Endothelial cGMP does not regulate basal release of endothelium-derived relaxing factor in culture NANDOR

MARCZIN,

UNA

S. RYAN,

AND JOHN

D. CATRAVAS

Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2300; and Washington University, St. Louis, Missouri 63110 Marczin, Nandor, Una S. Ryan, and John D. Catravas. Endothelial cGMP does not regulate basal release of endothelium-derived relaxing factor in culture. Am. J. Physiol. 263 (Lung CeLI. MOL. Physiol. 7):Lll3-Ll21, 1992.-Guanosine 3’,5’-cyclic monophosphate (cGMP) accumulation in single and cocultures of calf pulmonary arterial endothelial (CPAE) and rabbit pulmonary arterial smooth muscle cells (RPASM) was investigated to discover whether endothelial cGMP is involved in the feedback regulation of basally released endotheliumderived relaxing factor (EDRF). Endothelial cell-induced increases in smooth muscle cGMP levels were inhibited by competitive inhibitors of endothelial nitric oxide synthesis, NGmonomethyl-L-arginine and N”-nitro-L-arginine, in both longterm cocultures and short-term bioassay. Such treatment had no effect on endothelial content of cGMP. Coculture cGMP accumulation was stimulated (twofold increases) by endotheliurn-dependent vasodilators, bradykinin and acetylcholine. Bradykinin and acetylcholine did not elicit cGMP accumulation in single cultures of either smooth muscle or endothelial cells. To investigate the underlying mechanism(s) of dissociation in cGMP accumulation between cocultures and single endothelial cell cultures, the distribution profile of guanylate cyclase isoforms was determined by stimulating CPAE and RPASM cells with vasodilators activating selectively the soluble or particulate isoenzymes. Both nitrovasodilators, sodium nitroprusside and a putative EDRF, S-nitroso+cysteine, produced a 20-fold increase in cGMP content of RPASM cells only, having no effect on endothelial cells. Conversely, atriopeptin II caused 80-fold increases in endothelial cells. Exposure of the short-term bioassay system to 100 nM atriopeptin II, which caused 60-fold increases in CPAE cGMP levels, did not affect basal EDRF-induced smooth muscle cell cGMP accumulation, suggesting that a cGMP-mediated negative feedback mechanism does not appear to be involved in the regulation of basally released EDRF in culture. acetylcholine; atriopeptin II; bradykinin; endothelial cells; guanylate cyclase; nitrosocysteine; smooth muscle cells; sodium nitroprusside ENDOTHELIUM releases powerful humoral substances that modulate the tone of the underlying smooth muscle cells (6, 10, 30, 44) and prevent the adherence and aggregation of blood elements (30, 35). Endothelium-derived relaxing factor (EDRF) is generated by vascular endothelial cells from the amino acid L-arginine (33) and released either as nitric oxide (9,17,32) or a labile nitric oxide-containing substance (31, 42) not only in response to a variety of stimuli but also under basal conditions (27). Several lines of evidence suggest that continuous basal utilization of L-arginine as a substrate for the generation of nitric oxide is important in the regulation of vascular tone, local blood flow, and antithrombogenecity of the vessel wall. In the rabbit (38, 47), rat (48)) and guinea pig (I), inhibitory analogues of L-arginine induce a dose-dependent, long-lasting increase in blood pressure, increased adherence of blood cells to endothelium (29) and, in humans, a reduction in

