Abstract-Short-term treatment of the endothelium with dihydropyridine calcium antagonists resulted in an increased release in NO that is not due to a modulation of L-type calcium channels, because macrovascular endothelial cells do not express this channel. We investigated whether long-term (48 hours) treatment of porcine endothelial cell cultures with the dihydropyridine calcium antagonist nifedipine resulted in a similar enhanced NO liberation. Regarding to the underlying mechanism, we examined whether (1) nifedipine changed the mRNA and protein levels of the constitutive endothelial NO synthase (NOS) in endothelial cell cultures or (2) nifedipine exerts an NO protective effect via its antioxidative properties, as revealed in a cell culture model and with native endothelium from porcine coronary arteries. ihydropyridine (DHP)-type calcium antagonists are important drugs in the treatment of hypertension and coronary heart disease. They induce their specific pharmacological effects by binding to L-type calcium channels, 1,2 which results in a reduced calcium influx with impaired electromechanical coupling both in vascular smooth muscle cells and in the heart. 3 A few years ago, however, it was observed that removal of the endothelium or blockade of the guanylate cyclase of the vessel wall reduced the efficacy of the DHP-induced vasorelaxation, 4 which indicated an endothelium-responsive cGMP-mediated process as part of the DHP action. Because macrovascular endothelial cells lack voltage-operated L-type calcium channels, 5,6 the DHPs must exert these effects via other mechanisms. In this context, it is worth mentioning that DHPs may also exert antihrombotic 7,8 and antiatherosclerotic 9,10 effects in different experimental and clinical settings, of which the underlying signal transduction remains obscure.With this background, it is tempting to speculate that NO, one of the most prominent endothelium-derived factors, 11,12 which relaxes smooth muscle cells via the cGMP signal cascade, 13 might be involved in these DHP actions. In fact, in various models, evidence has accumulated that DHPs stimulate the endothelial NO release, 14 -19 which may mediate or at least contribute to the abovementioned calcium channel-independent effects.Up to now, however, all of these findings were obtained only after acute exposure to DHPs (lasting minutes to hours). Because patients usually take calcium antagonists for a longer period of time, we investigated whether long-term treatment of endothelial cell cultures with nifedipine may alter the basal endothelial NO release as well as the expression of the constitutive endothelial NO synthase (ecNOS) mRNA and protein. Furthermore, because NO is rapidly deactivated by reactive oxygen species (ROS) 20 and the DHPs may act as scavengers as known from different in vitro models, [21][22][23] we also determined the antioxidative potency of nifedipine in endothelial cell cultures as well as in native cells to reveal a potential NO-protection effect as an underlying mechanism of the increased ...

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.