Medroxyprogesterone abrogates the inhibitory effects of estradiol on vascular smooth muscle cells by preventing estradiol metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA

Metabolism of cAMP to Adenosine in the Renal Vasculature 1

ABSTRACT We recently demonstrated that cAMP added to the perfusate increased the renal venous recovery of adenosine in the isolated rat kidney, an effect blocked by inhibition of ecto-phosphodiesterase and ecto-5Ј-nucleotidase. Although our previous study established the cAMP-adenosine pathway, i.e., the conversion of cAMP to adenosine, as a viable metabolic pathway within the kidney, that study did not determine whether conversion of arterial cAMP to adenosine recoverable in the venous effluent occurred in the tubules versus nontubular sites. In the current study, we addressed this issue by determining the effects of blocking cAMP transport into the renal tubules with probenecid (0.1, 0.3 and 1 mM) on the increase in renal venous output of adenosine induced by adding cAMP (30 M) to the perfusate of isolated rat kidneys. Addition of cAMP to the perfusate caused a marked increase in renal venous secretion of adenosine, an effect that was augmented, rather than inhibited, by probenecid. To test the hypothesis that the renal vasculature supports a cAMP-adenosine pathway, cultured rat preglomerular vascular smooth muscle cells were incubated with cAMP (30 M) for 1 hr in the presence and absence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). Incubation with cAMP increased extracellular adenosine levels 41-fold, and this effect was abolished by 3-isobutyl-1-methylxanthine. In a third experimental series, addition of cAMP (0.3, 1, 3, 10 and 30 M) to the perfusate of isolated rat kidneys and mesenteric vascular beds increased the renal venous, but not mesenteric venous, output of AMP, adenosine and inosine. We conclude that the renal vasculature supports a cAMP-adenosine pathway, that administering cAMP into the renal artery and measuring adenosine in the venous effluent of the perfused rat kidney most likely monitors primarily the renal vascular cAMPadenosine pathway and that the quantitative importance of the cAMP-adenosine pathway is not equivalent in all vascular compartments. Renal adenosine participates importantly in the regulation of renin release, renal hemodynamics, tubuloglomerular feedback, erythropoietin production and tubular transport The existence of a cAMP-adenosine pathway in the kidneys is supported by two lines of evidence. First, infusion of IBMX (a phosphodiesterase inhibitor) into the renal cortical interstitium via a microdialysis probe decreases renal cortical interstitial levels of adenosine and inosine (a metabolite of adenosine) The above-mentioned studies in the isolated perfused rat kidney strongly suggest that in the kidney cAMP is converted to adenosine extracellularly. However, those studies do not determine whether conversion of perfusate cAMP to adenosine recoverable in the venous effluent occurs mostly in the tubules versus nontubular sites such as the renal vasculature. Several studies demonstrate that cAMP is efficiently transported by the probenecid-inhibitable organic anion transport system in the proximal tubul

Extracellular 3′,5′-cAMP-Adenosine Pathway Inhibits Glomerular Mesangial Cell Growth

Abnormal growth of glomerular mesangial cells (GMCs) contributes to the pathophysiology of many types of nephropathy. Because adenosine is an autocrine/paracrine factor that potentially could regulate GMC proliferation and because the extracellular 3′,5′-cAMP-adenosine pathway (i.e., the conversion of extracellular 3′,5′-cAMP to 5′-AMP and adenosine on the cell surface) could generate adenosine in the biophase of GMC receptors, we investigated the role of the 3′,5′-cAMP-adenosine pathway in modulating growth [cell proliferation, DNA synthesis ([3H]thymidine incorporation), collagen synthesis ([3H]proline incorporation), and mitogen-activated protein kinase activity] of GMCs. The addition of exogenous 3′,5′-cAMP to human GMCs increased extracellular levels of 5′-AMP, adenosine, and inosine, and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), 1,3-dipropyl-8-p-sulfophenylxanthine (ecto-phosphodiesterase inhibitor), and α,β-methylene-adenosine-5′-diphosphate (ecto-5′-nucleotidase inhibitor) attenuated the increases in adenosine and inosine. Forskolin augmented extracellular 3′,5′-cAMP and adenosine concentrations, and 2′,5′-dideoxyadenosine (adenylyl cyclase inhibitor) blocked these increases. Exogenous 3′,5′-cAMP and forskolin inhibited all indices of cell growth, and antagonism of A2 [(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine, KF17837] or A1/A2 (1,3-dipropyl-8-p-sulfophenylxanthine, DPSPX), but not A1 (8-cyclopentyl-1,3-dipropylxanthine), or A3{N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF5574}, adenosine receptors blocked the growth-inhibitory actions of exogenous 3′,5′-cAMP, but not the effects of 8-bromo-3′,5′-cAMP (stable 3′,5′-cAMP analog). Erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor) plus 5-iodotubercidin (adenosine kinase inhibitor) enhanced the growth inhibition by exogenous 3′,5′-cAMP and forskolin, and A2 receptor antagonism blocked this effect. In rat GMCs, down-regulation of A2B receptors with antisense, but not sense or scrambled, oligonucleotides abrogated the inhibitory effects of 3′,5′-cAMP and forskolin on cell growth. The extracellular 3′,5′-cAMP-adenosine pathway exists in GMCs and attenuates cell growth via A2B receptors. Pharmacological augmentation of this pathway could abate pathological glomerular remodeling

