Chide Han

14-3-3 Proteins regulate glycogen synthase 3β phosphorylation and inhibit cardiomyocyte hypertrophy

14‐3‐3 Proteins are dimeric phophoserine‐binding molecules that participate in important cellular processes such as cell proliferation, cell‐cycle control and the stress response. In this work, we report that several isoforms of 14‐3‐3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14‐3‐3 peptide inhibitor, R18, to disrupt 14‐3‐3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus‐expressing YFP‐R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3‐kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt – also called protein kinase B, PKB) at Ser473 and glycogen synthase 3β (GSK3β) at Ser9, but not extracellular signal‐regulated kinase 1/2 (ERK1/2). AdR18‐induced PKB and GSK3β phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14‐3‐3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3β and NFAT pathway.

Alteration of α1-adrenoceptor subtypes in aortas of 12-month-old spontaneously hypertensive rats

Alterations in α1-adrenoceptor subtypes in aortas from 12-month-old spontaneously hypertensive rats (SHR) were studied in functional studies and RNase protection assays. The norepinephrine-induced contraction, including maximum response and pD2 values, was not significantly different between the SHR and age-matched Kyoto Wistar (WKY) rats. The pA2 values of the α1D-adrenoceptor subtype-selective antagonist BMY7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dionedihydrochloride) were increased from 8.10±0.12 in WKY rats to 8.45±0.13 in SHR (P 0.05). Preincubation of preparations in 50 μM chloroethylclonidine for 30 min irreversibly inhibited the norepinephrine-induced response more profoundly in aortas from SHR than in aortas from WKY rats. The results of RNase protection assays showed that mRNAs for α1A- and α1B-adrenoceptor subtypes were decreased and that mRNA for the α1D-adrenoceptor subtype was increased in aortas from SHR compared with WKY rats. The results suggested that the α1A-adrenoceptor subtype was decreased and the α1D-adrenoceptor subtype was increased in aortas of 12-month-old SHR.

The antipsychotic drug sertindole is a specific inhibitor of α1A-adrenoceptors in rat mesenteric small arteries

We have investigated the adrenergic antagonist effect of the antipsychotic sertindole in rat resistance vessels and in membranes of HEK293 cells transfected with alpha1-adrenoceptors. Segments of rat mesenteric small arteries or rat aorta were mounted on a myograph for isometric tension recording. In mesenteric small arteries, specific alpha1A-adrenoceptor antagonists (5-methyl urapidil and WB-4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane)) inhibited phenylephrine responses with high affinity (pA2 9.1 and 9.5, respectively). Chlorethylclonidine (alpha1B- and alpha1D-adrenoceptor antagonist) and BMY7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4,5] decane-7,9-dione dihydrochloride, alpha1D-adrenoceptor antagonist) had little effect. This indicated that the adrenoceptor subtype in the mesenteric small arteries was of the alpha1A subtype. Sertindole inhibited the phenylephrine response of mesenteric small arteries (pA2 9.0), but had little effect on the phenylephrine response of aorta (which lacks alpha1A-adrenoceptors). The specific action of sertindole on alpha1A-adrenoceptors was supported by experiments with membranes of HEK293 (human embryonic kidney) cells transfected with the alpha1A-, alpha1B- and alpha1D-adrenoceptors. Here, with concurrent incubation, sertindole showed selective competitive inhibition of BE2254 (2-beta(4-hydroxyphenyl)-ethylaminomethyl)-tetralone) binding to alpha1A-adrenoceptors (Ki 8.9), compared to alpha1B-adrenoceptors (Ki 7.1) and alpha1D-adrenoceptors (Ki 6.8). Despite the apparent competitive action sertindole, it was not possible to wash out its antagonist effect within 6 h in the functional phenylephrine concentration-response experiments. Furthermore, in the membranes of HEK293 cells transfected with the alpha1A-adrenoceptors, binding of [125I]BE2254 was reduced by 45% following preincubation with sertindole (1 nM). We conclude that the alpha-adrenoceptors of rat mesenteric small arteries are of the alpha1A-type, and that sertindole is a specific pseudo-irreversible competitive antagonist of this adrenoceptor subtype.

