Activation of the JAK/STAT pathway in vascular smooth muscle by serotonin.

Serotonin (5-hydroxytryptamine, 5-HT) is a vasoconstrictor and mitogen whose levels are elevated in diabetes. Previous studies have shown the presence of 5-HT2A, 5-HT2B, and 5-HT1B receptors in vascular smooth muscle cells (VSMCs). There are currently no data regarding 5-HT2B and 5-HT1B receptor activation of the JAK/STAT pathway in VSMCs and resultant potential alterations in 5-HT signaling in diabetes. Therefore, we tested the hypothesis that 5-HT differentially activates the JAK/STAT pathway in VSMCs under conditions of normal (5 mM) and high (25 mM) glucose. Treatment of rat VSMCs with 5-HT (10(-6) M) resulted in time-dependent activation ( approximately 2-fold) of JAK2, JAK1, and STAT1, but not STAT3 (maximal at 5 min, returned to baseline by 30 min). The 5-HT2B receptor agonist BW723C86 and the 5-HT1B receptor agonist CGS12066A (10(-9)-10(-5) M, 5-min stimulation) did not activate the JAK/STAT pathway. Treatment with the 5-HT2A receptor antagonist ketanserin (10 nM) inhibited JAK2 activation by 5-HT. Treatment of streptozotocin-induced diabetic rats with ketanserin (5 mg.kg-1.day-1) reduced activation of JAK2 and STAT1 but not STAT3 in endothelium-denuded thoracic aorta in vivo. 5-HT (10(-6) M) treatment resulted in increased cell proliferation and increased DNA synthesis, which were inhibited by the JAK2 inhibitor AG490. Further studies with apocynin, diphenyleneiodonium chloride, catalase, and virally transfected superoxide dismutase had no effect at either glucose concentration on activation of the JAK/STAT pathway by 5-HT. Therefore, we conclude that 5-HT activates JAK2, JAK1, and STAT1 via the 5-HT2A receptors in a reactive oxygen species-independent manner under both normal and high glucose conditions.

Angiotensin II blockade prevents hyperglycemia-induced activation of JAK and STAT proteins in diabetic rat kidney glomeruli.

Clinical and animal studies show that treatment with angiotensin-converting enzyme (ACE) inhibitors or ANG II-receptor antagonists slows progression of nephropathy in diabetes, indicating ANG II plays an important role in its development. We previously reported that hyperglycemia augments both ANG II-induced growth and activation of Janus kinase (JAK)2 and signal transducers and activators of transcription (STAT) proteins in cultured rat mesangial cells. Furthermore, we demonstrated that the tyrosine kinase enzyme JAK2 plays a key role in both ANG II- and hyperglycemia-induced growth in these cells. We hypothesized that the ACE inhibitor captopril and the ANG II-receptor antagonist candesartan would hinder hyperglycemic-induced activation of JAK and STAT proteins in rat glomeruli, demonstrating that ANG II plays an important role in the activation of these proteins in vivo. Adult male Sprague-Dawley rats were given either streptozotocin (STZ; 60 mg/kg iv) or vehicle, and glomeruli were isolated 2 wk later. Activation of JAK and STAT proteins was evaluated by Western blot analysis for specific tyrosine phosphorylation. Groups of rats were given captopril (75-85 mg x kg(-1) x day(-1)), candesartan (10 mg x kg(-1) x day(-1)), or the JAK2 inhibitor AG-490 (5 mg x kg(-1) x day(-1)) for the study's duration. STZ stimulated glomerular phosphorylation of JAK2, STAT1, STAT3, and STAT5. Phosphorylation was reduced in rats treated with captopril, candesartan, and AG-490. Furthermore, both candesartan and AG-490 inhibited STZ-induced increases in urinary protein excretion. In conclusion, our studies demonstrate that hyperglycemia induces activation of JAK2 and the STATs in vivo via an ANG II-dependent mechanism and that these proteins may be involved in the early kidney damage associated with diabetes.

Arterial expression of 5-HT2B and 5-HT1B receptors during development of DOCA-salt hypertension.

