Department of Pharmacology, The University of South Alabama College of Medicine, Mobile, Alabama 36688
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ABSTRACT |
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Pulmonary
microvascular endothelial cells (PMVECs) form a more restrictive
barrier to macromolecular flux than pulmonary arterial endothelial
cells (PAECs); however, the mechanisms responsible for this intrinsic
feature of PMVECs are unknown. Because cAMP improves endothelial
barrier function, we hypothesized that differences in enzyme regulation
of cAMP synthesis and/or degradation uniquely establish an elevated
content in PMVECs. PMVECs possessed 20% higher basal cAMP
concentrations than did PAECs; however, increased content was
accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs,
responsiveness to -adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and
responsiveness to phosphodiesterase inhibition (rolipram) was increased
compared with those in PAECs. Although both types of endothelial cells
express calcium-inhibited adenylyl cyclase, constitutive
PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by
maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low
ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at
elevated levels compared with PAECs, which likely contribute to
enhanced microvascular barrier function.
adenyl cyclase; phosphodiesterase; signal transduction; calcium; pulmonary edema
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INTRODUCTION |
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MORPHOLOGICAL AND BIOPHYSICAL assessment of microvessels illustrates that endothelial cells form a semipermeable barrier to fluid and protein transudation in the noninflamed lung that limits accumulation in interstitial spaces (44). However, lung inflammation is associated with endothelial cell disruption that causes fluid and protein accumulation in the underlying tissue. Inflammation can be compartmentalized to either extra-alveolar or alveolar vessels (7, 26). Stimuli such as microthrombi and neurohumoral inflammatory mediators increase pulmonary macrovascular permeability, accompanied by perivascular cuffing (7, 46). Fluid, protein, and inflammatory infiltrates present in perivascular (arterial and venular) cuffs may be cleared through lymphatics without directly impacting the functional gas-exchange area. However, stimuli such as endotoxin- and oxidant-mediated lung injury from ischemia and reperfusion or activated neutrophils increase pulmonary microvascular permeability, accompanied by interstitial edema in the lung parenchyma (6, 18, 24, 25). Such accumulation of fluid, protein, and inflammatory infiltrates directly impacts the functional gas-exchange area, resulting, in severe cases, in refractory hypoxemia.
Because refractory hypoxemia represents a clinical complication lacking effective therapy (5), significant emphasis has recently been placed on elucidating the cellular and molecular mechanisms governing the pulmonary microvascular endothelial cell (PMVEC) compared with the pulmonary arterial endothelial cell (PAEC) response to inflammatory stimuli (27). Constitutive protein flux is greatly attenuated in PMVECs compared with that in PAECs (11, 12, 20, 34, 36). This enhanced barrier property is associated with increased expression of focal adhesion complexes that promote cell-cell tethering (36). Furthermore, whereas PAECs respond to inflammatory calcium agonists with a decrease in cell-cell and cell-matrix tethering and an increase in centripetal tension that produces intercellular gaps and protein permeability (14, 23, 27), PMVECs exhibit neither a visible change in surface morphology nor an increase in macromolecular permeability (7, 20). Thus, compared with conduit endothelial cells, PMVECs express a unique phenotype characterized by increased cell-cell and cell-matrix tethering and decreased centripetal tension development.
Given this unique phenotype of PMVECs, important questions remain as to what intracellular signals regulate focal adhesion complexes that govern enhanced barrier properties. cAMP, by activating protein kinase A, directly impacts focal adhesion complex formation (1, 15-17, 31, 35, 48). Elevated cAMP promotes cell-cell and cell-matrix association, ostensibly by phosphorylation of key complex-associated proteins (1, 15-17, 31, 35, 48). Therefore, increases in cAMP may account for enhanced barrier properties of PMVECs. Also, strategies aimed at elevating cAMP may reduce inflammatory permeability by promoting cell-cell contact (2, 8, 27, 28, 37, 41). Current studies were therefore undertaken to determine whether PMVECs possess elevated cAMP content compared with that in PAECs and to critically evaluate the mechanisms regulating cAMP production and hydrolysis in the two cell types.
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METHODS |
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Isolation and culture of lung endothelial cells. Main stem PAECs were isolated from the pulmonary truncus as previously described (26). Briefly, male Sprague-Dawley rats (CD strain, 350-400 g; Charles River) were anesthetized with pentobarbital sodium (50 mg intraperitoneally), a sternotomy was performed, and the heart and lungs were excised en bloc. The pulmonary artery was isolated, cut, and inverted, and endothelial cells were obtained by gentle intimal scraping with a plastic cell lifter. The cells were seeded onto a 100-mm petri dish and grown out as previously described (26). Cells ranging between passages 5 and 15 were used for these studies. Routine verification of endothelial cell purity was performed with 1,1'-dioctadecyl-1,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein (DiI-LDL)- and factor VIII-staining criteria.
