| lptakalim (Ipt), a novel chemical structural entity, designed and synthesized at Beijing Institute of Pharmacology and Toxicology. Our precious pharmacological studies demonstrated that the compound has several distinctive pharmacological and therapeutic advantages with potent and selectively antihypertensive actions, highly selective for small blood vessels. It shows stable, long-lasting anti-hypertensive action with little influence on heart rate. In addition to reducing blood pressure, the compound could reverse the hypertensive vascular, cardiac remodeling and exhibit the protective effects on hypertensive brain, renal damage. From available preclinicai observation, the future of iptakalim seems promising for hypertension therapy. To date, however, the molecular biology nature of iptakalim was not addressed.With conventional molecular biological or biochemical approaches, studies on regulation of expression can be conducted on only a small number of genes and proteins. Advances in molecular genetics and computational biology have led to the development of innovative methods to analyze differential gene expression profiles. DNA microarray technology represents a powerful tool for rapid, comprehensive, and quantitative analysis of gene expression profiles of different states.In present study, with the use of microarray, we analyzed and identified the genes with changed expression upon the administration of iptakalim in rat vital organs. Thismicroarray approach might contribute to an comprehensive analysis of the drug efficacy and side effect and molecular biology nature in vivo relevant to iptakalim.1. Effects of iptakalim-induced gene expression changes in rat hearts by cDNA microarray1.1 Characteristic expression profiles induced by iptakalim in rat heartsUsing BioStarR40s cDNA microarray, the present study evaluated the gene expression profile of rat hearts with iptakalim treatment for 2 weeks at a dose of 3 mg/kg/day by oral administration. Among 4,096 transcripts represented on the chip, mRNA levels of 236 transcripts were changed (100 increased and 136 decreased) in the iptakalim-treated rats compared to control rats.1.2 RT-PCR analysisSimilar to genechip results, RT-PCR also showed that iptakalim significantly increased expressions of myosin regulatory light chain, ADP-ribosylation factor 1, ADP-ribosylation factor 4, calreticulin, cyclase-associated homologue, guanine nucleotide-binding protein G-s, alpha subunit in heart tissues, compared with the control rats, whereas expressions of protein phosphatase 2, protein tyrosine phosphatase were significantly decreased.1.3 Effects of iptakalim on PKA activity in rat heartsAfter iptakalim treatment for 2 weeks at a dose of 3 mg/kg/day by oral administration, PKA activity increased significantly (p < 0.05 compared with the untreated normal control) .1.4 Effects of iptakalim on cardiovascular hemodynamics parameters in anestheti -zed ratsRats were randomly divided into normal control group and 4 iptakalim groups. Iptakalim was administered orally at doses of 1, 3, 9 mg/kg body weight per day for 2 weeks. The last group was orally administered at a dose of 9 mg/kg body weight per day for 2 weeks, and iptakalim was withdrawn for 10 days. We observe the effects of iptakalim on hemodynamics parameters in anesthetized rats after 24h at the end of thelast administration.Compared with control, oral administration of iptakalim at the doses of 1, 3, 9 mg/kg had no effects on heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MAP), systolic function and diastolic function in anesthetized rats. The results suggested that iptakalim had no pharmacological changes on cardiac function in vivo.1.5 Effects of iptakalim on cardiac function in isolated working heart of ratsIn isolated working rat hearts, isoprenaline 10 nmol/L had positive chronotropic and positive inotropic actions. The response of hearts to isoprenaline after iptakalim treatment of 1, 3, 9 mg/kg were identical to control group. The results suggested that iptakalim (1, 3, 9 mg/kg) didn t affect the response of hearts to isoprenaline in isolated working rat hearts.In isolated working rat hearts, pinacidil 10 umol/L had positive chronotropic and negative inotropic actions. The response of hearts to pinacidil after iptakalim treatment of 1, 3, 9 mg/kg were identical to control group. Iptakalim (1, 3, 9 mg/kg) didn t affect the response of hearts to pinacidil in isolated working rat hearts.1.6 Effects of iptakalim on myocardial morphological and ultra-structureObserved under light microscope, low or moderate or high dose of iptakalim (1,3, 9 mg/kg) had no effects on myocardial structure. Observed under electron microscope, low or moderate or high dose of iptakalim (1, 3, 9 mg/kg) had no effects on mitochondria and myofilament ultra-structure. The results suggested that iptakalim under the same experimental conditions had no pathological structure changes on myofilament.2. Effects of iptakalim-induced gene expression changes in rat liver by cDNA microarray 2.1 Characteristic expression profiles induced by iptakalim in rat liverUsing BiostarR40s cDNA microarray, the present study evaluated the gene expression profile of rat livers. Among 4,096 transcripts represented on the chip, mRNA levels of 6 transcripts were changed (6 increased) in the iptakalim-treated ratscompared to control rats. It includes CYP4alO, CYP8bl, carnitine pahnitoyltransferase 1 alpha, liver isoform (Cptla), enoyl hydratase-like protein, and peroxisomal (Echl). 2.2 RT-PCR analysisSimilar to genechip results, RT-PCR also showed significantly increased expressions of CYP4alO, CYP8bl, and enoyl hydratase-like protein, in livers of iptakalim- treated rats compared with the control rats.3. Effects of iptakalim-induced gene expression changes in rat brain by cDNA microarrayUsing BiostarR40s cDNA microarray, the present study evaluated the gene expression profile of rat brain tissues with iptakalim treatment for 2 weeks at a dose of 3 mg/kg by oral administration. Under this experiment conditions, there is no changed gene at transcriptional levels in normal rat brains.Therefore, we come to the conclusion:1. The new chemical entity iptakalim has selectivity on the changes of gene expression in rat heart, liver and brain. We use BiostarR40s microarray to analyze the gene expression changes in rat vital organs after treatment with iptakalim at a dose of 3mg/kg for 2 weeks. Results show that compared with control group, 236 genes are changed (100 increased and 136 decreased) in rat hearts and 6 transcripts (6 increased) in rat livers, there are no changes on gene expression in rat brains.2. After treatment with iptakalim, gene expression related to muscle contractile are changed, such as myosin regulatory light chain, smooth muscle alpha-actin. Signal transduction genes are also affected, such as G protein, cyclase-associated protein homologue.3. Repeatedly administration of iptakalim increases activity of protein kinase A.4. In anesthetized rats, iptakalim at doses of 1, 3, 9 mg/kg neither affect pharmacological effects on cardiovascular hemodynamics parameters nor pathological changes on myocardial morphological and ultrastructure. There are no pharmacological responses to isoprenaline and pinacidil in isolated working rat hearts after treatment with iptakalim. Under the same experimental conditions, iptakalim... |
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