Altered Expression of Cyclic Nucleotide Phosphodiesterase Isozymes during Culture of Aortic Endothelial Cells

doi:10.1016/S0006-2952(97)00287-6

Biochemical Pharmacology

Volume 54, Issue 10, 15 November 1997, Pages 1071-1079

https://doi.org/10.1016/S0006-2952(97)00287-6 Get rights and content

Abstract

Primary cultures of bovine aortic endothelial cells (BAEC) express cyclic nucleotide phosphodiesterase (CN PDE) isozymes of the PDE2, PDE4 and PDE5 gene families. We report here that the isozyme profiles of CN PDE and the amounts of each vary with the passage number of BAEC cultures. Characterization by anion-exchange chromatography and pharmacological criteria were used to study CN PDE in early (4–6), intermediate (6–10), and late (>17) passages of purified BAEC. PDE2 and a minor fraction of PDE5 accounted for cyclic GMP hydrolysis in early passages, but both isozymes were lost with cell passage. Cyclic AMP was hydrolyzed by both PDE2 and PDE4 isozymes in early passage endothelial cells, but PDE4 was increased dramatically in higher passage cells. Also appearing in the higher passage cells were prominent PDE1 and minor PDE3 activities. The ratios of cytosolic to particulate activities were similar at all passages. BAEC PDE isoforms in intact cells assessed by [³H]-adenine prelabeling showed that atriopeptin II decreased isoproterenol-induced cyclic AMP accumulation in early but not later passage cells, consistent with the loss of PDE2 expression. Enhancement of isoproterenol-induced cyclic AMP accumulation by rolipram, a PDE4 inhibitor, was also greatly diminished during culture passages. Changes in CN PDE isoform expression and consequent cyclic AMP turnover validate the importance of considering cell passage number when cultures of BAEC are used to study the regulation of endothelial cell cyclic nucleotide metabolism and processes mediated by cyclic nucleotides in this model system.

Section snippets

Chemicals

[2,8-³H]cAMP (sp. act. 27 Ci/mmol) and [8-³H]cGMP (sp. act. 16 Ci/mmol) were purchased from ICN and purified as described previously [21]. Benzamidine, tosyl-lysl-chloroketone (TLCK), aprotinin, and pepstatin A were obtained from the Sigma Chemical Co. and leupeptin from Peninsula Laboratories. DEAE-Trisacryl was supplied by IBF (Villeneuve La Garenne, France), and Dowex-1X8 (200–400) resin and MIX were from Aldrich. Other chemicals were obtained as follows: rolipram from Dr. I. Williams,

Characteristics of CN PDE during BAEC Passages: Distribution

The specific activities of the 100,000 g supernatant fractions from BAEC homogenates changed from 3- to 5-fold when early passage 4, 5, 6, 7, 8, 9activities were combined from three different primary preparations and compared with those of later passages 10, 11, 12, 13, 14, 15, 16, 17as shown in the upper panel of Fig. 1. Total cAMP hydrolysis was constant through early passages, increased near passage 10, and fell at passage 30 (data not shown) where the cells had ceased growth and spread

Discussion

cAMP and cGMP regulation of endothelial cell functions has been studied extensively in cells cultured from a variety of sources, including large vessels such as bovine aorta. Primary cultures are passed several times to achieve both purity and quantity of cells for biochemical analyses, and it is sometimes assumed that higher passage cells have maintained early passage parameters as models of EC functions. PDE activity has been reported to vary in ECs from different anatomic locations based on

Acknowledgements

This research was supported by USPHSG HL 46494.

References (34)

JR Vane et al.
Endothelial cell as a metabolic and endocrine organ
Trends Pharmacol Sci
(1987)
C Lugnier et al.
Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells
Biochem Pharmacol
(1990)
JD Pearson et al.
Prostacyclin release stimulated by thrombin or bradykinin in porcine endothelial cells cultured from aorta and umbilical vein
Thromb Res
(1983)
MM Bradford
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)
VG Manolopoulos et al.
Adenylyl cyclase isoforms are differentially expressed in primary cultures of endothelial cells and whole tissue homogenates from various rat tissues
Biochem Biophys Res Commun
(1995)
RT MacFarland et al.
High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells
J Biol Chem
(1991)
M Yanagisawa et al.
A novel potent vasoconstrictor peptide produced by vascular endothelial cells
Nature
(1988)
S Koga et al.
TNF modulates endothelial properties by decreasing cAMP
Am J Physiol
(1995)
RW Stout
Cyclic AMP: A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells
Diabetologia
(1982)
AL Schafer et al.
Regulation of endothelial cell function by cyclic nucleotides

JW Barnard et al.

