Propranolol has direct antithyroid activity: Inhibition of iodide transport in cultured thyroid follicles

The effect of propranolol on the process of thyroid hormone formation was studied in a physiological culture system. Porcine thyroid follicles were preincubated with propranolol for 24 h. Iodide transport, iodine organification, and de novo thyroid hormone formation were measured by incubating these follicles with the mixture of carrier-free 0·1 μCi Na ¹²⁵I and 50 nM NaI for 2 to 6 h at 37°C. A concentration of propranolol greater than 100 μM inhibited iodide transport in a dose-dependent manner; this inhibition was non-competitive with iodide and independent of thyrotropin (TSH). Reduced iodine organification and thyroid hormone formation was seen with 150 μM propranolol or greater. The inhibitory action of propranolol was not caused by beta-blocking activity, since D -propranolol (devoid of beta-blocking activity) inhibited iodide transport, and other beta-blockers (metoprolol, atenolol, and labetalol) did not inhibit iodide transport. The inhibition of iodide transport was most likely caused by membrane stabilizing activity since quinidine, which possess the same membrane stabilizing activity as propranolol, also inhibited iodide transport. TSH-mediated cAMP generation and Na ⁺K⁺ ATPase activity, membrane functions for iodide transport, were unaffected by propranolol. Our study has shown, for the first time, that propranolol has a direct antithyroid action, namely inhibition of iodide transport in the intact thyroid follicle.     

Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor

Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1-1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300-500 muU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic peptide provokes a dramatic increase in cyclic GMP formation and markedly inhibits muscarinic-stimulated Ca2+ mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca2+]i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca+1]i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 microM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca2+]i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca2+]i mobilisation with IC50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with EC50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>CNP>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant (Kd) and total receptor density (Bmax) values of the natriuretic peptide receptor for [125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic responses in the iris sphincter smooth muscle cells, and SV-CISM-2 cells can serve as an excellent model for investigating the cross talk between cGMP and the Ca2+ signalling system.     

Atrial natriuretic peptide negatively regulates follicle-stimulating hormone-induced porcine oocyte maturation and cumulus expansion via cGMP-dependent protein kinase pathway

This study examined the effect of atrial natriuretic peptide (ANP) on porcine cumulus-enclosed oocyte (CEO) maturation and cumulus expansion. ANP negatively regulated follicle-stimulating hormone (FSH)-stimulated germinal vesicle breakdown (GVBD; 90.1, 81.2 and 68.2% for FSH, FSH+10nM ANP and FSH+1 microM ANP, respectively), first polar body emission (PB1; 86.1, 75.3 and 53.3% for FSH, FSH+1 nM ANP and FSH+1 microM ANP, respectively) and cumulus expansion (CEI; 3.47, 3.16 and 2.43 for FSH, FSH+1 nM ANP and FSH+1 microM ANP, respectively) in a dose-dependent manner when CEOs were cultured in the maturation medium containing porcine follicular fluid (pFF). This negative effect showed a time-dependent manner after preincubation with 100 nM ANP for 5h (78.4% PB1), 10h (81.7% GVBD and 74.1% PB1), 20 h (78.5% GVBD and 68.9% PB1), and 44 h (75.3% GVBD and 60.5% PB1), respectively. ANP also significantly inhibited FSH-induced porcine oocyte GVBD (47.6% versus 83.8%) and PB1 emission (22.4% versus 45.2%) when CEOs were cultured in pFF-free maturation medium. cGMP analog 8-Br-cGMP (10 microM to 1mM) mimicked the effects of ANP on GVBD, PB1, and CEI. The negative effect of ANP was completely reversed by KT5823 (a specific inhibitor of cGMP-dependent protein kinase), while C-ANP-(4-23) (an analogue of ANP and specific binder for natriuretic peptide receptors-C) was ineffective in oocyte maturation. Neither ANP nor C-ANP-(4-23) had an effect on spontaneous porcine oocyte maturation and cumulus expansion. These results suggested that ANP negatively regulates FSH-activated porcine oocyte meiotic resumption, meiotic maturation and cumulus expansion. The function of ANP on porcine oocyte maturation is via the cGMP dependent protein kinase (PKG) pathway.     

