Calcineurin antagonists FK506 and CsA, administered to treat organ allograft rejection, exert specific effects on renal vasoconstriction and nephrotoxicity, possibly due to endogenous vasoconstrictor release such as ET-1. We investigated contribution of FK506 and CsA on regulation of prepro ET-1 gene transcription in HUVEC. To conclude on transcriptional regulation, ET-1 mRNA levels were quantified by Northern blot analysis upon stimulation with calcineurin antagonists, and newly transcribed luciferase gene, placed under the control of the rat ET-1 promoter, was quantified by reporter gene assays, where luciferase activity reflects ET-1 promoter activation. Calcium fluorometry was employed to examine calcium dependency of ET-1 promoter-dependent gene transcription. Northern blot analysis shows differential induction of prepro ET-1 mRNA in favour of CsA over FK506. Likewise, luciferase assays demonstrate stronger ET-1 promoter-dependent stimulation of the reporter gene by CsA than by FK506. Transcription of prepro ET-1 gene upon stimulation with both calcineurin antagonists is regulated by intracellular calcium levels. Lack of extra- or intracellular calcium prevents ET-1 promoter-dependent gene transcription and ET-1 mRNA induction. These observations demonstrate that calcineurin antagonists FK506 and CsA differ in quality to induce transcription of prepro ET-1 in HUVEC via calcium-dependent nuclear signalling events. To examine the contribution of ET-1 in nephrotoxicity upon CsA and FK506 immunosuppression the availability of endothelin receptor antagonists or endothelin converting enzyme inhibitors is required.

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In vascular endothelium, endothelium-derived relaxing factor, predominantly nitric oxide (NO), is synthesized by endothelial NO synthase (eNOS). While regulatory influences on eNOS enzyme activity are widely clarified, little is known about the regulation of the eNOS gene. We investigated the regulatory signaling mechanisms of eNOS mRNA expression and accumulated NO production in human endothelial cells. Northern blot analysis and NO assays demonstrate that the vasoconstrictor peptide endothelin-1 (ET-1) induces the eNOS gene and leads to accumulated NO production. Induction occurs via ETA receptor activation and depends on improved transcript stability. It is maintained for incubation periods of 30-90 min and tapers thereafter. Regulatory signaling mechanisms depend on de novo protein synthesis to control eNOS mRNA fate. Selectively blocking protein tyrosine kinases (PTK) and inhibiting protein kinase C (PKC) inhibit eNOS mRNA expression and accumulated NO secretion. These observations indicate that regulation of eNOS at the genomic level occurs via post-transcriptional mechanisms. Two protein-bound intracellular kinase pathways, PTK and PKC, regulate eNOS mRNA expression and accumulated NO production.

Plasma levels of alpha-human atrial natriuretic peptide (hANP) were measured in 17 patients with primary hypertension (11 females, 6 males, aged 22-61; blood pressure systolic 154 +/- 7 mmHg, diastolic 92 +/- 4 mmHg) and in 9 normotensive controls (4 males, 5 females, aged 20-71; blood pressure systolic 117 +/- 4 mmHg, diastolic 76 +/- 2 mmHg) during unrestricted sodium diet, at the 4th day of a low sodium intake (40-60 mEq/day) and at the 6th day of sodium loading (280-320 mEq/day) both after an overnight rest and after 4 h of upright posture. In the controls, plasma levels of hANP at 8:00 a.m. were lowered from 73 +/- 11 to 49 +/- 7 pg/ml during low sodium diet and increased to 128 +/- 37 pg/ml after high salt intake. Plasma ANP levels were significantly lower after 4 h of upright posture during unrestricted, low and high sodium intake. In the hypertensive group, plasma ANP levels were elevated during unrestricted diet (203 +/- 43 pg/ml), during the low sodium period (139 +/- 31 pg/ml), and after high sodium intake (267 +/- 63 pg/ml) compared to the controls. All levels were lowered by upright posture. The absolute decrease was more pronounced compared to the normotensives, the relative decline was similar in both groups. In the hypertensives, plasma ANP levels significantly correlate with systolic and diastolic blood pressure (r = 0.468, r = 0.448, P less than 0.05) and with urinary aldosterone during unrestricted diet (r = 0.536, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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