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Copyright

forearm blood flow (46). Furthermore, basal release of EDRF has been demonstrated in a variety of preparations in vitro, including isolated vessels and cultured vascular cells (11, 19, 26, 42). EDRF-induced vascular relaxation and inhibition of platelet adherence to endothelial cells are associated with stimulation of the soluble form of guanylate cyclase, resulting in elevation of guanosine 3’,5’-cyclic monophosphate (cGMP) in smooth muscle cells (14, 16, 36) and in platelets (35), suggesting a paracrine mechanism of action of EDRF. It has been suggested, however, that EDRF produced in endothelial cells stimulates guanylate cyclase and increases cGMP levels not only in the target (smooth muscle) cells but also at the site of its production within the endothelial cells themselves (5, 28). It was further proposed that endothelial cGMP might regulate the generation and release of EDRF via a negative feedback mechanism (8, 28). The current study was undertaken to investigate the relationship between basal release of EDRF and endothelial cGMP content. To elucidate the effects of EDRF on endothelial cGMP accumulation, basal release of EDRF was attenuated or prevented by inhibitors of endothelial nitric oxide synthesis or stimulated by endothelium-dependent vasodilators, and the effect of such modulations of EDRF release on endothelial cell cGMP content was determined in single cultures of endothelial cells. We also studied the distribution of guanylate cyclase isoform activity between cultured endothelial and smooth muscle cells, as reflected by cGMP accumulation in response to different vasodilators that stimulate the soluble or particulate enzyme selectively. The potential regulatory role of endothelial cGMP on basal release of EDRF was further investigated by determining the release of EDRF from vasodilator-stimulated endothelial cells. MATERIALS

AND METHODS

CeZl culture. Calf pulmonary arterial (CPAE), calf aortic (CAE), and rabbit aortic (RAE) endothelial cells and rabbit pulmonary arterial smooth muscle cells (RPASM) were harvested nonenzymatically and identified utilizing previously published procedures (39). Cells were mechanically subcultured for up to 24 times, using a rubber policeman. Smooth muscle cells were grown in T-75 tissue culture flasks (Corning, Corning, NY) in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 mM sodium pyruvate, IO mM minimum essential medium-nonessential amino acids (GIBCO), penicillin (Sigma, St. Louis, MO, 10,000 U/l), and streptomycin (Sigma, 10,000 U/l). Culture medium was changed twice weekly. Confluent smooth muscle cells were subcultured (1:6) onto 24-well tissue culture plates (Costar, Cambridge, MA). Endothelial cells were grown as monolayers in T-75 tissue culture flasks (Corning) in medium

0 1992 the American

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199 (Mediatech, Washington, DC), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin, and streptomycin. Culture medium was changed twice weekly. Endothelial cells were used together with smooth muscle cells in long-term cocultures and in short-term bioassay or alone in single endothelial cultures. To establish long-term cocultures, endothelial cells were seeded on top of a confluent smooth muscle cell layer. For short-term bioassay, endothelial cells were grown on glass cover slips (12 mm diameter circles, Fisher) in 24well tissue culture plates (Costar) in the media described above. Each subculture of endothelial cells was monitored for morphology with the use of phase-contrast microscopy, size, and angiotensin-converting-enzyme activity with [3H] benzoylPhe-Ala-Pro as a substrate (7). All cultures were allowed to grow at 37°C under 5% CO, in air. Experiments were performed between passages 5 and 24 and at 3-6 days postconfluence.

tylcholine (10 PM) for 5 min after 10 min of preincubation of the cells with IBMX. Distribution of guanylate cyclase isoforms among cultured vascular cells. To assess soluble or particulate guanylate cyclase activities present in cultured vascular cells, time- and concentration-dependent cGMP accumulation in response to different vasodilators was determined. Confluent single cultures of RPASM and endothelial cells from two species and anatomic location (CPAE, CAE, RAE) were exposed to sodium nitroprusside or a putative EDRF, S-nitroso+cysteine (l&31), to stimulate the soluble isoform of guanylate cyclase, and atriopeptin to activate the particulate isoform of the enzyme (13, 45). Time and concentration effects of vasodilators on cGMP levels were studied in the absence or presence of IBMX. S-nitroso-Lcysteine was kindly provided by Dr. James Bates (Dept. of Anesthesia, The University of Iowa).

Experimental

cGMP Determination by Radioimmunoassay (RIA) Monoclonal antibody for cGMP was a generous gift from Dr. Ferid Murad (Abbott Laboratories). The radioligand ( 1251succinyl cGMP-tyrosine methyl ester) was prepared in our laboratory. Stock solutions of the succinyl tyrosine methyl esters of cGMP (Sigma) were made up in 50 mM sodium acetate buffer, pH 4.75, and iodinated by the method of Hunter and Greenwood (15) with carrier-free 125I (Du Pont-New England Nuclear, Boston, MA). The iodination reaction products were separated by reverse-phase high-performance liquid chromatography (34). RIA was performed using the Gammaflow automated RIA system (4). Standard stock solutions of cGMP (20 ,uM) were prepared in 0.1 N HCl, and the absorbance of the solution was routinely monitored spectrophotometrically (Shimadzu, UV 160-A). Standard dilutions (0.63-80 nM) were made from the stock solution. The HCl extract containing cGMP was directly used for RIA.

