Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5-upstream region of the rat 25-hydroxyvitamin D 3 24-hydroxylase gene at ؊151/؊137 and ؊259/؊245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter ؊291/؉9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat ؊169/؊155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element ؊169/؊155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous -globin promoter. The fragment ؊291/؊102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element ؊169/؊155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.Vitamin D 3 exerts its biological activity after conversion into 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 )1 by successive hydroxylations at C-25 in the liver and C-1 in the kidney. 1,25-(OH) 2 D 3 mediates various biological activities, including calcium homeostasis, bone remodeling, cell growth and differentiation, and immune responsiveness (1-5).One of the targets of 1,25-(OH) 2 D 3 is the vitamin D 24-hydroxylase gene. The enzyme expressed by this gene catalyzes 24-hydroxylation of 25-hydroxyvitamin D 3 and 1,25-(OH) 2 D 3 into 24,25-dihydroxyvitamin D 3 and 1,24,25-trihydroxyvitamin D 3 , respectively (6). 24-Hydroxylation is considered an inactivation process of 1,25-(OH) 2 D 3 (3, 7-9). We have isolated rat 24-hydroxylase cDNA and determined the gene structure (10, 11). The expression of 24-hydroxylase mRNA was strikingly increased by vitamin D in vivo and in vitro (9, 12, 13). Reinhardt and Horst (14) Three groups have independently identified two functional VDREs in an antisense orientation in the rat 24-hydroxylase gene promoter; at Ϫ151/Ϫ137 (VDRE-1) (24, 25) and Ϫ259/ Ϫ245 (VDRE-2) (26). Zierold et al. (27) examined the interaction between the two VDREs by means of a reporter gene assay using heterologous promoters and by a gel mobility shift assay. They concluded that the two VDREs synergistically increased binding affinity for the receptor complex to elicit powerful promoter activity.In this study, we assessed the functions of the two VDREs in more detail using a native promoter containing site-specific mutations at the VDREs and determined their affinity for the receptor complex by gel retardation assays. We found that the two VDREs work addi...
The mfuslon of L-argmme mduces the productron of mtrtc oxrde and sttmulates the immediate secretron of msuhn To examme the relatronshrp between msulm reststance and endothelmm-dependent vascular relaxatton m patrents with essential hypertension, we evaluated the renal and msulm responses to L-argmme, 500 mg/kg infused mtravenously over 30 mmutes, m 23 patients with mild essenttal hypertensron who were neither obese nor diabetic and m 20 normotenstve control subJects We found no difference between the two groups m blood glucose or msulm m the fasting condrtton The renovascular relaxatron mduced by L-argmme was srgmficantly less m patients with essential hypertension than m normotensrve control subJects The mcrease m plasma cychc GMP in response to L-arginine was lower m hypertensive patients than m normotensive SubJeCtS Although the serum concentrattons of glucose m response to L-argmme were smular m the two groups, the serum msulm response of me essenhal hypertensrves was srgmficantly hrgher than that of the normotensive subJects In all subJects, the peak cychc GMP response to L-argmme was srgmficantly correlated wtth the peak Aglucose/ Amsulm ratto response to L-argmme (r= 69, P< 001) Fmdmgs suggested that an impanment of endothehum-dependent renal vascular relaxation and a reduced sensrtrvtty to msulm are present m patients with essential hypertension A lmk may be present between the abnormahty of the L-argmmelmtrrc oxide/cyclic GMP pathway and msulm resistance m patients with essential hypertension (Hypertension. The vasodilatory effect of msulm is reportedly mediated by the stimulation of the release of NO.26127 If so, there should be a dtrect physiologrcal correlation between msulm resistance and vascular endothehal dysfunction m patients with essential hypertension The mfusion of L-argmme induces the production of NO-cGMP and sttmulates the immediate secretron of msulm. An elevated plasma level of argmme is a particularly potent stimulus for insulin secretion, although the mechanism 1s not completely known 2829 We examined the responses to renal ctrculation and glucose/msulm to infused L-argmme to evaluate the relattonshtp between rnsulrn sensrtrvtty and endothebal function in patients with essential hypertension Subjects MethodsWe studied 23 Japanese mpattents with mild to moderate essential hypertension (16 males and 7 females, mean age 47 -t-3 years) and 20 normotensive control subJects (14 males and 6 females, mean age 4522 years) Hypertension was defined as a systolic or diastolic blood pressure of more than 160 mm Hg or/and 95 mm Hg, determmed m stttmg posmon on at least three different occastons Measurements were obtained m the outpatient clmtc of Hiroshima University School of Medrcme Patients with secondary forms of hypertenston were excluded by the appropriate chmcal and btochemtcal exammatrons None of the pattents had a hrstory of cardiovascular or cerebrovascular disease, drabetes melhtus, hypercholesterolemla, hver dtsease, or renal dtsease Normotenston was defined as a systohc blood ...