Cytochrome P-450 metabolites in endothelin-stimulated cardiac hormone secretion

We examined the role of cytochrome P-450-arachidonate (CYP450-AA) metabolites in endothelin-1 (ET-1)-stimulated atrial natriuretic peptide (ANP) and pro-ANP-(1-30) secretion from the heart. 17-Octadecynoic acid (17-ODYA, 10(-5) M) significantly inhibited ANP secretion stimulated by ET-1 (10(-8) M) in the isolated perfused rat atria and inhibited pro-ANP-(1-30) secretion stimulated by ET-1 (10(-8) M) or 20-hydroxyeicosatetraenoic acid in cultured neonatal rat ventricular myocytes (NRVM). In NRVM, 17-ODYA significantly (P < 0.05) increased secretion of cAMP but had no significant effect on the secretion of cGMP from NRVM. Staurosporine, an inhibitor of protein kinase C, completely blocked the inhibitory action of 17-ODYA, whereas a protein kinase A inhibitor, H-89 (5 x 10(-5) M), did not significantly attenuate the effects of 17-ODYA. The results show that the inhibitory action of 17-ODYA on ET-1-augmented ANP secretion is mediated through cAMP and suggest that CYP450-AA may play an important role in ET-1-induced cardiac hormone secretion.     

Defective thyroglobulin storage in LDL receptor-associated protein-deficient mice

The molecular chaperone receptor-associated protein (RAP) is required for biosynthesis of megalin, an endocytic receptor for follicular thyroglobulin (Tg), the thyroid hormone precursor. RAP also binds to Tg itself, suggesting that it may affect Tg trafficking in various manners. To elucidate RAP function, we have studied the thyroid phenotype in RAP-knockout (RAP-KO) mice and found a reduction of Tg aggregates into thyroid follicles. Serum Tg levels were significantly increased compared with those of wild-type (WT) mice, suggesting a directional alteration of Tg secretion. In spite of these abnormalities, hormone secretion was maintained as indicated by normal serum thyroxine levels. Because Tg in thyroid extracts from RAP-KO mice contained thyroxine residues as in WT mice, we concluded that in RAP-KO mice, follicular Tg, although reduced, was nevertheless sufficient to provide normal hormone secretion. Serum TSH was increased in RAP-KO mice, and although no thyroid enlargement was observed, some histological features resembling early goiter were present. Megalin was decreased in RAP-KO mice, but this did not affect thyroid function, probably because of the concomitant reduction of follicular Tg. In conclusion, RAP is required for the establishment of Tg reservoirs, but its absence does not affect hormone secretion. low-density lipoprotein; knockout mice     

PCB153 disrupts thyroid hormone homeostasis by affecting its biosynthesis, biotransformation, feedback regulation, and metabolism

PCB153, one of the 3 dominant congeners in the food chain, causes the disruption of the endocrine system in humans and animals. In order to elucidate the effects of PCB153 on the biosynthesis, biotransformation, regulation, metabolism, and transport of thyroid hormones (THs), Sprague-Dawley (SD) rats were dosed with PCB153 intraperitoneally (i.p.) at 0, 4, 16 and 32 mg/kg/day for 5 consecutive days and sacrificed 24 h after the last dose. Results showed that after treatment with PCB153, serum total thyroxine (TT4), total triiodothyronine (TT3), and thyrotropin releasing hormone (TRH) decreased, whereas serum thyroid stimulating hormone (TSH) concentration did not alter. The serum sodium iodide symporter (NIS), thyroid peroxidase (TPO), and thyroglobulin (Tg) levels decreased. The mRNA expressions of type 2 and 3 deiodinases (D2 and D3) reduced, but the type 1 deiodinase (D1) showed no significant change. The TSH receptor (TSHr) and TRH receptor (TRHr) levels declined. PCB153 induced hepatic enzymes, and the UDPGTs, CYP2B1, and CYP3A1 mRNA levels were significantly elevated. Taken together, the observed results from the present study indicated that PCB153 disrupted thyroid hormone homeostasis through influencing synthesis-associated proteins (NIS, TPO and Tg), deiodinases, receptors (TSHr and TRHr), and hepatic enzymes, and the decrease of D3 expression might be the compensatory response of body.     