Design

Effect of modulation of EDRF on endothelial content of cGMP. To determine the effect of substrate or inhibitors of endothelial nitric oxide synthesis (37) on endothelial cGMP content, growth medium from endothelial cultures was aspirated, and cells were washed with Earle’s balanced salt solution. After a 30-min preincubation of the cells with L-arginine (1 mM), NWnitro-L-arginine (100 PM), or both, cultures were further incubated in Earle’s balanced salt solution containing 1 mM 3-isobutyl- 1 -methylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor, to prevent the breakdown of accumulated cGMP. In other series of experiments NG-monomethylL-arginine (300 PM) was used as inhibitor of nitric oxide synthesis. After 15 min, the medium was rapidly aspirated, and 250 ~1 of 0.1 N HCl was added to each well to stop enzymatic reactions and to extract cGMP. Thirty minutes later, the HCl extract was collected, and acid-extracted monolayers were solubilized by 1.0 N NaOH and by scraping the wells with a rubber policeman. The HCl extract was analyzed for cGMP by radioimmunoassay, and the NaOH-solubilized samples were used for protein determination. To investigate the modulatory effects of L-arginine and inhibitory analogues of L-arginine on EDRF release, the endothelial cell-elicited cGMP accumulation in long-term cocultures or in short-term bioassay of endothelial and smooth muscle cells was determined, as described before (26). Briefly, smooth muscle cells were subcultured into 24 multiwell plates, and the next day endothelial cells were seeded on top of the subconfluent smooth muscle layer. Under these conditions, small islets of cobblestone endothelial cells were formed on top of the smooth muscle layer (26), and the activity of angiotensine-converting enzyme was increased (2-fold) in cocultures compared with matched single cultures of smooth muscle cells. Age-matched single cultures of smooth muscle and endothelial cells served as control. Cocultures were maintained for 4-5 days. After 3O-min incubations with L-arginine (1 mM), N”-nitro-L-arginine (100 PM), or both, cGMP accumulation during a 15-min IBMX incubation was determined and compared with cGMP levels in single cultures. In the short-term bioassay model, endothelial cells grown on glass cover slips were selectively pretreated with L-arginine or NG-monomethyl-L-arginine, and after wash, the cover slips with the endothelial cells (lo5 cells/cover slip) were placed into wells containing smooth muscle cells. After 15 min, the smooth muscle cells and endothelial cells were separated by removing the cover slips with the endothelial cells from the wells, and cGMP levels were determined in the smooth muscle cells only. To study the effects of an increased output of EDRF on endothelial cGMP content, single cultures of endothelial cells, smooth muscle cells, and age-matched cocultures were exposed to the endothelium-dependent vasodilators bradykinin and ace-

Protein Determination Protein content of the supernatant of the centrifuged (2,000 rpm for 5 min at room temperature) NaOH-solubilized samples was measured by the Bradford method using bovine albumin, fraction V (Sigma), as a standard (3). Standard and sample aliquots were combined with the protein-binding dye (Bio-Rad, Richmond, CA), and optical density was determined at 630 nm with a multiwell plate reader (Dynatech Laboratories). Data Analysis Data are presented as means t SE of the indicated number of individual cultures. Statistical comparisons between groups were performed using the one-way analysis of variance followed by the Newman-Keuls multiple-range test, unless indicated otherwise. Differences among means were considered significant at P < 0.05. RESULTS

