Tobias A. Marsen

Nifedipine Increases Endothelial Nitric Oxide Bioavailability by Antioxidative Mechanisms

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. Nifedipine induced a significant time- and concentration-dependent increase (132±47%, 1 &mgr;mol/L, 40 minutes’ incubation) in the basal NO liberation (oxyhemoglobin assay). This increased NO release was not due to elevated NOS (type III) mRNA (Northern blot analysis) and protein (Western blot analysis) levels. However, nifedipine (both short- and long-term treatment) significantly reduced the basal and glucose (20 and 30 mmol/L)-stimulated formation of reactive oxygen species (lucigenin assay) of endothelial cell cultures and native cells. We conclude that the calcium antagonist nifedipine enhances the bioavailability of endothelial NO without significantly altering the NOS (type III) mRNA and protein expression, possibly via an antioxidative protection. This increased NO availability may cause part of the vasodilation and might contribute to the antithrombotic, antiproliferative, and antiatherosclerotic effects of dihydropyridine calcium antagonists.

Thrombin Induces the Preproendothelin-1 Gene in Endothelial Cells by a Protein Tyrosine Kinase–Linked Mechanism

Thrombin stimulates synthesis and secretion of endothelin-1 (ET-1), a vasoactive peptide that triggers responses in the vascular endothelium and smooth muscle. We investigated the signal transduction pathways by which thrombin stimulates preproET-1 gene expression and ET-1 peptide secretion in macrovascular cells (human umbilical vein endothelial cells [HUVECs] and bovine pulmonary artery endothelial cells [BPAECs]) and microvascular cells (human microvascular endothelial cell line [HMEC-1]). Thrombin (4 U/mL) stimulated maximal induction of ET-1 peptide secretion and preproET-1 mRNA after 2 hours in HUVECs and BPAECs and after 1 hour in HMEC-1. A synthetic thrombin receptor activator peptide confirmed ligand-specific receptor actions to induce preproET-1 mRNA. Protein kinase C (PKC) activation by phorbol ester transiently induced preproET-1 mRNA but had no effect on ET-1 peptide synthesis. PKC inhibitors sangivamycin and calphostin C and PKC depletion failed to suppress thrombin-stimulated preproET-1 mRNA. Adenylate cyclase and cAMP-dependent protein kinase did not participate in thrombin-induced preproET-1 gene activation. Thrombin stimulated a rapid increase in phosphotyrosine-containing proteins, suggesting a role for tyrosine phosphorylation in thrombin signaling. These data demonstrate that thrombin induces the preproET-1 gene and ET-1 peptide synthesis by a PKC-independent PTK-dependent pathway in macrovascular and microvascular endothelial cells. Protein tyrosine kinase inhibitors herbimycin A and genistein blocked thrombin-stimulated preproET-1 mRNA and peptide secretion, whereas daidzein, which lacks inhibitory activity, did not suppress thrombin-induced ET-1.

Pharmacokinetics of omega-3-fatty acids during ingestion of fish oil preparations

An in vivo comparison of three dosages (3 g, 6 g, 12 g) of two different fish oil preparations in terms of plasma concentrations of their major active components eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was performed. The plasma accumulation was measured during 28 days of ingestion and an equally long wash out period. Data were scrutinized for bioavailability in order to distinguish between the efficiency of the two preparations. Rapid increases in EPA and DHA plasma concentrations can be demonstrated at all dosages during a 28-day ingestion period. EPA accumulated more during ingestion of high than of low dosages of fish oil. DHA revealed almost identical increases and peak values in plasma concentrations in all subgroups. The present data demonstrate dose dependent increases of EPA concentrations whereas DHA plasma concentrations are comparable in all dosages investigated. Measurable EPA and DHA plasma concentration levels are inappropriate means to explain clinical effectiveness. These results were found in both commercially available fish oil preparations. Direct comparison of both preparations revealed no differences in bioavailability.

Differentia] transcriptional regulation of endothelin-1 by immunosuppressants FK506 and cyclosporin A

Abstract— 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 FK.506. 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.

Cyclosporin A induces prepro endothelin-1 gene transcription in human endothelial cells

Cyclosporin A employed in treatment of organ allograft rejection, is associated with hypertension possibly due to endothelin-1. We studied transcriptional regulation of endothelin-1 by cyclosporin A in human endothelial cells using cell transfection experiments and reporter gene assays. Human umbilical vein endothelial cells were established expressing a fusion gene of the coding sequence of the firefly luciferase gene, placed under the control of the rat endothelin-1 promoter. Luciferase assays demonstrate 2.8-fold stimulation of the reporter gene by cyclosporin A (P < 0.01), and Northern blot analysis shows induction of prepro endothelin-1 mRNA. Transcription is tightly repressed in the absence of the immunosuppressant, its regulation occurs Ca(2+)-dependent. Lack of extra- or intracellular Ca2+ prevents cyclosporin A-dependent endothelin-1 gene transcription and mRNA induction. These data demonstrate transcriptional regulation of endothelin-1 over a range of several orders of magnitude in human umbilical vein endothelial cells by cyclosporin A via Ca(2+)-dependent mechanisms. They support the critical role of endothelin- in cyclosporin A-associated hypertension.

