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Am J Physiol Heart Circ Physiol 277: H1878-H1883, 1999;
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Vol. 277, Issue 5, H1878-H1883, November 1999

cAMP production by piglet cerebral vascular smooth muscle cells: pHo, pHi, and permissive action of PGI2

Charles W. Leffler, Liliya Balabanova, and K. Keven Williams

Laboratory for Research in Neonatal Physiology, Departments of Physiology and Pediatrics, The University of Tennessee, Memphis, Tennessee 38163


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In newborn pig pial arterioles and cocultures of cerebral microvascular endothelial and smooth muscle cells, hypercapnia increases cAMP. In the intact cerebral circulation, both the increase in cAMP and the accompanying vasodilation require the presence of PGI2. Using piglet cerebral microvascular smooth muscle in primary culture, we addressed the hypothesis that, in the presence of PGI2, hypercapnia-induced changes in extracellular pH cause increases in cAMP. The stable PGI2-receptor agonist iloprost did increase production of cAMP in response to combined extracellular pH and pHi (11 ± 6 vs. 32 ± 10% in the absence and presence of 10-10 M iloprost, respectively). However, there was no positive dose-response relationship between iloprost concentration and stimulation of cAMP production by acidosis (e.g., 58 ± 9 vs. 41 ± 5% in the presence of 10-12 and 10-9 M iloprost, respectively). Rapid decreases in pHi stimulate the cAMP production. Decreases in extracellular pH do not appear to contribute further. The G protein inhibitor pertussis toxin did not augment cAMP production in response to decreasing pHi. We conclude that PGI2 receptor activation permits another mechanism to enhance cAMP generation in response to intracellular, but not extracellular, acidosis and that the mechanism of the permissive effect of PGI2 does not involve inhibition of a pertussis toxin-sensitive G protein.

newborn; cerebrovascular circulation; acidosis; prostacyclin; intracellular pH; extracellular pH


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CEREBROVASCULAR DILATION IN response to hypercapnia is associated with activation of adenylyl cyclase and subsequent elevation of cAMP in newborn pigs (17). In newborn pigs, both the dilation and cAMP elevation are endothelium-dependent phenomena (14, 18). The key endothelial messenger appears to be PGI2 (15). However, although hypercapnia stimulates PGI2 synthesis by cerebral microvascular endothelium (4), only the presence, not increasing concentrations, of PGI2 is necessary for full vasodilatory and cAMP responses of the vascular smooth muscle to hypercapnia. The increased gain produced by PGI2 in the vascular smooth muscle cAMP response to hypercapnia is referred to as the "permissive role of PGI2" (15). Combined in vivo and in vitro results suggest that the permissive action involves coupling of the PGI2 receptor (IP) through phospholipase C, diacylglycerol, and protein kinase C to augment the cAMP production in cerebral vascular smooth muscle in response to hypercapnia (12, 21). In vivo results in newborn pigs do not support the hypothesis that inhibition of a Gialpha protein associated with adenylyl cyclase is involved in the protein kinase C action (28). In fact, a pertussis toxin-sensitive G protein may couple the IP receptor to phospholipase C.

CO2 simultaneously decreases pHi and extracellular pH (pHo; see Ref. 2). The relative contributions of pHi and pHo to cerebrovascular smooth muscle responses to hypercapnia remain poorly understood. In vivo, several studies appear to indicate that the decline in pHo is the predominant, if not sole, mediator of the vasodilation to hypercapnia (9, 11, 22, 25). The assumption inherent in these experimental designs was that decreased pHo with constant CO2 (i.e., fixed acid) would not markedly affect pHi. However, we found that, even in cerebral microvascular endothelial cells expected to be uniquely resistant to extracellular perturbations, changes in pHo caused by fixed acid load (metabolic acidosis) markedly affected pHi (2). Furthermore, contrary to presumption, a rapid decline in pHi rather than any change in pHo was responsible for stimulating PGI2 synthesis by cerebral microvascular endothelial cells.