Effect of modulation of EDRF release on endothelial content of cGMP. Basal cGMP levels of 8.6 t 0.9

pmol mg protein-l 15 min-l were found in single cultures of CPAE. As shown in Fig. IB, preincubation of CPAE with L-arginine and NW-nitro-L-arginine alone or in the presence of L-arginine had no significant effects on basal cGMP content of the endothelial cells (120.2 t 13.2, 115 t 9.7, and 137.1 t 15.7% of control for IV”-nitro-r.,arginine, L-arginine, and NW-nitro-L-arginine + L-arginine, respectively). Similarly, no significant changes in endothelial levels of cGMP were observed after exposure of CPAE to NG-monomethyl-L-arginine (87 t 12% vs. l

l

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Fig. 1. Accumulation of guanosine 3’,5’-cyclic monophosphate (cGMP) in single cultures and long-term cocultures of vascular cells. A: cGMP levels in single cultures of calf pulmonary arterial endothelial cells (CPAE), rabbit pulmonary arterial smooth muscle cells (RPASM), or in long-term coculture of CPAE + RPASM. B: effects of inhibitor (NWnitro+arginine, L-NNA, 100 PM) or substrate (L-arginine, L-ARG, 1 mM) of endothelial synthesis of nitric oxide on cGMP accumulation in CPAE and coculture. Bars, mean t SE of 3 individual cultures for single cultures and 6 individual determinations for cocultures. * P < 0.05 from single cultures for A and P < 0.05 from corresponding control values for B.

100 t 9.6% of control, Fig. ZB). cGMP levels greater than 2O-fold (101.1 t 7.2 pmolmg protein-l 15 min-l) were found in long-term cocultures of endothelial cells with smooth muscle cells more so than in single cultures of smooth muscle cells, alone (4.3 t 0.23 pmol mg protein-l 15 min-I, Fig. 1A). As with endothelial cells, L-arginine had no effect on basal smooth muscle cGMP levels but significantly stimulated coculture cGMP accumulation (154.5 t 20.4 pmolmg protein-l min- l, Fig. 1B). Preincubation with W-nitroL-arginine resulted in an almost complete inhibition of coculture cGMP production (13.2 t 0.8 pmol mg protein-l 15 min-l, Fig. 1B). Although L-arginine did not reverse the inhibitory effects of 100 PM N”-nitro+arginine at 1 mM (Fig. lB), it significantly but only partially (50%) prevented the action of N”-nitro-L-arginine at 5 mM (data not shown). Neither treatment affected basal smooth muscle cGMP levels. As in long-term coculture, endothelial cells produced a significant increase in smooth muscle cGMP content in short-term bioassay conditions, also. Intracellular cGMP levels of 5.8 t 0.3 pmol mg protein-l 15 mine1 were found in control smooth muscle cells (treated with cover slips devoid of endothelial cells), whereas cGMP content increased to 18 t 1 pmol mg protein-l. 15 min-l in response to endothelial cells (Fig. 2A). Selective pretreatment of endothelial cells with NG-monomethyl+argil

l

l

l

l

l

CONTROL

L-ARG

L-NMMA

L-NMMA +L-ARG

Fig. 2. Short-term bioassay of cultured vascular cells. A: cGMP levels in CPAE, in RPASM cells in absence of endothelial cells (RPASM), and in RPASM after 15-min bioassay with CPAE (RPASM + CPAE). B: effects of inhibitor (NG-monomethyl+arginine, L-NMMA, 300 PM) or substrate (L-arginine, L-ARG, 1 mM) of endothelial nitric oxide synthesis on cGMP accumulation in CPAE or in smooth muscle cells exposed to pretreated endothelial cells (Bioassay). Bars, means k SE of 3 individual cultures. * P < 0.05 from single cultures for A and from corresponding control values for B. “f P < 0.05 between L-NMMA and L-NMMA + L-ARG.

nine before bioassay virtually abolished (4.89 t 0.7 pm01 mg protein -lo 15 min-l) the action of endothelial cells to stimulate smooth muscle cGMP accumulation, and L-arginine, although it had no significant effects alone, partially reversed the inhibitory effects of Wmonomethyl+arginine (11.1 t 0.4 pmol mg protein-l -15 min-l, Fig. 2B). Figure 3 presents the effects of the endothelium-dependent vasodilators acetylcholine and bradykinin on cGMP accumulation in long-term cocultures and in single cultures of endothelial or smooth muscle cells. In these series of experiments, a threefold increase in coculture cGMP was observed when compared with basal smooth muscle cGMP levels (Fig. 3A). Both acetylcholine and bradykinin induced a further elevation in cGMP from basal coculture levels. However, endothelial content of cGMP was not affected by either agent in single cultures (Fig. 3B). l

l

Different modulation of cGMP accumulation among vascular ceUs. Exposure of CPAE to sodium nitroprusside (100 PM) for 30-300 s had no effect on cGMP levels