PMVECs were isolated with a modification of the technique developed by
Ryan et al. (33) as previously described by Kelly et al.
(20). Briefly, male Sprague-Dawley rats (CD strain, 350-400 g;
Charles River) were anesthetized with pentobarbital sodium (50 mg
intraperitoneally), a sternotomy was performed, and perfusion of a
physiological salt solution was established ex vivo. Once the lung was
cleared of blood, flow was established with a physiological salt
solution containing 50 mg of hyaluronidase I and collagen IV-coated
50-µm microspheres. Perfusate flow alternated between anterograde and
retrograde (0.03 ml · min1 · g
1),
and the effluent microbeads bound to endothelial cells were collected
on ice. The beads were washed, and the cells were cultured as
previously described (39). PMVECs were verified with a panel of
lectin-binding criteria, and all cells stained positive for DiI-LDL and
factor VIII.
Measurement of cAMP content. Assessment of cAMP content was performed with a standard radioimmunoassay (Biomedical Technologies, Stoughton, MA). The cells were seeded onto 2-cm2 24-well plates, PAECs at 150,000 cells/ml and PMVECs at 40,000 cells/ml, and grown to confluency over 3-4 days. Experiments were conducted with DMEM with physiological extracellular calcium concentrations unless otherwise noted and pH balanced to 7.4, with osmolality equal to 285-305 mosM. Agonists were added for 5 min, the cells were washed with DMEM, and the cells were solubilized (reactions were stopped) with 1 M NaOH. After assessment of cAMP concentrations, the results were standardized to cell counts (106 cells). Analysis of dose-response curves was performed with Prism software utilizing nonlinear regression ("Equation 1"). Derivatives were evaluated with Richardson's method.
Measurement of ATP-to-cAMP conversion. ATP-to-cAMP conversion was performed as described previously by Kelly et al. (19). Briefly, the cells were seeded onto 35-mm six-well plates, PAECs at 150,000 cells/ml and PMVECs at 40,000 cells/ml, and grown to confluence over 3-4 days. Experiments were conducted in DMEM with physiological extracellular calcium concentrations unless otherwise noted and pH balanced to 7.4, with osmolality equal to 285-305 mosM. The cells were incubated with 2 ml of [3H]adenine at a concentration of 2.0 µCi/ml for 1 h at 37°C to radiolabel the ATP pool for assessment of ATP-to-cAMP conversion. [3H]adenine was aspirated, and the cells were rinsed with DMEM. Agonists were added at the concentrations and for the time periods indicated. Fifteen microliters of 14C at a concentration of 0.01 mCi/ml was diluted in 100 ml of 5% ice-cold TCA, and 1 ml was added to stop the reactions. The wells were scraped, samples were collected, and the wells were washed with 200 µl of distilled H2O (dH2O). Two hundred microliters of sample were transferred to a scintillation vial containing 6 ml of scintillation fluid to evaluate total [3H]adenine and 14C counts. The remaining samples were centrifuged for 5 min, and the supernatants were poured over Dowex columns. The Dowex columns were then washed with 4 ml of dH2O to remove ATP from the column and then rewashed with 4 ml of dH2O to transfer cAMP onto alumina. To elute cAMP from alumina, 4 ml of 0.1 M imidazole (pH 7.1) were poured over the columns. Effluent containing cAMP was collected into scintillation vials, and 6 ml of scintillation fluid were added to the vials. Counts from purified samples were standardized to total counts. These studies were designed to evaluate the independent and combined effects of adenylyl cyclase and phosphodiesterase activity on cAMP accumulation. Studies of adenylyl cyclase turnover rates are not reported because our studies did not completely inhibit either phosphodiesterase activity or cAMP extrusion. Thus the value of percent ATP-to-cAMP conversion represents an estimate of cAMP accumulation.
Assessment of cytosolic calcium concentration. Cells seeded onto glass coverslips and grown to confluence as described in Measurement of cAMP content were loaded for 20 min with 3 µM Ca2+-sensitive fluorophore fura 2-AM (Molecular Probes, Eugene, OR) and 10% pluronic acid. The cells were rinsed with a physiological salt solution and incubated in a CO2 controlled incubator (37°C) to allow fluorophore deesterification. The cells were again washed and utilized to evaluate cytosolic calcium concentration ([Ca2+]i) as previously described (26). Experiments were routinely performed with a physiological salt solution containing physiological relevant extracellular calcium concentrations unless otherwise noted.