Reversal of pulmonary capillary ischemia–reperfusion injury by rolipram, a cAMP phosphodiesterase inhibitor

J Appl Physiol

(1994)

N Marczin et al.

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

Am J Physiol

(1992)

W Martin et al.

Endothelin-derived relaxing factor and atriopeptin II elevate cyclic GMP levels in pig aortic endothelial cells

Br J Pharmacol

(1988)

Y Kishi et al.

Atrial natriuretic peptide reduces cyclic AMP by activating cyclic GMP-stimulated phosphodiesterase in vascular endothelial cells

J Cardiovasc Pharmacol

(1994)

M Conti et al.

Recent progress in understanding hormonal regulation of phosphodiesterases

Endocr Rev

(1995)

JE Souness et al.

Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases

Biochem J

(1990)

Y Kishi et al.

Phosphodiesterases in vascular endothelial cells

Adv Second Messenger Phosphoprotein Res

(1992)

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We utilized rat fetal lung fibroblasts (RFL-6) to evaluate our PDE5 inhibitors at cellular level and observed a decrease in cGMP accumulation induced by sodium nitroprusside (SNP) and PDE5 inhibitors with passage. To further investigate this observation, we examined cGMP synthesis via soluble guanylyl cyclase (sGC) and degradation via phosphodiesterases (PDEs) at different passages. At passage (p)4, p9, p14, major cGMP and cAMP degradation activities were contributed by PDE5 and PDE4, respectively. The PDE5 activity decreased 50% from p4 to p14, while PDE4 activity doubled. The cGMP accumulation was evaluated in the presence of sodium nitroprusside (SNP) and/or PDE inhibitors in p4 and p14 cells. SNP together with sildenafil, a PDE5 inhibitor, induced dose-dependent increase in cGMP levels in cells at p4, but showed little effect on cells at p14. The possible down regulation of sGC at mRNA level was explored using real-time RT-PCR. The result showed the mRNA level of the α1 subunit of sGC decreased about 98% by p9, while the change on β1 mRNA was minimal. Consistently, sGC activities in cell lysate decreased by 94% at p9. Forskolin stimulated a dramatic increase in cAMP levels in cells at all passages examined. Our results show that sGC activity decreased significantly and rapidly with passage due to a down regulation of the α1 subunit mRNA, yet the adenylyl cyclase activity was not compromised. This study further emphasized the importance of considering passage number when using cell culture as a model system to study NO/cGMP pathway.
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Radioimmunoassay (RIA) was performed immediately using a modification of the methods previously described [36] with I125 labeled cAMP/cGMP-S-TME and specific antibodies. Crude 100,000 × g supernatants obtained from RPMVECs, forskolin activated cells, and RFM cells were used for immunoblot analysis as described previously [21,22] with the TM-PI buffer supplemented with EDTA (0.5 mM) and NaF (10 mM). Briefly, near confluent cells were collected as low-speed pellets and homogenized in TM-PI buffer as described above.
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¹: Current address: 1st Department of Medicine, Yokohama Minami Kyosai Hospital, 500 Mutsu-ura, Kanazawa-Ku, Yokoham-shi, Kanagawa-ken, 236 Japan.

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Original ArticlesAltered Expression of Cyclic Nucleotide Phosphodiesterase Isozymes during Culture of Aortic Endothelial Cells

Abstract

Section snippets

Chemicals

Characteristics of CN PDE during BAEC Passages: Distribution

Discussion

Acknowledgements

Trends Pharmacol Sci

Biochem Pharmacol

Thromb Res

Anal Biochem

Biochem Biophys Res Commun

J Biol Chem

A novel potent vasoconstrictor peptide produced by vascular endothelial cells

Nature

TNF modulates endothelial properties by decreasing cAMP

Am J Physiol

Cyclic AMP: A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells

Diabetologia

Regulation of endothelial cell function by cyclic nucleotides

Reversal of pulmonary capillary ischemia–reperfusion injury by rolipram, a cAMP phosphodiesterase inhibitor

J Appl Physiol

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

Am J Physiol

Endothelin-derived relaxing factor and atriopeptin II elevate cyclic GMP levels in pig aortic endothelial cells

Br J Pharmacol

Atrial natriuretic peptide reduces cyclic AMP by activating cyclic GMP-stimulated phosphodiesterase in vascular endothelial cells

J Cardiovasc Pharmacol

Recent progress in understanding hormonal regulation of phosphodiesterases

Endocr Rev

Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases

Biochem J

Phosphodiesterases in vascular endothelial cells

Adv Second Messenger Phosphoprotein Res

Original Articles
Altered Expression of Cyclic Nucleotide Phosphodiesterase Isozymes during Culture of Aortic Endothelial Cells