(Fig. 4A). To investigate whether the lack of responsiveness to sodium nitroprusside is the unique characteristic of this endothelial cell line, the responsiveness of other endothelial cells from the same species but from different anatomic locations or from different species was determined. Similar to calf pulmonary arterial endothelial cells, cGMP content of endothelial cells from either

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Ll16

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Fig. 3. Effects of endothelium-dependent vasodilators acetylcholine (ACh, 10 PM) and bradykinin (Bk, 10 PM) on cGMP accumulation in long-term cocultures (A) of CPAE with RPASM or in their single cultures (B). * P < 0.05 from single cultures of smooth muscle cells (RPASM, control, 100%). t P < 0.05 from basal coculture levels (RPASM + CPAE, control).

CPAE

I

RPASM

Fig. 4. Time course of nitrovasodilator-induced cGMP accumulation in CPAE, calf (CAE) and rabbit (RAE) aortic endothelial cells and in RPASM. A: effect of sodium nitroprusside (100 PM) in absence of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). B: effects of sodium nitroprusside and a putative EDRF, S-nitroso-Lcysteine (both 100 PM). cGMP accumulation during 5-min exposure to vasodilators after lo-min preincubation with 1 mM IBMX was determined. Bars, mean t SE of 3 individual cultures.

calf or rabbit aorta was not affected by sodium nitroprusside (Fig. 4A). Conversely, the nitrovasodilator induced a rapid and time-dependent increase in smooth muscle cell cGMP accumulation. Already at 15 s, cGMP was higher among endothelial and smooth muscle cells. Both the than control, and maximal response was achieved at 120 sensitivity and maximal cGMP response of smooth muss. To elucidate whether the observed differences in cGMP cle cells was approximately one order of magnitude less accumulation among cultured vascular cells were related than those of endothelial cells. to variances in phosphodiesterase activities present Effect of atriopeptin on basal release of EDRF. The rather than differences in guanylate cyclase activities, selective distribution of guanylate cyclase activities betime-course experiments were performed in the presence tween endothelial and smooth muscle cells allowed us to of phosphodiesterase inhibitor. IBMX significantly po- investigate the potential inhibitory role of endothelial tentiated cGMP responses to sodium nitroprusside in the cGMP on the release of EDRF. To estimate EDRF resmooth muscle cells; however, no increases were observed lease from atriopeptin-stimulated endothelial cells, the in endothelial cGMP levels. Like sodium nitroprusside, short-term bioassay was established in the presence or another nitrovasodilator, S-nitroso+cysteine, stimuabsence of atriopeptin. We reasoned that with this biolated smooth muscle cGMP levels @O-fold) but had no assay model the EDRF donor endothelial cells could be effect on CPAE cells (Fig. 4B). selectively exposed to atriopeptin and the contribution of Exposure of smooth muscle cells to atriopeptin II (100 EDRF to smooth muscle cGMP levels could then be nM) had no effect on cGMP accumulation in the absence determined. To study the direct effects of atriopeptin on of IBMX; however, a twofold increase was observed after the smooth muscle cells, smooth muscle cells were prein15 min incubation with the peptide in the presence of cubated with IBMX for 10 min, and basal or atriopeptinIBMX (Fig. 5A). Atriopeptin increased cGMP levels in a stimulated cGMP accumulation (15 min) was detertime-dependent manner in CPAE cells even without mined. A few smooth muscle cultures were exposed to phosphodiesterase inhibitor (4.8 t 0.4 pmol/mg protein control endothelial cells on glass cover slips or endothelial in control, 19.3 t 3.1 pm01 mg protein-l 2 min-l, 13.5 t cells previously pretreated with an inhibitor of endothe0.6 pmol . mg protein-l 5 min-l, 11.5 t 1.8 pmol mg pro- lial nitric oxide synthesis (A@‘-nitro+arginine, 100 PM, tein-l. 15 min-l). A 5O-fold increase in cGMP content 30 min). After 15 min, the endothelial cells were removed, (253.3 t 21.7 pmol mg protein-l. 2 min-l) was observed and smooth muscle cGMP levels were determined. EDRF in the presence of IBMX (Fig. 5B). Similar increases in activity was defined as the NW-nitro-L-arginine-inhibcGMP accumulation were observed with aortic endotheitable, CPAE-induced cGMP accumulation in the lial cells (Fig. 5, C and D). As shown in Fig. 6, concensmooth muscle cells. To determine the effect of atriopeptration-dependent responses to atriopeptin were different tin on EDRF release, the bioassay was repeated in the l

