A rise in blood pressure is the main side effect of erythropoietin (EPO) treatment in patients with renal anemia. The mechanisms, however, by which EPO may cause hypertension are still unclear. We therefore investigated the effects of EPO on endothelin (ET) synthesis and cytosolic free calcium concentration ([Ca2+]i) in vascular endothelial cells. Porcine endothelial cells were isolated from thoracic aorta, pulmonary artery, and vena cava. Studies were performed with cells of the first subculture. ET concentrations were measured radioimmunologically. Changes in [Ca2+]i were determined with the fluorescent probe fura-2. Cytotoxicity was assessed by sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)ben zene sulfonic acid hydrate (XTT) assay. ET synthesis was similar in cells of different vascular origins and was time-dependent, reaching approximately 2 pmol ET/mg protein within 12 h of incubation. EPO (12 to 200 U/mL) stimulated ET release time- and dose-dependently by up to 83.2% (P < .01) within 12 h in the absence of fetal calf serum and heparin. EPO induced an immediate significant rise in [Ca2+]i from 58 +/- 12 nmol/L to 495 +/- 85 nmol/L (P < .01) with a subsequent slow return to 257 +/- 3 nmol/L. During 2 h of incubation, the Ca-ionophore A 23187 (10(-8) mol/L) moderately but significantly stimulated endothelial ET synthesis. However, the Ca-channel blocker verapamil, the intracellular Ca-release blocker TMB-8, and nickel, an unspecific calcium channel blocker, had no consistent effects on [Ca2+]i or ET synthesis. The protein kinase C inhibitor H-7 stimulated basal [Ca2+]i and cellular ET synthesis. The tyrosine kinase inhibitor genistein suppressed the EPO-induced rise in [Ca2+]i and cellular ET synthesis. From these data we conclude that EPO may stimulate ET synthesis in vascular endothelial cells by activation of an EPO-receptor and via intracellular signalling mechanisms that comprise tyrosine kinase activation and a rise in [Ca2+]i. Therefore, the systemic hypertensive effects of EPO may be due at least in part to local stimulation of vascular endothelial ET synthesis via calcium mobilization.
In a new model of spontaneous hypertension, namely the Prague hypertensive rat (PHR), hypertension is transferred with a kidney transplanted from the PHR to its normotensive counterpart (PNR) by an as yet unknown mechanism. One candidate may be endothelin (ET), since this potent vasoconstrictor affects vascular tone, renal haemodynamics and renal excretory function, and all members of this peptide family are located within the kidney and act in an autocrine/paracrine fashion. In the present study we investigated, in the renal tissue of PHRs and PNRs: (1) preproET-1 and preproET-3 mRNAs as well as ET-1 and ET-3 peptide distribution, (2) endothelin-converting enzyme (ECE)-1 mRNA expression, and (3) ET receptors and their characteristics in membranes of glomeruli and papillae. In addition, plasma ET concentration and urinary ET excretion were determined. Quantitative measurements by competitive reverse transcription-polymerase chain reaction revealed ET-1 mRNA levels in the renal cortex from PHRs and PNRs of 1.09+/-0.13 and 1. 29+/-0.18 amol/microgram of total RNA respectively, and in red medulla of 2.72+/-0.82 and 3.30+/-0.68 amol/microgram respectively. In contrast, renal papilla from PHRs showed significantly lower levels of preproET-1 mRNA (1.81+/-0.64 amol/microgram of total RNA, compared with 4.25+/-0.82 amol/microgram in PNRs; each n=5; P<0.05). The ET-1 peptide concentration in papillary tissue was also significantly lower in PHRs than in PNRs (120.2+/-30.8 and 491.3+/-53.4 fmol/mg of protein respectively; n=5; P<0.01), whereas it was similar in cortex and medulla from PHRs and PNRs. The preproET-3 mRNA content in renal tissue was much lower than that of preproET-1 mRNA. It was significantly higher in red medulla from PHRs compared with that from PNRs (0.25+/-0.05 and 0.13+/-0.02 amol/microgram of total RNA respectively; P<0.05), but was similar in papillae of PHRs and PNRs (0.04+/-0.02 and 0.05+/-0.01 amol/microgram respectively; n=5). Cortical preproET-3 mRNA was at the lower limit of detection. Similarly, the ET-3 peptide concentration was slightly but significantly higher in the red medulla of PHRs compared with PNRs (15.4+/-2.0 and 8.8+/-0.8 fmol/mg of protein respectively; n=5; P<0. 05), whereas no differences in ET-3 peptide concentration were found in papillae from PHRs and PNRs. ECE-1 mRNA levels were similar in the renal cortex, red medulla and papillae from PHRs and PNRs, ranging between 0.34+/-0.03 and 0.56+/-0.12 amol/microgram of total RNA. Of the total ET receptors in glomerular membranes, 39% were ETA receptors, whereas papillary membranes contained exclusively ETB receptors. PHRs and PNRs showed similar Bmax and Kd values for ET-1 in renal glomerular membranes (Bmax, 6.5+/-1.3 and 4.9+/-1.2 pmol/mg of protein respectively; Kd, 0.69+/-0.10 and 0.56+/-0.10 nM respectively) and papillary membranes (Bmax, 9.7+/-1.1 and 11.3+/-1. 6 pmol/mg of protein respectively; Kd, 0.30+/-0.04 and 0.42+/-0.07 nM respectively). Plasma ET-1/2 concentrations (10.4+/-1.3 and 12. 2+/-1.2 fmol/ml in PHRs and PNRs re...
Mitogen-activated protein (MAP) kinases are important intracellular mediators for proliferation and hypertrophy and therefore may also regulate cardiomyoblast growth in hypertensive heart disease. Thus, the aim of the present study was to examine the activities of MAP kinases, namely extracellular signal-regulated kinase (ERK)1,2, c-Jun NH2-terminal kinases (JNK)1,2 and p38 MAP kinase, in myocardial tissue of 12-week-old Prague normotensive (PNR) and hypertensive rats (PHR), a model of genetic hypertension with marked cardiac hypertrophy. Systolic blood pressure was 121 ± 5 in PNR and 208 ± 15 mm Hg in PHR (p < 0.01). Total heart weight was 247 ± 4 in PNR vs. 316 ± 4 mg/100 g body weight in PHR (p < 0.01). Left and right ventricular weights were 121 ± 5 and 53 ± 3 in PNR vs. 168 ± 4 (p < 0.01) and 57 ± 2 mg/100 g body weight (n.s.) in PHR. Using anti-ERK2 Western blot analysis as well as immunocomplex ERK activity assay, we found no activation of ERK2 in left or right ventricular tissue of PHR and PNR. Similary, p38 MAP kinase phosphorylation and activity were not detectable. In contrast, Western blot analysis using antiphospho-JNK antibodies revealed in myocardial tissue of right and left ventricles significantly greater phosphorylation of JNK2 in PHR than in PNR. This finding was confirmed by immunocomplex JNK activity assay using ATF-2 as substrate, which demonstrated a significant increase in JNK activity in the left ventricle of PHR as compared to PNR (6.4 ± 1.5 vs. 2.5 ± 0.5 OD; each n = 5; p < 0.05). In conclusion, cardiac JNK2 seems to be regulated differently from ERK2 in this rat model. In PHR, as compared to PNR, we found enhanced activity of JNK2 in the left and right ventricles suggesting that JNK2 is involved in hypertensive cardiac disease. The rise in JNK in both ventricles may result indirectly from humoral stimuli, e.g., endothelin-1 and/or angiotensin II, and may contribute to ventricular hypertrophy in this model of spontaneous hypertension.
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