Thrombin-mediated ET-1 gene regulation involves CaM kinases and calcineurin in human endothelial cells

Summary: The preproendothelin-1 (preproET-1) gene is induced by thrombin after phosphorylation of nonreceptor protein tyrosine kinase pathways. This study investigated the contribution of Ca2+/calmodulin-dependent intracellular signaling cascades to this pathway and measured ET-1 mRNA levels by Northern blot analysis in human endothelial cells. Increased intracellular Ca2+ levels in response to Ca2+ ionophore or Ca2+ ATPase inhibitors fert-butylhydroquinone and thapsigargin mimicked thrombin actions on ET-1 mRNA induction. Thrombin-mediated activation of ET-1 mRNA was reduced by specific calmodulin antagonists W7 or calmida-zolium and after inhibition of CaM kinase II by KN-62. Inhibition of calcium/calmodulin-dependent phosphatase calcineurin by cyclosporin A, however, stimulated ET-1 mRNA in human endothelial cells. Phosphotyrosine immunoblot assays show that calcium/calmodulin-dependent signaling pathways precede thrombin-induced tyrosine phosphorylation, and that the calcium/ calmodulin-dependent phosphatase calcineurin also exerts its effects via activation of protein tyrosine kinases. These observations demonstrate that thrombin stimulates the preproET-1 gene in human endothelial cells through calcium-dependent activation of CaM kinase and protein tyrosine kinases, and that calcineurin may also participate in regulation of the prepro ET-1 gene.

Enalapril and losartan augment endogenous nitric oxide release in Takayasu's arteritis--a case report.

Prognosis in Takayasus arteritis is limited owing to renovascular hypertension. The authors report a patient with Takayasus arteritis who had been unilaterally nephrec tomized and presented with malignant hypertension due to renal artery stenosis. Hypertension was refractory to conventional antihypertensive treatment, and stenosis was not accessible by interventional angioplasty. Initiation of enalapril and losartan therapy was successful in improving blood pressure without deterioration of renal function due to ischemic failure. Antihypertensive treatment resulted in dramatically stimulated endogenous nitric oxide (NO) synthesis, while elevated plasma endothelin-1 levels were unchanged. Renovascular hypertension in Takayasus arteritis is associated with an imbalance of vasoconstrictor peptide endothelin-1 and vasodilator peptide NO. Successful treatment of hypertension by enalapril or losartan results in improved endoge nous NO synthesis, which putatively counterbalances excessive vasoconstrictor actions and may retard the progression of renal failure.

Spontaneous tendon rupture after ofloxacin treatment in renal transplant recipients on high-dose corticosteroids

Abstract Acute tendon rupture without any history of trauma, a rare complication occurring mainly in metabolic diseases, is increasingly reported after treatment with fluoroquinolones. We report here on the occurrence of Achilles tendinitis and Achilles tendon rupture after ofloxacin treatment for uncomplicated urinary tract infection in four patients receiving high-dose corticosteroid treatment after renal organ transplantation and compare them with a control group without this complication. Patients experiencing tendon rupture had been on regular dialysis treatment for 54.5 ± 39.5 months before receiving cadaveric kidney transplants. All had secondary hyperparathyroidism, with parathyroid hormone levels ranging from 247 to 707 ng/L, had low or normal serum phosphate levels (1.21 to 2.69 mg/dL), and had moderately elevated alkaline phosphatase levels (75 to 285 U/L). Tendon ruptures occurred at a median of 49 days after organ transplantation and 15.3 ± 9.6 days after initiation of quinolone treatment. Mean time of treatment was 5 days. All patients received triple-regimen immunosuppression consisting of methylprednisolone, cyclosporin A, and mycophenolate mofetil. Transplant function was stable, and none had experienced transplant rejection. Mean ingestion of prednisolone ranged from 6 to 16 mg/d, and mycophenolate mofetil from 1 to 2 g/d; cyclosporin A serum levels were 208 ± 76 μg/L. After kidney transplantation, spontaneous large tendon rupture after quinolone treatment is deleterious during the early posttransplantion phase. Secondary risk factors are high-dose corticosteroid treatment and history of secondary hyperparathyroidism. Quinolones should be restricted for treatment of severe urinary tract infection only, which failed to respond to other antibiotic regimens in the early posttransplantation period after kidney transplantation under high-dose corticosteroid treatment.