l

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Fig. 5. Time course of atriopeptin-induced cells. Cells were exposed to atriopeptin (+IBMX) phosphodiesterase inhibitor. (0 min, -1BMX).

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cGMP accumulation in RPASM (A), CPAE (B), RAE (C), and CAE (D) for indicated time after lo-min preincubation without (-IBMX) or with Bars, mean t SE of 3 individual cultures. * P < 0.05 from control cultures

presence of atriopeptin. The magnitude of cGMP accumulation in the endothelial cells during the 15min exposure to atriopeptin was assessed in parallel endothelial cultures. As shown in Fig. 7, atriopeptin alone caused a 56% increase (4.6 t 0.4 to 7.2 t 0.5 pmol. mg protein-la 15 min-l) in cGMP content of smooth muscle cells treated with cover slips devoid of endothelial cells (Fig. 7, bars labeled RPASM). When the smooth muscle cells were exposed to NW-nitro+arginine-pretreated endothelial cells, smooth muscle cGMP levels were not different from those in smooth muscle cells exposed to coverslips devoid of endothelial cells (3.7 t 0.1 pmol mg protein-l 15 min-l and 8.25 t 0.5 pmol mg protein-l. 15 min-l) for vehicle or atriopeptin, respectively (Fig. 7, bars labeled RPASM+CPAE+L-NNA). In this series of experiments, 13-fold higher cGMP was found in endothelial cell-exposed smooth muscle cells than in empty cover slip-treated smooth muscle cells (64.08 t 6.7 pmol. mg protein-l. 15 min-I). EDRF activity (defined as the NWnitro+arginine-inhibitable, CPAE-induced cGMP accumulation) of 60.4 t 6.7 pmol mg protein-l 15 mine1 was calculated in the absence of atriopeptin. As shown in Fig. 7, in the presence of atriopeptin, endothelial cells stimulated smooth muscle cGMP accumulation up to 85.6 t 5.7 pmolmg protein-l 15 min. The release of EDRF (calculated as the difference of smooth muscle cGMP l

2

[min]

accumulation in response to control or NW-nitro-L-arginine-pretreated endothelial cells) was 77.36 t 5.7 pmol. mg protein-l 15 min-l, in the presence of atriopeptin. As shown in the inset, under identical conditions atriopeptin produced comparable 30-fold increases in the cGMP content of both control and NWnitro+arginine-pretreated endothelial cells. Because the release of EDRF from atriopeptin-stimulated endothelial cells was not different from that of control endothelial cells, and at the same time atriopeptin increased endothelial cGMP levels, elevated endothelial cGMP levels did not affect basal release of EDRF. To provide elevated endothelial cGMP levels at the start of the bioassay, endothelial cells were pretreated with the peptide for 2 min before establishment of the bioassay. In this experiment, the calculated contribution of endothelial cells to elevation of smooth muscle cGMP was 21.9 t 1.2 pmol mg protein-l 15 min-l, again not different from control values, in the absence of atriopeptin (23.3 t 1.7 pm01 mg protein-‘. 15 min-l). l

l

l

l

DISCUSSION

The purpose of this study was to investigate the relationship between endothelial cell content of cGMP and release of EDRF. Because EDRF is the only endogenous endothelial factor capable of stimulating soluble guanyl-

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Fig. 6. Effects of increasing concentrations of atriopeptin on cGMP accumulation in RPASM (A), CPAE (B), RAE (C), and CAE (D) cells. Cells were exposed to atriopeptin for 5 min after lomin preincubation with IBMX. Points, mean ~fr SE of 3 individual cultures. * P < 0.05 from control cultures (Control).