In light of these uncertainties, we undertook to simplify the system to only the vascular smooth muscle cells themselves, using cerebral microvascular smooth muscle explants in primary culture. The overall working hypothesis is, in the presence of PGI2, hypercapnia-induced changes in pHo cause increases in cAMP. Specifically, in this study, we address three hypotheses. 1) IP receptor activation augments stimulation of cAMP production in response to the combination of declining pHi and pHo. 2) Acidic stimulation of adenylyl cyclase is caused by declining pHo. 3) The permissive action of IP receptor stimulation on cAMP production is accomplished by inhibition of Gialpha .

Data presented in this study support the first hypothesis but are not consistent with hypotheses 2 and 3.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and Culture of Microvascular Smooth Muscle Cells from Newborn Pig Brain

Procedures for collection of cells to culture were reviewed and approved by the Animal Care and Use Committee at The University of Tennessee, Memphis. All procedures were done using sterile techniques.

Primary cultures of cerebral microvascular smooth muscle cells were grown as explants from isolated cerebral microvessels as described previously (4, 18). Briefly, brains were removed from newborn pigs 1-3 days old. Newborn pig brain cortex was removed under ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im) anesthesia, placed into a beaker with 40 ml cold isolation solution containing medium 199 (M199), 0.015 M HEPES, 1 U/ml heparin sodium, and antibiotic-antimycotic solution (100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B). The dura mater and attached vessels were removed from the tissue, and the tissue was washed three times with M199 isolation solution. The tissue was minced into tiny pieces using two scalpels in 20 ml of M199 isolation solution, transferred to a 40-ml Dounce homogenizer, and homogenized with 10 strokes of loose-fitting pestle. The homogenate was passed through a 300-µm nylon mesh screen. The passage was refiltered over a 60-µm nylon mesh screen. The screen was removed and placed in a 50-ml centrifuge tube containing 50 ml of M199 isolation solution. Microvessels (60-300 µm) were washed off by agitation and scraping and then were centrifuged at 500 g for 5 min. The pellet was resuspended in culture medium consisting of DMEM containing 20% FBS, 2 mg/ml sodium bicarbonate, 1 U/ml sodium heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. Isolated microvessels were directly seeded onto Matrigel-coated 12-well Costar plates. Microvessels that grew out from the ends of the vessels were grown to confluence under 5% CO2 in air at 37°C (12-14 days). These cells stain positively for alpha -smooth muscle actin, form hills and valleys characteristic of vascular smooth muscle, and demonstrate phenotypic characteristics of vascular smooth muscle ultrastructurally. At this age in culture, these cells retain the contractile phenotype as they show the classical alignment of action-myosin complexes and dense bodies, a smooth muscle contractile unit (1). To achieve quiescence, cells were exposed to serum-free medium for 15-24 h before experimentation.

Measurement of cAMP

At the end of exposure to treatments (see experimental design below) the incubation medium was aspirated, mixed with EDTA-Tris solution, pH 7.4 (final concentration 5 mM EDTA), and stored at -60°C before extracellular cAMP determinations. To determine the intracellular level of cAMP, cells were extracted with 1 ml 0.1 N HCl for 2 h at room temperature. The contents of each well were sonicated using a high-intensity cell disrupter. Aliquots were taken for protein determination, and cell homogenates were centrifuged for 2 min to remove cell debris. Cell extracts were stored at -60°C before cAMP and protein determinations.

cAMP contents in cell extracts neutralized with 1 N NaOH, and in cell media, were determined by RIA using 125I-labeled cAMP as a radioligand. Acetylation with a 2:1 mixture of triethylamine and acetic anhydride was performed immediately before the assay to increase sensitivity. cAMP content was normalized to protein content of each well, which was determined by the Bradford assay with BSA as a standard.