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RESULTS |
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Constitutive pulmonary endothelial cell
cAMP. Constitutive PMVEC and PAEC cAMP contents were
assessed in six independent experiments. cAMP content was 20% higher
in PMVECs than in PAECs (Fig. 1). Intact
cell prelabeling was used to probe the mechanisms responsible for
elevated cAMP content in PMVECs. cAMP conversion
studies showed that constitutive ATP-to-cAMP conversion in PMVECs was
7% of the conversion rate seen in PAECs.
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Stimulation of adenylyl cyclase.
Stimulation of cAMP accumulation was tested with the nonselective
-adrenergic agonist isoproterenol over a range of doses. Intrinsic
activity of isoproterenol was higher in PAECs than in PMVECs, although
the sensitivity to isoproterenol was greater in PMVECs
(EC50 73 nM;
R2 = 0.97) than
in PAECs (EC50 = 3 M;
R2 = 0.89; Fig.
2). To determine whether increased agonist
stimulation in PAECs was an intrinsic feature of the adenylyl cyclase
complex, studies were repeated in response to the direct adenylyl
cyclase activator forskolin. Intrinsic activity was
increased in PAECs compared with that in PMVECs, whereas sensitivity
was greater in PMVECs (EC50 = 200 nM; R2 = 0.97)
compared with that in PAECs
(EC50 = 9.6 µM;
R2 = 0.97; Fig.
3). Therefore, the data indicate that PMVEC
-adrenergic and direct adenylyl cyclase stimulation of cAMP
production is suppressed. Further experiments were conducted to
determine whether suppression was due to an intrinsic feature of the
adenylyl cyclases or an enhanced activity of phosphodiesterases.
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Inhibition of phosphodiesterase.
PMVECs have been shown to express predominantly phosphodiesterase 4 family isoforms absent of cGMP-hydrolyzing activities (27). Therefore,
regulation of PAEC versus PMVEC cAMP accumulation was studied with
rolipram over a range of doses. The intrinsic activity of rolipram was increased in PMVECs compared with that in PAECs, whereas the
sensitivity was greater in PAECs
(EC50 = 230 nM;
R2 = 0.99) than
in PMVECs (EC50 = 1.7 µM;
R2 = 0.99; Fig. 4). These data indicated that low
constitutive and reduced -adrenergic- and adenylyl
cyclase-stimulated cAMP accumulation in PMVECs could be due to high
phosphodiesterase 4 activity.
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To address this issue, forskolin stimulation of cAMP content was
assessed after rolipram treatment of the cells to inhibit phosphodiesterase 4 activity in PMVECs and PAECs. Rolipram
synergistically increased forskolin responses in both cell types
(P < 0.05 vs. Figs. 3 and 4). The
intrinsic activity of forskolin was similar among the cells, although
the sensitivity to forskolin was greater in PAECs
(EC50 = 3.2 µM;
R2 = 0.96) than
in PMVECs (EC50 = 30 µM;
R2 = 0.99; Fig.
5). Thus whereas stimulation of cAMP content in PAECs is
principally regulated by receptor-coupled G protein
activation of the adenylyl cyclase activity, stimulation of cAMP
accumulation in PMVECs is more effectively regulated by
phosphodiesterase 4 inhibition.
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Calcium regulation of adenylyl cyclase and
cAMP. Stevens and colleagues (40, 41) have previously
established that physiological rises in
[Ca2+]i,
especially due to activation of store-operated calcium entry, inhibit
PAEC adenylyl cyclase activity and reduce global cellular cAMP content.
To assess the regulation of PMVEC adenylyl cyclase activity by calcium,
thapsigargin was administered to both cell types to activate
store-operated calcium entry, and ATP-to-cAMP conversion was measured.
Activation of store-operated calcium entry inhibited cellular cAMP,
ranging from 20 to 50% in PAECs in six separate experiments, but had
no discernable effect on cAMP in PMVECs (Fig. 6).
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The inability of calcium to regulate PMVEC cAMP could be due to the
insensitivity of adenylyl cyclase to calcium, a reduced calcium
response to thapsigargin, or an intrinsic feature of the activation
state of adenylyl cyclase. A prior study by
Stevens et al. (40) indicated that PMVECs express type VI
calcium-inhibited adenylyl cyclase. We therefore next tested whether
store-operated calcium entry was suppressed in rat PMVECs compared with
that in PAECs. Thapsigargin was applied over a range of doses to
measure the
[Ca2+]i
response to activation of store-operated calcium entry.