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ate cyclase (19), the release of EDRF was evidenced as endothelium-dependent increases in smooth muscle cGMP levels in long-term cocultures and in short-term bioassay. In this model cGMP responses, even in the absence of known pharmacological stimulants of EDRF generation, were large enough to assess the effects of various experimental conditions on the regulation of basal production of EDRF (25). Our study suggests that under basal conditions calf pulmonary endothelial cells release considerable amounts of EDRF in culture. The magnitude of endothelial-stimulated increases in smooth muscle cGMP levels was comparable to that seen after exposure of smooth muscle cells to micromolar concentrations of nitrovasodilators. This is in agreement with previous findings utilizing shortand long-term interactions between endothelial and smooth muscle cells to modulate cGMP generation (11, 19). This study demonstrates that the endotheliuminduced smooth muscle cGMP accumulation is completely dependent on endothelial synthesis of nitric oxide from L-arginine, as inhibitory analogues of L-arginine virtually abolished cGMP responses, and in the long-term

-9

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cocultures L-arginine alone potentiated cGMP accumulation. Previously we demonstrated that the action of these analogues is restricted to the endothelial cells, as no changes in basal or nitrovasodilator-elicited cGMP accumulation were observed in single cultures of smooth muscle cells (25). More specifically, selective pretreatment of endothelial cells with the inhibitors of nitric oxide synthesis before short-term bioassay blunted EDRF release. Although high amounts of EDRF were basally released from endothelial cells and the release of EDRF was abolished by N”-nitro-L-arginine and NG-monomethyl+ arginine, the simultaneous production of cGMP within the endothelial cells, albeit low, remained unchanged. This finding clearly suggests that basally released EDRF is not involved in the modulation of endothelial cGMP content in culture. This is further supported by the inability of acetylcholine and bradykinin to increase endothelial cGMP accumulation, although they significantly stimulated EDRF output. The similar results bf Loeb et al. (22), Ganz et al. (1 l), and Kuhn et al. (21) also support our conclusion.

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PREINCUBATION:

RPASM+CPAE +L-NNA

Fig. 7. Effects of atriopeptin II on basal or EDRF-stimulated intracellular cGMP content of RPASM cells in short-term bioassay. RPASM: smooth muscle cells were exposed to cover slips devoid of endothelial cells and incubated in absence (VEHICLE) or presence of atriopeptin (ANP, 100 nM, 15 min). RPASM + CPAE: smooth muscle cultures were exposed to cover slips with control CPAE cells (-LNNA) or to L-NNA (100 PM, 30 min) preincubated CPAE (+ L-NNA) with or without atriopeptin. Inset: effects of atriopeptin on cGMP accumulation in control or L-NNApretreated endothelial cells under identical conditions. Bars, mean t SE of 4 individual cultures. * P < 0.05 between corresponding cultures with control (RPASM + CPAE-L-NNA) and L-NNA-pretreated endothelial (RPASM + CPAE + L-NNA).

