessels, the perfusion of S1P caused a significant decrease in Lp to 37 ± 7% of the original control value. In the other six microvessels, the mean Lp was 1.5 ± 0.1 x 10–7 cm·s–1·cmH2O–1 (ranging between 1.1 and 1.8 x 10–7 cm·s–1·cmH2O–1). When each of these six microvessels was perfused with S1P, there was a slight but not statistically significant reduction of the baseline Lp to a mean value of 1.3 ± 0.1 x 10–7 cm·s–1·cmH2O–1. In the 10 venular microvessels, perfusion of PAF (10 nM) did not increase Lp in the presence of S1P; mean peak Lp was 1.3 ± 0.5 and 1.3 ± 0.02 times that of the two different levels of basal Lp. The reversibility of the inhibitory effect of S1P on the PAF response was examined in 2 of the 10 microvessels. After a 30-min perfusion with albumin-Ringer solution to wash S1P and PAF from the vessel lumen, a second perfusion of PAF alone increased Lp to 6.5 ± 0.2 times the control value. Figure 1A shows the time course and the magnitude of changes in Lp from one of the experiments. Figure 1B shows the comparison of the mean responses of PAF in the presence and absence of S1P.

S1P increases endothelial [Ca2+]i but does not modify PAF-induced increase in endothelial [Ca2+]i. To test whether S1P prevented the PAF-induced increase in Lp by inhibiting the PAF-induced increase in endothelial [Ca2+]i, changes in endothelial [Ca2+]i in response to PAF or S1P or S1P plus PAF were measured in eight venular microvessels in eight rats. PAF (10 nM) transiently increased endothelial [Ca2+]i, in the four microvessels studied, from a mean baseline [Ca2+]i of 72 ± 11 nM to a mean peak value of 415 ± 16 nM; [Ca2+]i fell to 178 ± 16 at 15 min. Four additional experiments were conducted to examine the effect of S1P on the PAF-induced increase in endothelial [Ca2+]i. Perfusion with S1P increased [Ca2+]i from a basal value of 89 ± 6 nM to a mean peak value of 193 ± 22 nM within 1 min; [Ca2+]i subsequently returned to the baseline value within 5 min. When each venule was perfused with PAF in the presence of S1P, the mean [Ca2+]i increased to 386 ± 24 nM, which was not significantly different from the PAF response in the absence of S1P. Figure 2A shows the time course of the changes in endothelial [Ca2+]i of an individual experiment, and Fig. 2B summarizes the mean peak increases in [Ca2+]i in response to PAF in the presence and absence of S1P.

Pertussis toxin has no effect on basal Lp and PAF-induced increase in Lp. S1P has been demonstrated in cultured cells to tighten the endothelial barrier, as assessed by measurements of electrical resistance and albumin permeability, via a signaling pathway involving the inhibitory G protein, Gi (5). In the present study, pertussis toxin, which causes the ADP-ribosylation of Gi and, thus, inhibits the protein, was used to examine the potential involvement of the Gi protein in the inhibitory effect of S1P on the PAF-induced increase in Lp. We first evaluated the effect of pertussis toxin alone on basal Lp and on the PAF-induced increase in Lp, considering PAF can induce cellular events via Gi and Gq (3, 4). The mean control Lp measured in four microvessels was 1.7 ± 0.3 x 10–7 cm·s–1·cmH2O–1. Basal Lp was not changed immediately or after 3 h of perfusion with pertussis toxin (0.1 µg/ml); mean Lp at 3 h was 1.8 ± 0.5 x 10–7 cm·s–1·cmH2O–1. Each microvessel was then exposed to PAF (10 nM) in the presence of pertussis toxin. The mean peak increase in Lp was 5.7 ± 1.0 times that of the control value, which was not significantly different from the mean peak increase in Lp measured when PAF was perfused in the absence of pertussis toxin. These results demonstrated that pertussis toxin has no effect on basal Lp and the increase in Lp induced by PAF. Figure 3A shows the paired measurements of Lp in the same venular microvessel in the absence and presence of pertussis toxin, and Fig. 3B summarizes the results.

Pertussis toxin attenuates inhibitory effect of S1P on PAF-induced increase in microvessel Lp. To determine whether the Gi protein-signaling pathway was involved in the inhibitory effect of S1P on the increased Lp induced by PAF, four microvessels were pretreated with pertussis toxin (0.1 µg/ml) for 3 h and then perfused with S1P for 20 min, followed by PAF. Baseline Lp was 1.4 ± 0.1 x 10–7 cm·s–1·cmH2O–1, and Lp did not change after pretreatment with pertussis toxin (1.5 ± 0.2 cm·s–1·cmH2O–1) or the subsequent perfusion of S1P (1.4 ± 0.1 cm·s–1·cmH2O–1). When each venular microvessel was exposed to PAF in the presence of pertussis toxin and S1P, Lp increased to a mean peak value of 4.9 ± 0.4 times the control value. Therefore, pretreatment with pertussis toxin significantly attenuated the inhibitory effect of S1P on the PAF-induced increase in Lp. Figure 4A shows the time course of changes in Lp in a single experiment, and Fig. 4B summarizes the results.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We hypothesized that S1P would play an important role in maintaining endothelial barrier function in intact microvessels, as has been demonstrated in vitro using endothelial cell monolayers (5). To test this hypothesis, the barrier function of intact vascular endothelia lining individually perfused venules in rat mesentery was assessed by the measurement of Lp. PAF was used as a representative receptor-mediated inflammatory mediator to induce a transient increase in Lp in mesenteric venular microvessels (21).