The iv administration of L-arginine, a precursor of endothelium-derived relaxing factor/nitric oxide, is known to decrease blood pressure in humans by its direct vasodilatory effects. The purpose of the present study was to determine whether L-arginine infusion modifies the renin-angiotensin (Ang)-aldosterone system as well as blood pressure and renal hemodynamics. L-Arginine and saline vehicle were iv administered to 10 healthy male subjects in random order on different days. L-Arginine infusion (500 mg/kg over 30 min) decreased mean blood pressure (from 81.2 +/- 2.7 to 74.0 +/- 2.5 mm Hg; P < 0.001) and renal vascular resistance (from 0.085 +/- 0.007 to 0.074 +/- 0.006 mm Hg/mL.min; P < 0.01) and increased heart rate (from 60.3 +/- 2.7 to 69.7 +/- 2.1 beats/min; P < 0.001) and renal plasma flow (from 616.6 +/- 37.8 to 701.0 +/- 49.2 mL/min; P < 0.05). L-Arginine reduced serum Ang-converting enzyme activity (from 10.4 +/- 0.6 to 8.9 +/- 0.5 nmol/mL.min; P < 0.05) and plasma Ang-II (from 19.3 +/- 3.3 to 12.7 +/- 2.8 pg/mL; P < 0.001), but had no effect on PRA or the glomerular filtration rate. The saline vehicle did not alter any of these parameters. The iv administration of L-arginine (endothelium-derived relaxing factor/nitric oxide) may reduce the plasma Ang-II concentration by inhibiting Ang-converting enzyme. The mechanism by which L-arginine infusion decreases blood pressure can be at least in part explained by inhibition of the renin-Ang system.
Results suggest that in VSMCs, capacitative Ca2+ entry is reduced by external Mg2+. This mechanism may explain in part the inhibitory effect of external Mg2+ on Ca2+ handling.
Objective-To examine coronary flow reserve immediately after emergency coronary angioplasty in patients with acute myocardial infarction.Design-A 3 F coronary Doppler catheter was used to measure coronary blood flow velocity in the infarct artery and in the non-infarct artery. Maximal hyperaemia was produced by 10 mg of intracoronary papaverine and coronary flow reserve was calculated.Patients-l patients with acute myocardial infarction undergoing both emergency coronary angioplasty (4 7 (3 6) h after the onset of chest pain (mean (SD))) and at follow up catheterisation 16 (4) days after angioplasty.Setting-Hiroshima City Hospital. Patients and methods PATIENTSWe examined coronary flow reserve in 11 patients (10 men and a woman, 53 (14) years (mean (SD))) with acute myocardial infarction undergoing both emergency coronary angioplasty and follow up cardiac catheterisation.Chest pain consistent with ongoing myocardial ischaemia that persisted for > 30 minutes and caused at least 1 mm ST elevation in at least two leads was diagnosed as acute myocardial infarction. Each patient underwent emergency cardiac catheterisation within 12 hours of the onset of symptom (4 7 (3 6) h). Creatine kinase was measured every three hours after hospital admission and peak serum creatine kinase concentration was more than twice of the upper limit of the normal range (3127 (1330) IU/l).
platelets; mag-fura 2; fura 2 IN THE LAST SEVERAL DECADES, abnormal Ca 2ϩ handling in many cell types from human subjects and animal models of primary hypertension has been reported and proposed as a factor in the pathogenesis of hypertension. Platelets are often used in the study of cellular cation metabolism in hypertension, because they are readily available for study and are thought to share a number of features with vascular smooth muscle cells (20). Most investigators have reported that basal levels of cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] i ) are higher in human subjects with essential hypertension than in normotensive subjects (5,8,11,16,30) MATERIALS AND METHOD Subjects.We studied 30 patients with essential hypertension (15 men, 15 women, mean age 51 Ϯ 11 yr) and 30 sex-and age-matched normotensive controls (15 men, 15 women, mean age 50 Ϯ 13 yr). Normotensive controls were recruited from healthy subjects who underwent annual physical examinations. Hypertension was defined as systolic blood pressure Ն160 mmHg and/or diastolic blood pressure Ն95 mmHg on each of three consecutive clinical visits. We measured blood pressure with a mercury sphygmomanometer in sitting subjects at least five times during each clinical visit and used the average value of these measurements. The blood pressure in normotensives was consistently Ͻ140/90 mmHg. None of the hypertensives or normotensives had received any medication for at least 4 wk before the study. Subjects with secondary forms of hypertension were excluded by careful clinical examination. Hypertensive patients and normotensive controls were maintained on a regular diet with an intake of 170 mmol/day NaCl to allow stabilization of the systemic Na ϩ balance, and they ingested constant amounts of K ϩ (2,000 mg/day), Ca 2ϩ (500 mg/day), and calories (40 kcal/kg) for 7 days before the study. Venous blood was collected from fasting and resting subjects, slowly and steadily via a 19-gauge needle into a syringe containing 3.8% trisodium citrate (1:9 by vol, total 30 ml), using a two-syringe method (21)
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