Measurement of pHi

Isolated microvessels were directly seeded onto 9 × 35-mm Matrigel-coated coverslips within Leighton tissue culture tubes (16 × 93 mm). The pHi of adherent smooth muscle cells was measured using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Molecular Probes, Eugene, OR). Cells attached to the coverslip were incubated for 30 min at 37°C in 1 ml artificial cerebrospinal fluid (aCSF; in mM: 3.0 KCl, 0.6 MgCl2, 2.0 CaCl2, 3.7 glucose, 6.7 urea, 127 NaCl, and 27.4 NaHCO3; pH 7.4, PCO2 36 mmHg) containing 8.35 µM BCECF-AM. Stock solutions of 1.44 mM BCECF-AM were made in DMSO. For experimentation, BCECF stock was thawed and added to loading media. After 30 min of loading, the cells were washed one time in fresh control aCSF for 15 min. Next, the coverslip containing the cells was placed in a 1-ml cuvette contained in an LS-50B spectrofluorophotometer (Perkin-Elmer) at a 45° angle to the excitation beam. Cells were then superfused at 3 ml/min with aCSF for 10 min before excitation. The temperature of the experimental chamber was kept at 37°C for all of the experiments. Measurement of the fluorescence intensity of BCECF was performed with an excitation wavelength pair of 490/440 nm (slit, 15 nm) and an emission wavelength of 540 nm (slit, 10 nm). After a relatively constant fluorescence ratio was observed, cells were superfused for an additional 5 min with aCSF to obtain basal values for pHi. The medium was then changed to an experimental medium for 15 min.

At the end of each experiment, calibration of the fluorescence ratio to pHi was performed on each coverslip by using the monovalent cation ionophore nigericin to equilibrate pHi with pHo when intracellular K+ concentration ([K+]) equaled extracellular [K+] (10, 23). The calibration solution contained 145 mM KCl, 5 mM HEPES, 20 µM nigericin, 5 mM NaCl, and 2.5 mM CaCl2. The ratio of the fluorescence intensities at 490 to 440 nm was linearly related to the pHi.

Experimental Design

All experiments were performed on confluent quiescent cells (15-24 h serum free). For the experiment, the medium was replaced with an incubation medium with a composition similar to that of cortical cerebrospinal fluid in vivo (in mM: 3.0 KCl, 1.5 MgCl2, 1.5 CaCl2, 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO3). The incubation medium was preequilibrated with 5% CO2-21% O2-74% N2 for 1 h and typically showed pH, PCO2, and PO2 of 7.40 (control pH), 32-36 mmHg, and 100-120 mmHg, respectively. Cells were incubated under the experimental conditions for 15-min periods before media and cell collection.

Experiment 1 (hypothesis 1). Cells were treated for 1 h with 1 µM indomethacin and were divided into the following two groups for 15-min experimental treatments: 1) control aCSF and 2) aCSF with pH reduced to 7.0 by addition of HCl and 80 mM sodium propionate. Such treatment causes rapid and sustained decline in pHi (Table 1 and Ref. 3), in addition to pHo. In earlier studies on endothelial cells, this treatment produced pHi/pHo changes similar to those produced by changing CO2 concentration from 5 to 14% (2). In each of the two groups, one-half of the wells was exposed simultaneously to iloprost (10-8 M; gift from Schering, Berlin, Germany).

                              
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  		Table 1.   pHi of cerebromicrovascular smooth muscle cells

In addition to the above two group experiments, the dose dependence of iloprost action was evaluated by an additional two groups of experiments, with the doses of iloprost in the treatment groups of 10-9, 10-10, 10-11, and 10-12 M.

Experiment 2 (hypothesis 2). Cells were divided into the following four groups for 15-min experimental treatments: 1) pHo 7.4, control aCSF, 2) pHo 7.4 plus 80 mM sodium propionate, which causes rapid decline in pHi, 3) pHo 7.0, which causes a slower but similar decline in pHi, and 4) pHo 7.0 plus 80 mM sodium propionate, which causes a rapid and sustained decline in pHi along with the decrease in pHo (Table 1). In each group, one-half of the wells were simultaneously exposed to iloprost.

Experiment 3 (hypothesis 3). Cells were divided into one-half treated overnight with pertussis toxin (1 µg/ml) and one-half not treated with pertussis toxin. Untreated and pertussis toxin-treated cells received experimental stimulation (15 min) of control aCSF (pH 7.4) or propionate (80 mM, pH 7.4).