BACKGROUND: 5-hydroxytryptamine (5-HT)2B and 5-HT1B receptors are upregulated in arteries from hypertensive DOCA-salt rats and directly by mineralocorticoids. We hypothesized that increased 5-HT2B and 5-HT1B receptor density and contractile function would precede increased blood pressure in DOCA-high salt rats. We performed DOCA-salt time course (days 1, 3, 5 and 7) studies using treatment groups of: DOCA-high salt, DOCA-low salt, Sham and Sham-high salt rats. RESULTS: In isolated-tissue baths, DOCA-high salt aorta contracted to the 5-HT2B receptor agonist BW723C86 on day 1; Sham aorta did not contract. The 5-HT1B receptor agonist CP93129 had no effect in arteries from any group. On days 3, 5 and 7 CP93129 and BW723C86 contracted DOCA-high salt and Sham-high salt aorta; Sham and DOCA-low salt aorta did not respond. Western analysis of DOCA-high salt aortic homogenates revealed increased 5-HT2B receptor levels by day 3; 5-HT1B receptor density was unchanged. Aortic homogenates from the other groups showed unchanged 5-HT2B and 5-HT1B receptor levels. CONCLUSION: These data suggest that functional changes of 5-HT2B but not 5-HT1B receptors may play a role in the development of DOCA-salt hypertension.

S100B-RAGE-mediated augmentation of angiotensin II-induced activation of JAK2 in vascular smooth muscle cells is dependent on PLD2.

Angiotensin II (Ang II), a vasoactive peptide that is also considered a growth factor, has been implicated in both normal and diabetic cellular proliferation. We recently found that activation of janus kinase 2 (JAK2) is essential for the Ang II-induced proliferation of vascular smooth muscle cells (VSMCs) and that high glucose augments Ang II-induced proliferation of VSMCs by increasing signal transduction through activation of JAK2. Here, we demonstrate that S100B, a ligand for the receptor of advanced glycation end products (RAGEs), augmented both Ang II-induced tyrosine phosphorylation of JAK2 and cell proliferation in VSMCs in a receptor-dependent manner. We also found that S100B-RAGE interaction triggered intracellular generation of reactive oxygen species (ROS), VSMC proliferation, and JAK2 tyrosine phosphorylation via activation of phospholipase D (PLD)2. These results provide direct evidence for linkages between PLD2, ROS production, and S100B-RAGE-induced enhancement of Ang II-induced cell proliferation and activation of JAK2 in VSMCs.

NADPH oxidase-derived superoxide augments endothelin-1-induced venoconstriction in mineralocorticoid hypertension.

Deoxycorticosterone acetate (DOCA)-salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ET(A) receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ET(B) receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ET(A) receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10(-9) mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1-induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1-treated vena cavas of normal rats. Finally, in vivo ET(A) receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1-induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.

5-Hydroxytryptamine(2B) receptor function is enhanced in the N(omega)-nitro-L-arginine hypertensive rat.

Arterial hyperresponsiveness to serotonin (5-hydroxytryptamine, 5-HT) is observed in experimental models and human forms of hypertension. Presently, we test the hypothesis that the 5-HT(2B) receptor is up-regulated and necessary for maintaining elevated blood pressure in a rat made hypertensive by the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine (LNNA; 0.5 g/l). After 2 weeks of treatment, thoracic aorta were removed from LNNA hypertensive (systolic blood pressure = 189 +/- 5 mm Hg) and sham normotensive rats (121 +/- 1 mm Hg), denuded, and mounted into isolated tissue baths for measurement of isometric contraction. In sham tissues, 5-HT-induced contraction was mediated by the 5-HT(2A) receptor as evidence by a parallel rightward shift in response to 5-HT by the 5-HT(2A/2C) receptor antagonist ketanserin (10 nM) and lack of shift by the 5-HT(2B) receptor antagonist 6-methyl-1,2,3,4-tetrahydro-1-[3,4-dimethoxyphenyl)methyl]-9H-pyrido[3,4-b]indole hydrochloride (LY272015) (10 nM). In contrast, LY272015 produced a 4-fold rightward shift to 5-HT in aorta from LNNA hypertensive rats, and blockade by ketanserin did not occur at low concentrations of 5-HT. Maximal contraction to the 5-HT(2B) receptor agonist 1-[5-(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine hydrochloride was significantly greater in LNNA hypertensive rats (percentage of phenylephrine contraction in sham = 7 +/- 4, and in LNNA = 61 +/- 7%). 5-HT(2B) receptor protein was present in aortic homogenates from sham and LNNA rats, but the density of 5-HT(2B) receptor protein in LNNA homogenates was 300% that in sham. Importantly, the 5-HT(2B) receptor antagonist LY272015 reduced blood pressure of the LNNA hypertensive but not the sham normotensive rats. Thus, these data suggest that the up-regulated 5-HT(2B) receptor in the LNNA hypertensive rats is physiologically activated to maintain elevated blood pressure.

Anticoagulants prevent monocrotaline-induced hepatic parenchymal cell injury but not endothelial cell injury in the rat.