Purine Metabolites in Tumor-Derived Exosomes May Facilitate Immune Escape of Head and Neck Squamous Cell Carcinoma

Body fluids of patients with head and neck squamous cell carcinoma (HNSCC) are enriched in exosomes that reflect properties of the tumor. The aim of this study was to determine whether purine metabolites are carried by exosomes and evaluate their role as potential contributors to tumor immune escape. The gene expression levels of the purine synthesis pathway were studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer database. Exosomes were isolated from supernatants of UMSCC47 cells and from the plasma of HNSCC patients (n= 26) or normal donors (NDs;n= 5) using size exclusion chromatography. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to assess levels of 19 purine metabolites carried by exosomes. In HNSCC tissues, expression levels of genes involved in the purinergic pathway were upregulated indicating an accelerated purine metabolism compared to normal tissues. Exosomes from supernatants of UMSCC47 cells contained several purine metabolites, predominantly adenosine and inosine. Purine metabolite levels were enriched in exosomes isolated from the plasma of HNSCC patients compared to those isolated from NDs and carried elevated levels of adenosine (p= 0.0223). Exosomes of patients with early-stage disease and no lymph node metastasis contained significantly elevated levels of adenosine and 5 '-GMP (p= 0.0247 andp= 0.0229, respectively). The purine metabolite levels in exosomes decreased in patients with advanced cancer and nodal involvement. This report provides the first evidence that HNSCC cells shuttle purine metabolites in exosomes, with immunosuppressive adenosine being the most prominent purine. Changes in the content and levels of purine metabolites in circulating exosomes reflect disease progression in HNSCC patients

2′-AMP and 3′-AMP Inhibit Proliferation of Preglomerular Vascular Smooth Muscle Cells and Glomerular Mesangial Cells via A2B Receptors

Studies show that kidneys produce 2′,3′-cAMP, 2′,3′-cAMP is exported and metabolized to 2′-AMP and 3′-AMP, 2′-AMP and 3′-AMP are metabolized to adenosine, 2′,3′-cAMP inhibits proliferation of preglomerular vascular smooth muscle cells (PGVSMCs) and glomerular mesangial cells (GMCs), and A2B (not A1, A2A, or A3) adenosine receptors mediate part of the antiproliferative effects of 2′,3′-cAMP. These findings suggest that extracellular 2′,3′-cAMP attenuates proliferation of PGVSMCs and GMCs partly via conversion to corresponding AMPs, which are metabolized to adenosine that activates A2B receptors. This hypothesis predicts that extracellular 2′-AMP and 3′-AMP should exert A2B receptor-mediated antiproliferative effects. Therefore, we examined the antiproliferative effects (cell counts) of 2′-AMP and 3′-AMP. In PGVSMCs and GMCs, 2′-AMP and 3′-AMP exerted concentration-dependent antiproliferative effects. 3′-AMP was equipotent with and 2′-AMP was 3-fold less potent than 5′-AMP (prototypical adenosine precursor). In PGVSMCs, the effects of 2′-AMP and 3′-AMP were mimicked by adenosine, and 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine (MRS-1754) (A2B receptor antagonist) equally blocked the antiproliferative effects of 2′-AMP, 3′-AMP, and adenosine but less effectively blocked the effects of 2′,3′-cAMP. Similar results were obtained in GMCs except that MRS-1754 also incompletely blocked the effects of 3′-AMP. We conclude that in PGVSMCs, 2′-AMP and 3′-AMP are antiproliferative, the antiproliferative effects of 2′-AMP and 3′-AMP are mediated nearly entirely by adenosine/A2B receptors, and some of the antiproliferative effects of 2′,3′-cAMP are independent of adenosine/A2B receptors. Similar conclusions apply to GMCs except that 3′-AMP also has actions independent of adenosine/A2B receptors. Because A2B receptors are renoprotective, 2′-AMP and 3′-AMP may provide renoprotection by generating adenosine that activates A2B receptors