Our results demonstrated that S1P (1 µM) had no effect on basal Lp, unless basal Lp was slightly elevated. However, perfusion of S1P for 20 min blocked the sixfold increase in Lp induced by PAF (10 nM). The inhibitory effect of S1P was reversible, as a second perfusion of PAF, following washout of S1P and PAF for 30 min, increased Lp by 6.5-fold. Pretreatment with pertussis toxin (0.1 µg/ml) prevented this inhibitory action of S1P on the PAF response but had no effect on the response to PAF alone. In addition, S1P (1 µM) transiently increased the level of intracellular Ca2+ from 89 to 193 nM, but S1P had no effect on the magnitude or time course of the PAF-induced increase in endothelial [Ca2+]i. Therefore, we have demonstrated for the first time that S1P prevents the increase in permeability induced by an inflammatory mediator in intact microvessels. We conclude that this activity of S1P involves the Gi protein. Furthermore, the inhibitory action of S1P is not mediated by prevention of the PAF-induced increase in endothelial [Ca2+]i.

In the present study, S1P had no effect on Lp under basal conditions when Lp was in the normal range of ~2 x 10–7 cm·s–1·cmH2O–1 or lower (2, 11). However, in a few venular microvessels, basal Lp was double that of normal, or 3.6 to 4.1 x 10–7 cm·s–1·cmH2O–1, and in those venules, S1P reduced by 63% the basal value of Lp. Previous in vitro studies have demonstrated that S1P enhances the baseline barrier function of endothelial cell monolayers (5). It is understandable that S1P affects basal permeability under cell culture conditions considering that cultured, endothelial cell monolayers are less restrictive than endothelial cells in intact vessels to the paracellular passage of protein and water. The reduction of basal Lp in venular microvessels with high basal values of Lp probably reflects in some way the role of S1P in maintaining endothelial barrier function.

PAF induced a transient sixfold increase in Lp in venular microvessels, and S1P prevented this PAF response in the present study. The inhibitory activity of S1P on the PAF-induced increase in Lp was pertussis toxin sensitive; however, the PAF response was not sensitive to pertussis toxin. Pretreatment with pertussis toxin, which causes ADP ribosylation and inhibits Gi, has also been shown to block the S1P-induced increase in basal electrical resistance across endothelial cell monolayers (5). Similar results have been obtained by depletion of the S1P-1 (Edg-1) or the S1P-3 (Edg-3) receptor or Gi{beta}{gamma} with antisense technology (5).

The cellular mechanism that S1P initiates to inhibit the increase in microvessel Lp induced by PAF is not clear at the present time. S1P prevents and reverses the decrease in endothelial electrical resistance induced by thrombin, which like PAF activates the Gq protein (5). Fluorescent staining of actin with a fluorochrome conjugated to phalloidin reveals at the cell periphery that thrombin disassembles actin (15), whereas S1P enhances the appearance of actin (5). PAF increases the production of nitric oxide in individually perfused intact microvessels (20), and nitric oxide as well as reactive oxygen species has been reported to mediate the PAF-induced increase in microvascular permeability (12). Interestingly, S1P also stimulates within 5 min the phosphorylation of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide in bovine lung microvascular endothelial cells (16). However, there is no evidence to link eNOS, nitric oxide, or reactive oxygen species to the barrier-enhancing activity of S1P. S1P also increases in vitro the activity of the small GTPase, Rac1, and this increased activity can be inhibited by pertussis toxin, implicating signaling via the Gi protein (5). Rac1 has been implicated in the regulation of endothelial barrier function, although it is not understood whether this regulation takes place at the level of adherens junction proteins and/or at the cortical actin network (reviewed in Ref. 14).

In the present study, S1P increased endothelial [Ca2+]i from 89 to 193 nM but had no effect on the magnitude and time course of the larger increase in endothelial [Ca2+]i induced by PAF. Previous studies in venular microvessels from the frog, hamster, and rat have demonstrated a close relationship between the magnitude of the initial increase in endothelial [Ca2+]i and the magnitude of the initial increase in microvessel permeability (7–9). Agonists that demonstrate this close relationship include PAF, ATP, vascular endothelial growth factor, and calcium ionophore (1, 8, 9, 20). Attenuation of agonist-induced Ca2+ influx through conductive pathways by reduction of the electrochemical driving force reduces the agonist-induced increase in microvessel permeability (7, 9). Therefore, to our knowledge, S1P is the first physiological agent that enhances the barrier function of intact microvessels in conjunction with an increase in endothelial [Ca2+]i.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-56237 (to P. He) and HL-68079 (to F. L. Minnear).


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. L. Minnear, Dept. of Physiology and Pharmacology, PO Box 9229, Robert C. Byrd Health Sciences Center, West Virginia Univ., Morgantown, WV 26506 (E-mail: fminnear{at}hsc.wvu.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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