RPASM+CPAE -L-NNA

The mechanism underlying the aforementioned difference in cGMP accumulation between endothelial cells and cocultures appears to involve a selective distribution of guanylate cyclase isoform activities present in cultured vascular cells. We found no inducible soluble guanylate cyclase activity in endothelial cells from two species and anatomic location. The possibility that the lack of endothelial cGMP accumulation might have resulted from high phosphodiesterase activities, which facilitate the breakdown of generated cGMP, was dismissed because high concentrations of phosphodiesterase inhibitor had no effect either on basal or on nitrovasodilator-induced cGMP levels, suggesting that soluble guanylate cyclase (if at all present) may exist in an inactive and noninducible form in these cells. These results are in agreement with those of Bennett et al. (2), who demonstrated that although bovine aortic endothelial cells are capable of metabolizing nitrovasodilators, they do not respond with elevation of cGMP. On the basis of cGMP responses to nitrovasodilators or endothelium-dependent vasodilators, however, others have reported soluble guanylate cyclase activities in endothelial cells (43) . Although the reason for the discrepant results obtained by different groups of investigators on different types of endothelial cells is not clear, species differences and also culture conditions such as passage number or the presence of contaminating cell types have been suggested as responsible mechanisms. Primary cultures of pig aortic (2841) and human umbilical vein endothelial cells (5) have been reported to produce cGMP in response to nitric oxide and endotheliumdependent vasodilators . Although it was indicated that with increasing culture times vascular cells lose their ability to transduce biological responses (12) and, more specifically, guanylate cyclase activity appeared to be labile in smooth muscle cells (22), the activity of soluble guanylate cyclase in RPASM cells was well preserved in our experiments for at least up to 25 subcultures. Similar to other reports (40), we have found substantial activities of guanylate cyclase in endothelial cells, as high amounts of cGMP were accumulated in response to atriopeptin, an agent known to selectively stimulate particulate guanyl-

ate cyclase. Furthermore, binding sites and transduction mechanisms appeared to function for acetylcholine, bradykinin, atriopeptin, and isoproterenol in these cells, even at higher passages (29, probably due to our mechanical culturing method rather than repeated exposure of vascular cells to proteolytic enzymes. With regard to the selective loss of soluble guanylate cyclase in cultured endothelial cells, similarly to our experience with the lowest subculture used in this study (no. 3.), others (2, 13) were also unable to detect cGMP accumulation to nitrovasodilators, even in primary cultures. Therefore, we have concluded that cultured endothelial cells selectively express the particulate isoform of guanylate cyclase. Our coculture results also suggest that, under experimental conditions (e.g., primary cultures) when contaminating cell types possessing guanylate cyclase activities may be present (such as fibroblasts or smooth muscle cells), the potential exists to obtain elevated cGMP levels in the absence of any change in endothelial content of cGMP. On the basis of the observed inhibition of endotheliumdependent vasorelaxation to some vasodilator substances by 8bromoguanosine 3’,5’-cyclic monophosphate, the hypothesis has been proposed that increased endothelial cGMP would inhibit EDRF release (8, 28). Endothelial cells are strategically located at the interface between bloodstream and vascular smooth muscle cells; therefore, they are likely targets of circulating atria1 natriuretic peptides. We investigated whether atriopeptin stimulation of endothelial cGMP is associated with inhibition of basally released EDRF. Although atriopeptin produced large increases in endothelial cGMP levels, no inhibition of EDRF-induced smooth muscle cGMP accumulation was observed. Whereas cultured endothelial cells may lack some of the signal transduction mechanisms for cGMP (24)) atriopeptin-elicited cGMP accumulation has been reported to modulate permeability of endothelial monolayer (23) and inhibit endothelin release (20), suggesting intact cGMP mediated regulatory mechanisms in culture. Therefore, it appears that the basal activity of endothelial nitric oxide synthase is not under the control of such a mechanism.

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L120

MODULATION

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In summary, under the conditions of this study, endothelial accumulation of cGMP was dissociated from basal release of EDRF due to the selective distribution of guanylate cyclase activities among cultured vascular cells. Furthermore, atriopeptin elicited in endothelial cells cGMP increases that failed to affect EDRF action, suggesting that cGMP is not involved in the regulation of basal release of EDRF. We are pleased to acknowledge Connie Snead, Jim Parkerson, Mary Ann Roupp for her expert This work was supported HL-21568, and HL-44204 from Institute. Address for reprint requests: and Toxicology, Medical College Received

30 September

the expert technical assistance of and Livia Jozsa Marczin. We thank preparation of the manuscript. by Grants HL-35953, HL-31422, the National Heart, Lung, and Blood J. D. Catravas, Dept. of Georgia, Augusta,

1991; accepted

in final

form

of Pharmacology GA 30912-2300. 31 January

1992.

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Endothelial cGMP does not regulate basal release of endothelium-derived relaxing factor in culture.

Guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in single and cocultures of calf pulmonary arterial endothelial (CPAE) and rabbit pulmonary a...
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