Statistical Analyses

Data are presented as means ± SE of total (intracellular + media accumulation) or media cAMP per milligram protein at 15 min of treatment. Data were compared by ANOVA with Fisher's protected least-significant difference or t-test for comparisons of only two populations. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of experiment 1 are shown in Fig. 1. Combination treatment with medium of pH 7.0 containing 80 mM propionate, which rapidly decreased pHi from ~7.3 to 6.9, did not alter cAMP production by the quiescent cerebral vascular smooth muscle cells. Propionate causes rapid decreases in pHi (Ref. 3 and the results of experiment 2) and was, therefore, coupled with low medium pH to simulate the effect of hypercapnia. Addition of 10-8 M iloprost, in the absence of propionate and in pH 7.0 medium, did not significantly effect cAMP production (P = 0.45, n = 48 wells, 8 experimental runs). However, in the presence of iloprost, pH 7.0 medium plus propionate increased cAMP production (Fig. 1). The effect of iloprost on the cAMP response to acidosis was independent of the concentration of iloprost used (Fig. 2).


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  		Fig. 1.   cAMP production (total intracellular + media) by piglet cerebral microvascular smooth muscle cells in normal pH/PCO2 conditions (pH 7.4) and acidic conditions (pH 7.0, propionate) in the absence (vehicle) and presence (iloprost) of iloprost (10-8 M). Each bar represents 16 wells. * P < 0.05 compared with pH 7.4.



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  		Fig. 2.   Effect of iloprost dose on permissive role of iloprost. Data are presented as cAMP released to media in the absence and presence of iloprost at 10-9 M (each bar is 4 wells; A), 10-10 to 10-11 M (each bar is 11 wells; B), and 10-12 M (each bar is 12 wells; C). * P < 0.05 compared with normal acid/base conditions.

Experiment 2 was done to determine if the elevation of cAMP production observed in the presence of iloprost when both pHi and pHo declined was due to the drop of pHi, pHo, or both. In the absence of iloprost, propionate (pHo = 7.4), which caused a rapid (see below) decrease in pHi to ~7.1 (Table 1), did not affect cAMP production (Fig. 3). However, in the presence of iloprost, propionate stimulated cAMP production. Decreasing media pH from 7.4 to 7.0, without propionate, had no effect on cAMP production whether or not the cells were simultaneously treated with iloprost. This was true even though the minimal pHi attained, 7.1 (Table 1), was similar to the other groups. A major difference, as reported previously for endothelial cells, is that propionate and CO2 cause rapid declines in pHi, whereas that caused by fixed acid extracellularly is much slower. Thus, in the present experiments, treatment with 80 mM propionate, regardless of pHo, caused a decrease in pHi that was maximal in 96 ± 9 s. In contrast, in the absence of propionate, decreasing pHo from 7.4 to 7.0 caused a similar total decline in pHi (Table 1), but 344 ± 76 s were required to reach the minimal pHi. Finally, when extracellular media pH was decreased from 7.4 to 7.0 and propionate was also added, the results were identical to those produced by treatment with propionate and a constant pHo of 7.4. Treatment with pHo 7.0/propionate did not increase cAMP production unless iloprost was present, in which case the increase was similar to that produced by propionate alone in the presence of iloprost.


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  		Fig. 3.   Contribution of extracellular pH (pHo) vs. pHi changes in the cAMP response to acidosis by piglet cerebral microvascular smooth muscle cells (total production: cells + media). A: pHi was decreased (propionate), but extracellular pH was maintained at 7.4 (each bar is 8 wells). B: set pHo was decreased to 7.0 by reducing HCO-3 concentration in the media (each bar is 4 wells). C: treatment with pH 7.0 media and propionate (each bar is 16 wells). * P < 0.05 compared with previous normal pH conditions.

The third experiment was designed to test the hypothesis that the ability of iloprost to promote cAMP production in response to decreased pHi results from inhibition of a Gialpha coupled to adenylyl cyclase. If this hypothesis has merit, inhibition of Gialpha should have an effect similar to iloprost and should permit the increase in cAMP production in response to propionate. However, pertussis toxin had absolutely no effect on the response to pHi. In these cells, propionate increased cAMP 23 ± 9% (n = 28 wells) in cells not treated with pertussis toxin and 26 ± 7% (n = 28) in cells pretreated with pertussis toxin (both increases were significant but not different from each other). We cannot exclude the possibility that pertussis toxin was ineffective in blocking an inhibitory G protein involved in the response, but a high dose and long treatment time were used for pertussis toxin experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major new findings of the present study are 1) iloprost increases vascular smooth muscle cAMP production in response to combined pHo/pHi, 2) a rapid decrease in pHi, but not pHo, stimulates cAMP production in the presence of IP agonist, and 3) the mechanism by which IP agonists permit cAMP stimulation in response to a decrease in pHi does not appear to involve inhibition of Gialpha . The finding in the present study of the absence of any dose-response relationship between iloprost concentration and cAMP production upon stimulation with acidosis is consistent with the permissive concept in that only a particular minimum concentration of PGI2 agonist is necessary. Higher concentrations have no further effect until doses are reached that directly increase cAMP via coupling to adenylyl cyclase, above which the typical iloprost dose to cAMP response relationship occurs.

