The Antihypertensive Effect Of M-Nisoldipine On Spontaneously Hypertensive Rats And Its Underlying Mechanisms

m-Nisoldipine(m-Nis), a new calcium antagonist which is the isomer of Nisoldipine(Nis), was first developed and synthesized by Hebei Medical university. Our previous studies signified that m-Nis could inhibit vascular smooth muscle contraction, dilate corornary artery, decrease after load and oxygen consumption of heart. It is expected to become an ideal medicine in treating hypertension, ischemic heart disease, coronary artery disease, and atherosclerosis. The objective of the present study was to investigate the antihypertensive effect of m-Nis on conscious SHR and its possible mechanisms from intact animal to cells level. Part one. Antihypertensive effect of m-Nisoldipine on conscious SHR Objective: To evaluate the antihypertensive effects of m-Nis and provide experimental evidence for its clinical application, we studied the influence of m-Nis on blood pressure (BP) and heart rate (HR) in conscious spontaneously hypertensive rats (SHR) by single or multiple oral doses administration. Methods: Fifty qualified SHR aged for 14 weeks were divided randomly into 5 groups (n=10, 5 each in ♂and ♀): â‘´m-Nis7.5mg/kg group; ⑵m-Nis2.5mg/kg group; ⑶m-Nis 0.8mg/kg group; â‘·N is7.5mg/kg group; ⑸s olvent control group. m-Nis, Nis or solvent was given by oral administration of single dose for the acute hypotensive test and of multiple doses once daily at 8 am throughout the 14 days treatment for the chronic hypotensive test. Before administration, BP and HR were measured as control value at 0h or 0d. Then BP and HR of SHR in all groups were measured at 24h after dosing during 0d (as control value), 1d, 3d, 5d, 7d, 10d and 14d in the chronic antihypertensive experiment. On 2d and 4d after withdrawal of drugs, their BP and HR were measured again. In acute hypotensive experiment, BP and HR of SHR in all groups were measured at 0h (as control value), 1h, 2h, 3h, 4h, 6h, 8h, 12h and 24h after first medication, and the correlation analysis between dosage and BP was made at the maximal hypotensive effect time of m-Nis. Result: The significant reductions of BP in SHR occured at 1h after administration(P<0.001)in m-Nis groups compared with at 0h, its maximal hypotensive effect reached at 2h and BP showed a close negative correlation with dosage(r=-0.8541, P<0.01). Lower BP was kept for 12 hours in 0.8 mg/kg group, 24 hours in 2.5mg /kg group and 7.5mg/kg group. The maximal hypotension in Nis 7.5mg/kg group reached at 1h after dosing, and lasted for 12 hours. We also found that there were atransient elevation of HR at 1～2h in m-Nis 7.5 mg/kg and Nis 7.5 mg/kg groups compared with at 0h(p<0.05). Throughout 14 days chronic experiment, there was no obvious increase in HR and BP was decreased gradually. In m-Nis 0.8mg/kg group, a significant reduction of BP ( P<0.001) occurred on 7th day after administration and reached peak effect on 10th day. In m-Nis2.5mg/kg and 7.5mg/kg groups, reduction of the BP (p<0.05) were found on the third day and reached peak effect on 10th day. Effect of Nis 7.5mg/kg on BP was similar to that of m-Nis 7.5mg/kg group. After withdrawal of drugs, their hypotensive effects lasted nearly 4 days in all administration groups. Conclusion: Either single or multiple oral administration, m-Nisoldipine possesses a potent antihypertensive effect and a longer hypotensive duration without the changes of HR. Nis had the similar action. Key words: m-Nisoldipine; Nisoldipine; blood pressure; heart rate; spontaneously hypertensive rat Part two. Effects of m-Nisoldipine on hemodynamics and heart remodering in SHR Objective: To examine effects of m-Nis on hemodynamics and myocardium pathomorphology in SHR. Methods: Forty qualified SHR aged for 16 weeks were divided randomly into five groups (n=8, 5 each in ♂and ♀):â‘´m-Nis7.5mg/kg group; ⑵m-Nis2.5mg/kg group ; ⑶m-Nis 0.8mg/kg group; â‘·Nis7.5mg/kg group; ⑸solvent control group. They were given by intragastric administration once daily at 8 am. After treatment of 8 weeks, 2 SHR of each groups were retained for the proliferation and the cytosolic free calcium tests in aortic smooth muscle cells (AVSMC). The orthers was placed on operation table after abdominal anesthesia by sodium pentobarbital (40mg/kg), made a median incision on cervical part, and separated the right common carotid artery. A cardiac catheter was inserted into the left ventricular cavity via the right carotid artery and connected to MS4000U-1 system for examining hemodynamics parameters including HR, MBP, LVSP, LVEDP, and ±dp/dtmax.. Then hearts was quickly removed, Left ventricle was isolated and left ventricular mass index (LVMI) were calculated. Pathomorphological changes of the left ventricle were observed through hematoxylin-eosin staining and transverse diameters of myocardium (TDM) were measured by eyepiece micrometer under microscope (×400). Result: In hemodynamics experiment, no obviously change in HR was found, but MBP in all treatment groups had been significantly decreased, which were 19.2±1.4kpa in solvent control group, 15.6±2.5 kpa(P<0.05)in m-Nis 0.8mg/kg group,14.7±1.3 kpa in m-Nis 2.5mg/kg group(P<0.05),13.2±2.0kpa in m-Nis 7.5 mg/kg group(P<0.05),and 14.3±1.5 kpa in Nis 7.5 mg/kg group(P<0.05)respectively. LVSP were 36.2±3.1 kpa in solvent control group, 32.2±4.0 kpa in m-Nis 0.8 mg/kg group(P<0.05), 31.5±2.6 kpa in m-Nis 2.5 mg/kg group(P<0.05),30.4±4.2kpa in m-Nis7.5mg/kg group(P<0.05), and 29.5±3.7 kpa in Nis7.5mg/kg group(P<0.05), respectively. LVEDP were 2.8±0.4 kpa in the solvent control group, 2.1±0.3 kpa in m-Nis 0.8mg/kg group(P<0.05), 1.8±0.5kpa in m-Nis2.5mg/kg group(P<0.05), 1.7±0.2kpa in m-Nis7.5 mg/kg group(P<0.05),and 1.8±0.2kpa in Nis7.5mg/kg group(P<0.05), respectively. All of these were significantly lowered. There were little changes in ±dp/dtmax in all m-Nis groups , other than in Nis7.5mg/kg group, in which there was a significant decrease in ±dp/dtma(xP<0.05). In HE staining, the pathological damages of left ventricle in solvent control group were characterized by severe proliferation of left ventricular myocardium, myocyte hypertrophy, nucleus increased, cardiac muscle fiber twisted, and significant interstitial fibrosis. After 8 weeks adminstratiion, the pathological changes of left ventricle were greatly improved in different degree, especially in m-Nis2.5mg/kg group, 7.5 mg/kg group, and Nis7.5mg/kg group. Left ventricle mass index (LVMI) was 3.05±0.21mg/kg in 0.8mg/kg group, which had no significant decrease compared with solvent control group (3.29±0.27mg/kg) (P>0.05), but obvious decreases (P<0.05) exsisted in m-Nis2.5mg/kg group (3.02±0.16 mg/kg), in 7.5 mg/kg group(2.90±0.20mg/kg) , and in Nis7.5mg/kg group (2.96±0.25mg/kg). Transverse diameter of myocardium (TDM) in m-Nis2.5mg/kg group and 7.5mg/kg group were significantly decreased compared with solvent control group (P<0.05), butthere was no change in m-Nis0.8mg/kg group (P>0.05); TDM in Nis 7.5 mg/kg group was also significantly decreased (P<0.05). Conclusion: m-Nisoldipine could relieve left ventricular hypertrophy, decrease interstitial fibrosis, improve cardiac systolic and diastolic function. Key words: hemodynamics; spontaneously hypertensive rat; m-Nisoldipine; Nisoldipine; pathomorphology Part three. Effects of m-Nisoldipine on the proliferation and the cytosolic calcium content of aortic vascular smooth muscle cells in SHR Objective: To investigate the effects of m-Nis on proliferation and the cytosolic free calcium content of aortic smooth muscle cells (AVSMC) in SHR. Methods: 1 Three qualified SHR weighed 250～300g were used to isolate and culture AVSMC from aortic explants under sterile condition. Two to four generation cells were used in experiments. Cultured AVSMC were divided into 5 groups: m-Nis 10^-7mol/L group, 10^-6mol/L group, 10^-5mol/L group, Nis10^-6mol/L group, and the blank control group. m-Nis of different concentration (10^-7mol/L,10^-6mol/L,10^-5 mol/L) and 10^-6mol/L Nis were seperately added into cultured AVSMCs. The proliferation of AVSMC was detected by MTT method(OD544),and the growth curve of AVSMC was made. Fluo-3/AM labled cytosolic free Ca²⁺ and confocal laserscanning microscopy (LASM) were used to detect the cytosolic free calcium content in AVSMC of all groups. 2. Retained 10 SHR administrated for 8 weeks from each groups in part two were used to isolate and culture AVSMC according to the procedure above to observe directly the effects of m-Nis during long term administration on proliferation of AVSMC and the cytosolic free calcium content in AVSMC. Results: MTT test manifested that, after pretreatment with different concentration of m-Nis (10^-7mol/L, 10^-6mol/L, 10^-5mol/L),significant decreases of MTT values(OD544)were found in m-Nis 10^-5mol/L group at 3, 4, 5, 6 day during the experiment(P<0.05), and at 5, 6 day in 10-6mol/L group (P<0.05), but did not in m-Nis10-7mol/L group (P>0.05), compared with the blank control group. There was also a significant decrease ( P<0.05 ) at 4, 5, 6 day during the experiment period in Nis 10^-6 mol/L group. MTT test of AVSMC from SHR administrated for 8 weeks showed that OD₅₄₄ in m-Nis7.5mg/kg group was significantly decreased at 4, 5, 6 day during the experiment(P<0.05), at 6 day in m-Nis 2.5 mg/kg group, and at 5, 6 day in Nis7.5mg/kg group(P<0.05), but OD544 value of m-Nis0.8mg/kg group had little change, compared with corresponding solvent control group. The cytosolic free calcium content of AVSMC were 91.7±12.5 in m-Nis10-7mol/L group(P<0.05), 85.1±9.2 in m-Nis 10^-6 mol/L group(P<0.01),75.3±9.8 in m-Nis 10^-5 mol/L group(P<0.01), and 82.7±11.0 in Nis 10^-6 mol/L group(P<0.01),...