Triiodothyronine induces an increase in cyclic GMP in bullfrog tadpole tissues

Cyclic GMP (cGMP) and cyclic AMP (cAMP) were determined in bullfrog tadpole liver and tail fin using 125I-RIA. cGMP increased approx. 100% 1-6 h after the injection of T3 (3 X 10(-10) mol/g body wt.). Reducing the dose of T3 to 1, 3, and 10 X 10(-11) mol/g body wt. provided increases in cGMP of 50-100% above the control value after 2 h. In contrast, only small increases (less than 20%) in cAMP were observed 2-24 h after T3 injection. We conclude that T3 produces a rapid and significant increase in cGMP in the liver and tail fins of premetamorphic tadpoles. These results suggest that thyroid hormones in amphibia may not be an exclusively nucleus-mediated hormone.     

Natriuretic peptides: a new lipolytic pathway in human fat cells

Human fat cell lipolysis was considered until recently to be an exclusive cAMP/protein-kinase A (PKA)-regulated metabolic pathway under the control of catecholamines and insulin. Moreover, exercise-induced lipid mobilization in humans was considered to mainly depend on catecholamine action and interplay between fat cell beta- and alpha2-adrenergic receptors controlling adenylyl cyclase activity and cAMP production. We have recently demonstrated that natriuretic peptides stimulate lipolysis and contribute to the regulation of lipid mobilization in humans. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) stimulate lipolysis in human isolated fat cells. Activation of the adipocyte plasma membrane type A guanylyl cyclase receptor (NPR-A), increase in intracellular guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels and activation of hormone-sensitive lipase mediate the action of ANP. ANP does not modulate cAMP production and PKA activity. Increment of cGMP induces the phosphorylation of hormone-sensitive lipase and perilipin A via the activation of a cGMP dependent protein kinase-I (cGK-I). Plasma concentrations of glycerol and non-esterified fatty acids are increased by i.v. infusion of ANP in humans. Physiological relevance of the ANP-dependent pathway was demonstrated in young subjects performing physical exercise. ANP plays a role in conjunction with catecholamines in the control of exercise-induced lipid mobilization. This pathway becomes of major importance when subjects are submitted to chronic treatment with a beta-blocker. Oral beta-adrenoceptor blockade suppresses the beta-adrenergic component of catecholamine action in fat cells and potentiates exercise-induced ANP release by the heart. These findings may have several implications whenever natriuretic peptide secretion is altered such as in subjects with left ventricular dysfunction, congestive heart failure and obesity.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Immunoreactive atrial natriuretic peptide in the thyroid gland

Human thyroid follicles and primary cell cultures derived from them demonstrated atrial natriuretic peptide (ANP)-like immunoreactivity when stained with a monoclonal antibody raised against rat alpha-ANP (ANP 1-28). In thyroid sections the staining was most intense in the tall cuboidal epithelium of small follicles. The intracellular distribution of immunoreactive (ir)-ANP in primary cultures of thyroid follicular cells consisted of discrete granules with a largely perinuclear distribution. The granule density increased with time in culture but was unaffected by exogenous ANP, suggesting an intrinsic synthesis of the immunoreactivity. Thyroid stimulating hormone (TSH; thyrotropin) failed to alter the distribution of ir-ANP after either short-term (6 h) or long-term (1-12 day) exposure. Epinephrine or norepinephrine treatment, however, caused a reduction in the ir-ANP granularity compared with controls in what might represent a stimulated release of the immunoreactivity. The present results suggest that the peptide ANP coexists with thyroid hormones in follicular cells and that the two endocrine activities might be under separate control mechanisms.     

Involvement of oxidative reactions and extracellular protein chaperones in the rescue of misassembled thyroglobulin in the follicular lumen

Reactive oxygen species (ROS) are involved in many pathological processes through modifications of structure and activity of proteins. ROS also participate in physiological pathways such as thyroid hormone biosynthesis, which proceeds through oxidation of the prothyroid hormone (thyroglobulin, Tg) and iodide. Regarding the colloidal insoluble multimerized Tg (m-Tg), which bears dityrosine bridges and is present in the follicular lumen, a mild oxidative system generated different soluble forms of Tg, more or less compacted by hydrophobic associations, and linked with Grp78 and Grp94. In vitro, the combined action of ROS and PDI, in the presence of free glutathione (reduced/oxidized), increased the solubility of this misassembled Tg and partially restored the ability of Tg to synthesize hormones. Our results show that protein chaperones escape from the ER and are involved with ROS in thyroid hormone synthesis. Therefore, we propose a model of roles of m-Tg in the follicular lumen.     