Endothelium-derived relaxing factors, including nitric oxide (NO), PGI2, and endothelium-derived hyperpolarizing factors, have been considered as transducers of intravascular signals to the vascular smooth muscle. However, another potentially integral mechanism of functioning of endothelium-derived relaxing factors is to provide information regarding the functional integrity of the vascular wall (12). In these instances, the signals are permissive in that the presence of the permissive factor, not progressively increasing concentrations, is necessary to allow a response to occur to an alternative stimulus. For example, in the newborn pig cerebral circulation, vasodilations in response to hypercapnia (15), histamine (14), and epoxyeicosatrienoic acids (13) require the presence of a IP agonist and as such are blocked by treatment with indomethacin and are restored by subdilator concentrations of topical iloprost. Furthermore, in adult rats, vasodilation in response to hypercapnia requires a minimum level of NO; vasodilation to hypercapnia is strongly inhibited by NO synthase inhibitors and is restored by low concentrations of NO that restore cGMP to apparently normal levels (5, 6, 16, 26). Of interest in this instance is that the source of the NO appears to be neuronal NO synthase of perivascular neurons (16, 20, 27), suggesting that the permissive signal may convey information of the functional integrity of microvascular innervation.

The question of whether pHi, pHo, or both are involved in cerebral vasodilation in response to hypercapnia has been debated and is still uncertain. Initial reports suggested that the response was to pHo (9, 11, 22). These studies were based on similar vasodilation in response to hypercapnia and fixed acid. The assumption was made that changes in pHo had little if any effect on pHi when the change was caused by a fixed acid. Later experiments in other species suggested that similar affects could be obtained on cerebral vessels using decreased HCO-3 in solution and elevated PCO2 (25). However, Hsu et al. (2) found that the elevation of PGI2 by cerebral microvascular endothelial cells occurred in response to a rapid decline in pHi and did not occur either with slow decline in pHi or upon producing a decrease in pHo alone. These studies coupled with the earlier studies suggested possibly that the endothelial signal was elevated by a change in pHi, whereas the ultimate vasodilation in the vascular smooth muscle cell may include a response to pHo. With the discovery that the endothelial mechanism influencing the adjacent vascular smooth muscle was a permissive one (14, 15, 24), rather than a direct vasodilation caused by elevation of the endothelial mediator and increasing the second messenger in the adjacent vascular smooth muscle cell, the possibility arose that pHo signals on the vascular smooth muscle could be ultimately responsible for producing the vasodilation in the presence of the permissive mediator. Therefore, in the present experiment, we took a different approach to this question. We examined the vascular smooth muscle second mediator signal in the presence of the permissive agonist when 1) pHi was reduced but pHo was not, 2) when pHo was reduced with pHi slowly following, and 3) when both pHi and pHo were decreased rapidly. In these studies, only when pHi was rapidly decreased was there an elevated cAMP production by the cells. We could find no evidence of a role for pHo in this signal. Thus, when pHo was decreased with fixed acid and pHi decline was comparatively slow, there is no elevation of cAMP production by the vascular smooth muscle cells, and the cAMP production in response to concomitant, rapid pHi and pHo declines was no greater then that produced by a rapid decrease in pHi when pHo was maintained constant. The results have similarities to the signal necessary to increase PGI2 synthesis by endothelial cells (2). In the endothelial cells, PGI2 synthesis was only stimulated by a rapid decline in pHi, as with hypercapnia or propionate, but not by a slow decline in pHi to the same level, as with simulated metabolic acidosis. Likewise, in the present experiments, the cellular response of vascular smooth muscle seems to be influenced by the rate of pH decline more than by the absolute pHi achieved. In both cases, the mechanisms responsible for the greater response to rapidly changing pHi have been illusive. It does appear that, at least in isolated vascular smooth muscle cells, a role for an extracellular H+ receptor can be excluded. One must remember, however, that responses in vivo tend to be far more robust than those in vitro. The possibility of additional mechanisms contributing to various responses in the intact vessel that do not function in isolated cells must not be ignored.