Responsiveness and sensitivity to thapsigargin were suppressed in
PMVECs (EC50 = 65 nM;
R2 = 0.85)
compared with those in PAECs (EC50 = 31 nM; R2 = 0.95; Fig. 7). Thus it was possible that
decreased calcium entry accounted for the inability of thapsigargin to
decrease constitutive ATP-to-cAMP conversion in PMVECs.
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To further address this issue, thapsigargin was added to PAECs and
PMVECs in the presence of 100 nM extracellular calcium, and then
extracellular calcium was replenished. On readdition of extracellular
calcium, this protocol represented an efficient manner of increasing
[Ca2+]i
in PMVECs to equal to or greater than that observed in PAECs (Fig.
8). Sensitivity to the readdition of
extracellular calcium was similar in both cell types (PMVECs:
EC50 = 260 µM,
R2 = 0.94; PAECs:
EC50 = 195 µM,
R2 = 0.94). With
the use of this protocol to equilibrate the
[Ca2+]i
responses to thapsigargin, activation of store-operated calcium entry
decreased constitutive cAMP content, ranging from 18 to 35% in PMVECs
(Fig. 9), indicating that reduced
store-operated calcium entry uncouples calcium from regulation of
adenylyl cyclase activity in PMVECs.
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Because constitutive ATP-to-cAMP conversion in PMVECs is only
7% of that observed in PAECs and adenylyl cyclase sensitivity to
regulation by cytosolic mediators depends on the activation state of
the enzyme, we next examined whether low constitutive turnover
contributed to insensitivity of adenylyl cyclase to calcium. Rolipram
was applied to increase ATP-to-cAMP conversion, and then the cells were
challenged with thapsigargin. Inhibition of phosphodiesterase 4 activity increased the ATP-to-cAMP conversion in PMVECs, and unmasked
calcium inhibition of adenylyl cyclase (Fig.
10).
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DISCUSSION |
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Although collectively lung endothelium forms a semipermeable barrier to fluid and macromolecules, extra-alveolar endothelial cells are phenotypically distinct from alveolar endothelial cells. When compared with PAECs, PMVECs are constitutively less permeable and resistant to agonist-evoked macromolecular permeability largely due to increased expression of cell-cell adhesion proteins (11, 12, 20, 34, 36). Intracellular signal transduction mechanisms responsible for the maintenance of enhanced focal adhesion complexes regulating cell-cell adhesion proteins in PMVECs are unknown, although focal adhesion complex formation is promoted by elevated cAMP. The present studies were therefore undertaken to examine the enzymatic regulation of cAMP in PMVECs and PAECs.
Assessment of cAMP content in the two cell types indicated higher constitutive levels in PMVECs. However, the elevated content was accompanied by decreased ATP-to-cAMP conversion. Measurements of the conversion were made over a fixed time period, and although the turnover rates were not evaluated, the data are interpreted to indicate that the balance of cAMP synthesis versus degradation was higher in PAECs than in PMVECs. Thus low adenylyl cyclase and/or high phosphodiesterase activity establishes a set point for elevated cAMP content in PMVECs. cAMP concentrations typically differ between tissues and cell types. cAMP accumulation and turnover rates do not directly relate to content, indicating enzymatic regulation of cAMP production, and degradation establishes cell-specific set points. The biochemical mechanisms underlying establishment of constitutive set points are poorly understood. Nonetheless, increased PMVEC cAMP content is consistent with the hypothesis that increased constitutive cAMP promotes focal adhesion complex formation in these cells.
-Adrenergic stimulation of adenylyl cyclase is utilized clinically
to reverse airway inflammation in asthma (29, 32, 45) and peripheral
edema in urticaria (30, 47) and experimentally to prevent or reverse
pulmonary edema (2, 8, 27, 28, 37, 41).
-Adrenergic agonists elevate
endothelial cell cAMP to promote cell-cell apposition that decreases
macromolecular permeability. Although inflammatory agonists of
different etiologies target selective segments in the pulmonary
circulation, e.g., macrovascular versus microvascular (7), it is
unclear whether the biochemical response to
-adrenergic agonists
differs between macrovascular and microvascular endothelial cells. Our
data indicate that PAECs, which are sensitive to calcium agonist-evoked
increases in permeability (20), are highly responsive to
-adrenergic stimulation of cAMP, whereas PMVECs, which are insensitive to calcium
agonist-evoked increases in permeability (20), are unresponsive to
-adrenergic stimulation of cAMP.
