Activation of Src kinase in platelet-derived growth factor-B-dependent tubular regeneration after acute ischemic renal injury.

We previously reported that the platelet-derived growth factor B-chain (PDGF-B)/PDGF receptor (PDGFR) axis is involved in tubular regeneration after ischemia/reperfusion injury of the kidney. In the present study, we examined the activation of Src tyrosine kinase, a crucially important signaling molecule for PDGFR, and assessed the role of Src in PDGF-B-dependent renal tubular regeneration afterischemia/reperfusion injury. Immunoblot using clone 28, a monoclonal antibody specific for the active form of Src kinases, demonstrated increased active Src expression in the injured rat kidney 6 hours after reperfusion with peak activation at 12 hours. In vitro kinase assay confirmed increased Src activity that concurred with PDGFR-beta activation as detected by the increment of receptor-phosphorylated tyrosine. Immunohistochemistry using clone 28 demonstrated that active Src was preferentially expressed in the S3 segment of the proximal tubule in reperfused kidney, where it is not normally expressed. This enhanced expression of active Src was co-localized with the increased PDGFR expression in the tubular cells that were undergoing cell proliferation cycle. Trapidil administration suppressed Src and PDGFR-beta activation in the reperfused kidney and resulted in deteriorated renal function. These findings suggest that active Src participates in PDGF-B-dependent regeneration of tubular cells from acute ischemic injury.

Effects of trapidil on ATPase, lipid peroxidation, and correlation with ultrastructure in experimental spinal cord injury.

The present study was performed to investigate the effect of trapidil on ischemic damage of cells after spinal cord injury. The injury was produced by extradural clip compression of the exposed spinal cord in rats according to Rivlin and Tator. The ten rats in group 1 were used to determine normal findings without any surgery or medication. On the 15 rats in group 2, only six-level laminectomy was performed to determine the influence of the total laminectomy on the biochemical factors measured and the, light and ultrastructural findings. The 15 rats each in groups 3 and 4 were used as trauma and trapidil (40 mg/kg) treatment groups, respectively. The injury actually produced a significant decrease in Na+-K+/Mg+2 ATPase activity of the injured segments as early as 10 min after trauma. Trapidil attenuated Na+-K+/Mg+2 ATPase inactivation in the traumatized rats for 120 min after treatment (P<0.05) and significantly reduced the malone dialdehyde content below that in the traumatized group at all determined times (P<0.05). Light and electron microscopic findings supported the biochemical results.

Paracrine effect of human vascular endothelial cells on human vascular smooth muscle cell proliferation: transmembrane co-culture method.

To investigate the role endothelial cells have on underlying smooth muscle cell proliferation, human aortic vascular smooth muscle cells were co-cultured with human aortic endothelial cells at different cell densities, using a transmembrane co-culture method. Subconfluent endothelial cells subseeded at low (0.5 x 10(4) cells/well) and medium densities (2.0 x 10(4) cells/well) stimulated smooth muscle cell proliferation by 43 +/- 14% (P < 0.01) and 39 +/- 8% (P < 0.02), respectively. However, this stimulatory effect on smooth muscle cell proliferation was not evident in confluent endothelial cells subseeded at high cell density (8.0 x 10(4) cells/well). Treatment of smooth muscle cells with trapidil, at 10(-6) M, for anti-platelet derived growth factor (PDGF) effect or with endothelin-1 receptor blocker FR 139317, at 10(-6) M failed to inhibit this stimulatory effect. These results imply that subconfluent human endothelial cells are able to exert a stimulatory effect on human smooth muscle cell proliferation, and that this endothelial paracrine growth effect may not be mediated by endothelin or PDGF.

[Biotransformation of the trapidil (rocornal) derivative AR 12463 in the rat]. / Biotransformation des Trapidil (Rocornal)-Derivates AR 12463 bei der Ratte.

After p.o. administration of 5-piperidino-7-[N-pentyl-N-(beta- hydroxyethyl)]amino-s-triazolo[1,5-a]pyrimidine (1; AR 12463) more than 15 metabolites were isolated from urine and feces of male Wistar rats. Only small amounts of unchanged 1 were observed. The structure of 12 metabolites was elucidated or proposed on the basis of UV-, 13C NMR- and mass spectra. Main metabolites are 5-piperidin-4'-olyl-7-[N-pentyl-N-(beta- hydroxyethyl)]amino-s-triazolo[1,5-a]pyrimidine and 5-piperidin-4'-olyl-7-[N-pent-4-olyl-N-(beta-hydroxyet hyl)]amino-s- triazolo[1,5-a]pyrimidine. The other metabolites are mainly hydroxy- or ketopentyl derivatives and piperidinoles or piperidinones, respectively. Conjugates of most of the metabolites were identified, but the ratio phase-I/II metabolites was about 3:1. In contrast to trapidil, 5-methyl-7-diethylamino-s- triazolo[1,5-a]pyrimidine, no hydroxy derivatives of the bicyclic system were observed. The major part of unchanged 1 and metabolites is excreted via kidneys.

Trapidil and other 5-triazolo-(1, 5-alpha)-pyrimidine derivatives as calcium channel blockers in 108CC5 cells.

The action of trapidil (RocornalR) and its derivatives AR 12-456 and AR 12-160 on the inward calcium current (ICa) was studied in mouse neuroblastoma x rat glioma hybrid cells of the line 108CC5 under voltage clamp conditions by means of a suction pipette method. A dissociation constant of the calcium channel-trapidil complex of 277 microM was estimated for the initial inhibition of ICa by trapidil. Half maximal block of ICa was produced by 80 +/- 20 microM AR 12-456 and 500 +/- 150 microM AR 12-160.

[The bioavailability of trapidil from suppositories (author's transl)]. / Bioverfügbarkeit von Trapidil aus Suppositorien.

In a study with five healthy persons designed to compare the bioavailability of Trapidil from Rosupol U suppositories with that from Witepsol H 15 suppositories, the availability of Trapidil from the Rosupol U suppositories was inferior in four of the five test subjects. The difference in the amounts of released drug observed in vivo was smaller than that found in vitro.

[HPLC of trapidil (Rocornal) and metabolites (author's transl)]. / HPLC von Trapidil (Rocornal) und Metaboliten.

Trapidil (1; Rocornal) and 10 metabolites can be separated by a gradient technique on reversed phases (RP-8 instant columns) using the eluents methanol/water and acetonitrile/phosphate buffer (pH = 5.4); ultraviolet detection. Furthermore, HPLC techniques for purity testing and for determining 1 in serum are described.