Characterization of subtype of α1-adrenoceptor mediating vasoconstriction in perfused rat hind limb

The subtype of alpha1-adrenoceptor mediating the exogenous noradrenaline-induced vasopressor response in perfused rat hind limb was determined by functional measurements and radioligand binding assays. The potencies (pA2 values) of alpha1A-adrenoceptor-selective antagonists, RS-17053 (N-[2-(2-cyclopropylmethoxy-phenoxy) ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), WB 4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4 benzodioxane), 5-methyl-urapidil, and the alpha1D-adrenoceptor-selective antagonist, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4.5]de cane-7,9-dione), to inhibit the noradrenaline-induced vasopressor response determined by Schild plot were 9.47 +/- 0.21, 9.48 +/- 0.19, 8.10 +/- 0.27 and 6.66 +/- 0.14, respectively, with no slope significantly different from unity. The affinities (K(i) values) of these antagonists were determined by displacement of 125I-BE 2254 (2-beta(4-hydroxyphenyl)-ethylaminomethyl)-tetralone) binding from the cloned alpha1a-, alpha1b-, alpha1d-adrenoceptor, stably expressed in human embryonic kidney (HEK) 293 cells. The pA2 values of the above antagonists correlated well with the binding K(i) values only for alphaIA-adrenoceptors (r = 0.93), but not for alpha1B-adrenoceptors (r = 0.51) and alpha1D-adrenoceptors (r = 0.13). The concentration-vasopressor response curve for noradrenaline was not significantly affected by pretreatment with 50 microM chloroethylclonidine for 30 min. The results suggest that only alpha1A-adrenoceptors mediate the noradrenaline-induced vasopressor response in perfused rat hind limb.

Different roles of α1-adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal rats

1. Three different α1‐adrenoceptor subtypes, designated α1A, α1B and α1D, have been cloned and identified pharmacologically in cardiomyocytes. In vitro studies have suggested that α1‐adrenoceptors play an important role in facilitating cardiac hypertrophy. However, it remains controversial as to which subtype of α1‐adrenoceptors is involved in this response. In the present study, we investigated the different role of each α1‐adrenoceptor subtype in mediating cardiomyocyte protein synthesis, which is a most important characteristic of cardiac hypertrophy in cultured neonatal rat cardiomyocytes.

Gene expression profiles in response to the activation of adrenoceptors in A7r5 aortic smooth muscle cells.

1. Vascular adrenoceptors play an important role in vascular physiology and pathophysiology, such as hypertension, atherosclerosis and restenosis after angioplasty. To define the changes in the ene expression in vascular smooth muscle cells in response to the activation of α1‐ or β‐adrenoceptors, a DNA microarray was used.

Alterations of α1-adrenoceptor subtypes in the hearts of thyroxine-treated rats

Abstract Alterations in the cardiac α1-adrenoceptor and its subtypes in thyroxine-treated rats were studied by means of radioligand binding assays, measurement of contractile response and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that in thyroxine-treated rats the cardiac α1-adrenoceptor density (Bmax) was reduced from 51.6 ± 6.0 fmol/mg in control to 40.9 ± 3.7 fmol/mg (P

Role of K+ channels in neuropeptide Y-induced vasoconstriction in rabbit cerebral basilar artery

The role of K+ channels in the direct vasoconstrictive response induced by neuropeptide Y was investigated in isolated basilar arteries of rabbits and in vivo in rats. K+ channel openers, either BRL38227 or diazoxide, caused dose-dependent and complete relaxation of isolated arteries precontracted by neuropeptide Y. Exposure to both BRL38227 and diazoxide shifted the concentration-response curves for neuropeptide Y to the right without changing the maximal response. However, BRL38227 antagonized the angiotensin II-induced vasoconstriction noncompetitively. In vivo, the pressor responses produced by neuropeptide Y were significantly inhibited by pretreatment with BRL38227 in anesthetized rats. These results show that K+ channel openers antagonize neuropeptide Y-induced vasoconstriction in a competitive manner and suggest that blockade of K+ channels contributes, at least in part, to the direct vasoconstrictive effect of neuropeptide Y.