To assess whether decreased PMVEC responsiveness to -adrenergic
stimulation of cAMP was due to receptor function or altered adenylyl
cyclase activity, studies were performed with forskolin. Responsiveness
to forskolin was decreased in PMVECs, indicating either reduced
intrinsic activity of PMVEC adenylyl cyclase complexes or increased
phosphodiesterase activity. Both cell types express similar isoforms of
adenylyl cyclase, including types II, IV, VI, and VII (8, 40).
Expression of types VIII and IX have not been rigorously tested, and
quantitative estimates of cyclase abundance have yet to be performed.
Thus it is possible that PMVECs express a lower abundance of enzyme or,
alternatively, possess unique regulatory properties that directly
decrease enzyme intrinsic activity.
However, increased responsiveness to rolipram in PMVECs suggests that phosphodiesterase activity importantly dictates cAMP content in these cells to a greater extent than in PAECs and may contribute to the apparent lack of responsiveness to adenylyl cyclase activation in PMVECs. Indeed, when responsiveness to the combination of rolipram and forskolin was assessed, similar synergistic increases in cAMP were observed in both cell types. Activation of adenylyl cyclase, therefore, most effectively increases PAEC cAMP, whereas inhibition of phosphodiesterase activity most effectively increases PMVEC cAMP. It is unclear why phosphodiesterase 4 activity may be constitutively higher or more responsive to increases in cAMP in PMVECs than in PAECs (3, 21, 22, 38). Phosphodiesterase 4 consists of A-D subfamilies with 15-20 splice variants; each exhibits Michaelis-Menten kinetic behavior (13). Molecular characteristics and the associated biochemical properties of these related products are poorly characterized in endothelial cells and, in particular, in phenotypically distinct endothelial cells.
Physiological rises in
[Ca2+]i
inhibit type VI adenylyl cyclase in PAECs and reduce global cAMP to
promote intercellular gaps and increase macromolecular permeability (9,
10, 41). Although PMVECs express the type VI enzyme, activation of
store-operated calcium entry did not reduce constitutive cAMP. Because
[Ca2+]i
responses were suppressed in PMVECs relative to those in PAECs, we
determined whether suppressed calcium entry uncoupled calcium from the
inhibition of adenylyl cyclase. Studies were designed to increase
calcium entry in PMVECs to levels similar to those achieved in PAECs.
Under these conditions, stimulation of calcium entry inhibited cAMP in
PMVECs. We also evaluated whether sensitivity of adenylyl cyclase to
calcium could be enhanced in PMVECs by increasing the activation state
of the enzyme. Prior studies indicated conditional regulation of
certain adenylyl cyclase isoforms based on the presence of cytosolic
regulators like Gs and protein
kinase C (42, 43). Because rolipram increased ATP-to-cAMP conversion, we determined whether such increased conversion rates would unmask calcium regulation of adenylyl cyclase, and, indeed, increased conversion was associated with calcium inhibition of ATP-to-cAMP conversion. Thus calcium conditionally inhibits PMVEC cAMP accumulation when either adenylyl cyclase activity is increased or calcium entry is elevated.
Taken together, cAMP is regulated by distinct mechanisms in PMVECs and PAECs. PMVECs are unresponsive to adenylyl cyclase stimulation and highly responsive to phosphodiesterase inhibition, whereas PAECs are highly responsive to adenylyl cyclase stimulation and unresponsive to phosphodiesterase inhibition. Because inflammatory mediators of different etiologies selectively target macrovascular versus microvascular segments within the pulmonary circulation, it is conceivable that rational pharmacological therapies could be developed to selectively increase cAMP in macrovascular (e.g., adenylyl cyclase activation) versus microvascular (e.g., phosphodiesterase inhibition) endothelial cells to serve an adjunctive anti-inflammatory therapy. In support of this notion, rolipram reverses microvascular dysfunction in reperfusion pulmonary edema (4) but is only partially effective at reducing macrovascular dysfunction in thapsigargin-induced pulmonary edema (P. M. Chetham, unpublished observations).
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ACKNOWLEDGEMENTS |
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We thank Dr. Paul Babal for assistance in isolating the rat pulmonary arterial endothelial cells and Linn Ayers for assistance in the ATP-to-cAMP conversion studies.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56050, HL-60024 (both to T. Stevens), and HL-46494 (to W. J. Thompson).
T. Stevens is a Parker B. Francis Pulmonary Fellow.
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: T. Stevens, Dept. of Pharmacology, Univ. of South Alabama, College of Medicine-MSB 3130, University Blvd, Mobile, AL 36688-0002 (E-mail: tstevens{at}jaguar1.usouthal.edu).
Received 8 December 1998; accepted in final form 9 March 1999.
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