Biosynthesis of thyroglobulin in the endostyle of larva (ammocoetes) of a fresh water lamprey, Lampetra planeri B1]

The biosynthesis of thyroglobulin (Tg) in larva of a fresh-water lamprey, Lampetra planeri B1. has been established. This glycoprotein presents the same characters as in thyroid follicles of adult lampreys, as shown by its 18-19 S sedimentation coefficient and by the incorporation (in vivo and in vitro experiments of 4, 12, 72 h) of 125I, 3H-leucine and 3H-galactose. 3-8 S fractions and a 12 S monomer are the precursors of the 18-19 S protein. Total I % of Tg is very low (0.002 %) ; about 5 % of 125I are present in thyroid hormones (T3 and T4) in the 125I-labeled protein. The biosynthesis of 18-19 S Tg proceeds in larvs before the morphological differentiation of thyroid cells and follicles after metamorphosis. However, the biosynthesis of this protein is much slower in the endostyle of larvs, in which a primitive mechanism of storage is poorly efficient, compared to the accumulation of Tg in the colloid of the follicles of adults.     

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells.

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.     

Actions of atrial natriuretic peptide (ANP) on cyclic nucleotide concentrations and phosphatidylinositol turnover in ventricular myocytes

Atrial natriuretic peptide (ANP) stimulates cGMP production in isolated rabbit ventricular myocytes incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (1mM). Half maximal activation was found at 10(-8)M ANP. Cellular cGMP concentrations of around 0.6 pmol/10(6) cells were elevated 4-6 fold by ANP (10(-6)M), 3-4 fold by carbachol (1mM) and around 10 fold by sodium nitroprusside (1mM). ANP had no effect on basal or isoprenaline-stimulated cAMP concentrations or on basal or noradrenaline-stimulated turnover of phosphatidylinositol. From these results we conclude that ANP receptors, coupled to particulate guanylate cyclase, exist in cardiac ventricular muscle. This indicates that ANP may also have a physiological action on ventricular muscle contractility during volume expansion.     

Receptors for atrial natriuretic peptide (ANP) and regulation of thyroglobulin secretion by ANP in human thyroid cells

Specific binding sites for atrial natriuretic peptide (ANP) were identified and characterized in primary cultures of human thyroid cells. Saturation analysis using [125I] alpha rat ANP as the ligand showed a single class of high affinity binding (Kd = 0.2 nM) which was inhibited by atriopeptin I and the alpha -human form of ANP, but not by a C-terminal fragment of the peptide. The number of ANP binding sites in these cultures was not altered by the thyroid hormone concentration of the medium. In a dose-response experiment, thyro-globulin secretion was significantly reduced in the presence of 0.01 nM ANP and was maximally reduced (to 25% of control value) with 10 nM ANP. Cyclic GMP production was increased threefold in the presence of 100 nM ANP, but was unchanged with lower doses (0.01 and 0.1 nM) of the peptide. The finding of receptors in thyroid follicular cells suggests a hitherto unrecognized role of ANP in the thyroid gland.     

Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells

In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.     

The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse

Ischemia-reperfusion (IR) causes human lung injury in association with the release of atrial and brain natriuretic peptides (ANP and BNP), but the role of ANP/BNP in IR lung injury is unknown. ANP and BNP bind to natriuretic peptide receptor-A (NPR-A) generating cGMP and to NPR-C, a clearance receptor that can decrease intracellular cAMP. To determine the role of NPR-A signaling in IR lung injury, we administered the NPR-A blocker anantin in an in vivo SWR mouse preparation of unilateral lung IR. With uninterrupted ventilation, the left pulmonary artery was occluded for 30 min and then reperfused for 60 or 150 min. Anantin administration decreased IR-induced Evans blue dye extravasation and wet weight in the reperfused left lung, suggesting an injurious role for NPR-A signaling in lung IR. In isolated mouse lungs, exogenous ANP (2.5 nM) added to the perfusate significantly increased the filtration coefficient sevenfold only if lungs were subjected to IR. This effect of ANP was also blocked by anantin. Unilateral in vivo IR increased endogenous plasma ANP, lung cGMP concentration, and lung protein kinase G (PKG(I)) activation. Anantin enhanced plasma ANP concentrations and attenuated the increase in cGMP and PKG(I) activation but had no effect on lung cAMP. These data suggest that lung IR triggered ANP release and altered endothelial signaling so that NPR-A activation caused increased pulmonary endothelial permeability.