3-(4-Pyridyl)-Alanine

3-(4-Pyridyl)-Alanine

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts.[Pubmed:18475191]

Transplantation. 2008 May 15;85(9):1322-31.

BACKGROUND: Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. METHODS: IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. RESULTS: After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2'-deoxyuridine labeling and increases of graft weight were only approximately 3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. CONCLUSION: Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.

Cyclic stretch stimulates vascular smooth muscle cell alignment by redox-dependent activation of Notch3.[Pubmed:21169401]

Am J Physiol Heart Circ Physiol. 2011 May;300(5):H1770-80.

Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alan ine-2-aminoethyl amide (TAPI-1; 50 mumol/l) to inhibit TNF-alpha-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 mumol/l) to inhibit gamma-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H(2)O(2), inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H(2)O(2) activity [H(2)O(2) or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.

Orthovanadate-induced vasocontraction is mediated by the activation of Rho-kinase through Src-dependent transactivation of epidermal growth factor receptor.[Pubmed:25505586]

Pharmacol Res Perspect. 2014 Apr;2(2):e00039.

Orthovanadate (OVA), a protein tyrosine phosphatase (PTPase) inhibitor, exerts contractile effects on smooth muscle in a Rho-kinase-dependent manner, but the precise mechanisms are not elucidated. The aim of this study was to determine the potential roles of Src and epidermal growth factor receptor (EGFR) in the OVA-induced contraction of rat aortas and the phosphorylation of myosin phosphatase target subunit 1 (MYPT1; an index of Rho-kinase activity) in vascular smooth muscle cells (VSMCs). Aortic contraction by OVA was significantly blocked not only by Rho kinase inhibitors Y-27632 [R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cyclohexanecarboxamide] and hydroxyfasudil [1-(1-hydroxy-5-isoquinolinesulfonyl)homopiperazine] but also by Src inhibitors PP2 [4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine] and Src inhibitor No. 5 [4-(3'-methoxy-6'-chloro-anilino)-6-methoxy-7(morpholino-3-propoxy)-quinazoline], and the EGFR inhibitors AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline] and EGFR inhibitor 1 [cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide]. OVA induced rapid increases in the phosphorylation of MYPT1 (Thr-853), Src (Tyr-416), and EGFR (Tyr-1173) in VSMCs, and Src inhibitors abolished these effects. OVA-induced Src phosphorylation was abrogated by Src inhibitors, but not affected by inhibitors of EGFR and Rho-kinase. Inhibitors of Src and EGFR, but not Rho-kinase, also blocked OVA-induced EGFR phosphorylation. Furthermore, a metalloproteinase inhibitor TAPI-0 [N-(R)-[2-(hydroxyaminocarbonyl) methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine amide] and an inhibitor of heparin-binding EGF (CRM 197) not only abrogated the OVA-induced aortic contraction, but also OVA-induced EGFR and MYPT1 phosphorylation, suggesting the involvement of EGFR transactivation. OVA also induced EGFR phosphorylation at Tyr-845, one of residues phosphorylated by Src. These results suggest that OVA-induced vasocontraction is mediated by the Rho-kinase-dependent inactivation of myosin light-chain phosphatase via signaling downstream of Src-induced transactivation of EGFR.

Orthovanadate-Induced Vasoconstriction of Rat Mesenteric Arteries Is Mediated by Rho Kinase-Dependent Inhibition of Myosin Light Chain Phosphatase.[Pubmed:26521832]

Biol Pharm Bull. 2015;38(11):1809-16.

Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces vasoconstriction in a Rho kinase-dependent manner. The aim of this study was to determine the mechanism underlying OVA-induced vasoconstriction of rat mesenteric arteries. OVA-induced constriction of mesenteric arterial rings treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), a nitric oxide synthase inhibitor, was significantly blocked by the Rho kinase inhibitor Y-27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 10 microM), extracellular signal-regulated kinase 1 and 2 (Erk1/2) inhibitor FR180204 (5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine, 10 microM), Erk1/2 kinase (MEK) inhibitor PD98059 (2'-amino-3'-methoxyflavone, 10 microM), epidermal growth factor receptor (EGFR) inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline, 10 microM), and Src inhibitor PP2 (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 3 microM). However, the myosin light chain kinase inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-homopiperazine, 10 microM) did not affect OVA-induced constriction. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1, an index of Rho kinase activity) was abrogated by inhibitors of Src, EGFR MEK, Erk1/2, and Rho kinase. OVA-stimulated Erk1/2 phosphorylation was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2 but not affected by an inhibitor of Rho kinase. OVA-induced Src phosphorylation was abrogated by an Src inhibitor but not affected by inhibitors of EGFR, MEK, Erk1/2, and Rho kinase. In addition, the metalloproteinase inhibitor TAPI-0 (N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alan ine amide, 10 microM) and an inhibitor of heparin/epidermal growth factor binding (CRM 197, 10 microg/mL) did not affect OVA-induced contraction of rat mesenteric arterial rings. These results suggest that OVA induces vasoconstriction in rat mesenteric arteries via Src, EGFR, MEK, and Erk1/2 activation, leading to the inactivation of myosin light chain phosphatase through phosphorylation of MYPT1.

Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization.[Pubmed:27509198]

J Med Chem. 2016 Sep 8;59(17):8061-7.

Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.