Although not significant in any of the individual groups, acidosis in the absence of iloprost tends to increase production of cAMP (Fig. 3). If all three groups are combined, a small increase in cAMP can be demonstrated. These results are similar to those we reported previously in response to increased CO2 affects on smooth muscle cells (18). We suggested then and suggest now that decreased pH has a small stimulatory effect to increase cAMP, the gain of which is markedly increased via permissive action from PGI2.

Finally, we investigated a potential mechanism that could be involved in the permissive effect of PGI2 on vascular smooth cells. It is unlikely that the role of the IP agonist is to cause a basal level of cAMP tone for the following reasons. 1) Other mediators that cause an increase in cAMP are totally ineffective at substituting for PGI2 agonists in producing the permissive response (14). 2) The levels of PGI2 necessary to allow vasodilation to occur in response to hypercapnia, histamine, and epoxyeicosatrienoic acids are not sufficient to cause a detectable increase in cAMP either in cortical periarachnoid fluid or in vascular smooth muscle cells (19, 17). 3) Protein kinase C stimulation can substitute for IP receptor activation (21), further suggesting that receptor activation of adenylyl cyclase may not be the mechanism.

This is in contrast to the cerebral circulation of the adult rat in which several studies indicate that the permissive role of NO is to provide a specific level of "cGMP tone" that is necessary to allow secondary vasodilation to occur in response to hypercapnia (26).

The fact that phorbol esters can substitute for IP agonists suggests that the mechanism involved in PGI2's permissive role may involve a kinase. Possible targets for phosphorylation include 1) an inhibitory G protein coupled to adenylyl cyclase, inhibition of which would promote greater production of cAMP upon stimulation by an alternative stimulus (7), 2) phosphorylation of the adenylyl cyclase itself (8), 3) phosphorylation of a stimulatory G protein coupled to adenylyl cyclase, and 4) possible inactivation of a phosphodiesterase that would tend to promote cAMP accumulation. The latter possibility seems unlikely because previous experiments suggest that inhibition of phosphodiesterase augments vasodilation in response to hypercapnia and augments the production of cAMP in response to hypercapnia, but both responses are inhibited by indomethacin (19). One would expect that, if inhibition of a phosphodiesterase were involved, inhibition of phosphodiesterase activity would produce vasodilation, tend to inhibit vasodilation to hypercapnia, and prevent blockade to vasodilation of hypercapnia by indomethacin treatment. Previous studies in vivo suggested that inhibition of Gialpha was not involved in the response, as pertussis toxin did not restore vasodilation in response to hypercapnia after treatment with indomethacin (28). In fact, such treatment prevented renewal of vasodilation to hypercapnia by IP-receptor antagonist while still allowing vasodilation to occur after treatment with phorbol ester, suggesting that the action of protein kinase C is downstream of the permissive effect of the IP receptor. The present study further suggests that inhibition of Gialpha is not involved in isolated smooth muscle cells in the permissive role of IP activation for promoting elevation of cAMP in response to a decrease in pHi. The remaining possibilities remain viable, mainly increasing the gain in the cAMP system either by direct effects on the enzyme adenylyl cyclase or via phosphorylation of a coupled stimulatory G protein.


    ACKNOWLEDGEMENTS

We thank Danny Morse and Laura Malinick for graphic assistance and Barbara Rawls for secretarial assistance. Water-soluble indomethacin was a gift from Merck (Rahway, NJ), and iloprost was a gift from Schering (Berlin, Germany).


    FOOTNOTES

This research was supported by the National Institutes of Health.

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: C. W. Leffler, Dept. of Physiology, The Univ. of Tennessee, Memphis, 894 Union Ave., Memphis, TN 38163. (E-mail: cleffler{at}physio1.utmem.edu).

Received 19 January 1999; accepted in final form 23 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(5):H1878-H1883
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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