Monocrotaline (MCT) is a pyrrolizidine alkaloid plant toxin that produces hepatotoxicity in humans and animals. Human exposure to MCT occurs through consumption of contaminated grains and herbal medicines. Administration of MCT to rats stimulates activation of the coagulation system and fibrin deposition in the liver. Fibrin deposition occurs simultaneously with endothelial cell damage and prior to hepatic parenchymal cell injury. Accordingly, the hypothesis that activation of the coagulation system is required for MCT-induced liver injury was tested. Treatment of rats with either heparin or warfarin significantly reduced MCT-induced activation of the coagulation system and the increase in alanine aminotransferase activity in the plasma, a biomarker of hepatic parenchymal cell injury. Histopathological examination of liver sections revealed that heparin decreased parenchymal cell necrosis but did not affect central venular endothelial cell damage, congestion and dilation of the sinusoids, or hemorrhage in the liver. Morphometric analysis revealed that 28% of the area of livers from MCT-treated rats contained regions of coagulative necrosis, whereas less than 5% of the area of livers from rats treated with MCT and heparin contained these regions. By contrast, neither heparin nor warfarin prevented MCT-induced damage to endothelial cells in the liver as estimated by increased plasma hyaluronic acid concentration. These results suggest that activation of the coagulation system is required for MCT-induced parenchymal cell injury but not endothelial cell injury in the liver.

Upregulation of arterial serotonin 1B and 2B receptors in deoxycorticosterone acetate-salt hypertension.

Previous studies have established a role for 5-hydroxytryptamine (5-HT)(2B) and 5-HT(1B) receptors in mediating enhanced contraction to serotonin (5-HT) in arteries from hypertensive deoxycorticosterone acetate (DOCA)-salt rats. To determine whether the observed increase in responsiveness was due to upregulation of 5-HT receptors, we used Western analysis to measure 5-HT(1B) and 5-HT(2B) receptor protein density. In endothelium-denuded aortas from hypertensive DOCA-salt rats (mean systolic blood pressure 192 +/- 6 mm Hg), 5-HT(1B) and 5-HT(2B) receptor proteins were upregulated approximately 2-fold compared with the response in the aortas of sham-operated control rats (mean systolic blood pressure 119 +/- 2 mm Hg). Contraction to 5-HT(2B) receptor agonists was also enhanced in arteries from Wistar-Furth rats given DOCA and salt. This strain is relatively resistant to the hypertensive effects of DOCA and salt treatment. A common factor between the model of DOCA-salt hypertension and the DOCA-salt--treated Wistar-Furth rats is the presence of mineralocorticoids. Therefore, we tested the hypothesis that mineralocorticoids can upregulate 5-HT(1B) and 5-HT(2B) receptors. Aortas from normal Sprague-Dawley rats were incubated with aldosterone (100 nmol/L) for 8, 12, 24, and 48 hours. The expression of 5-HT(2B) and 5-HT(1B) receptor proteins was significantly increased (approximately 2- fold over vehicle treatment) by 8 hours. 5-HT(2B) and 5-HT(1B) receptors were upregulated by aldosterone in a concentration-dependent manner, and incubation with spironolactone (10 micromol/L) blocked this upregulation. These data support the conclusion that the increased expression of 5-HT(1B) and 5-HT(2B) receptors observed in arteries from DOCA-salt rats may be partially due to mineralocorticoids acting via the mineralocorticoid receptor to modulate gene expression.

Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces hepatotoxicity in people and animals. Human exposure to PAs occurs through consumption of contaminated grains and herbal remedies. Injection (ip) of MCT in rats produced dose-dependent hepatic parenchymal cell injury that was significant at 200 mg/kg. Injection of 300 mg/kg MCT produced time-dependent hepatotoxicity with significant injury beginning by 12 h after treatment. Histopathologic examination of liver sections revealed coagulative hepatocellular necrosis, widening of sinusoids and hemorrhage in centrilobular regions. MCT-induced damage to central venular endothelial cells (CVECs) and sinusoidal endothelial cells (SECs) in the liver was quantified using immunohistochemical staining and by increased plasma hyaluronic acid concentration. MCT damaged CVECs and SECs in the liver by 8 h after treatment. Extensive endothelial cell injury was restricted to centrilobular regions. To determine if damage to endothelial cells in the liver stimulated activation of the coagulation system, fibrin deposition was quantified using immunohistochemistry. Extensive fibrin deposition occurred in the liver after MCT treatment and was restricted to centrilobular regions. Interestingly, both endothelial cell damage and fibrin deposition preceded the onset of hepatic parenchymal cell injury. These results suggest that endothelial cell damage and fibrin deposition in centrilobular regions of the liver are prominent features of MCT-induced liver injury.