Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis

This is the final version. Available on open access from Springer via the DOI in this record.CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, full length CCN-2 is primarily eliminated by the liver via a fast RAP-sensitive, probably LRP1-dependent pathway.FibroGen, Inc

No effect of synesthetic congruency on temporal ventriloquism

A sound presented in temporal proximity to a light can alter the perceived temporal occurrence of that light (temporal ventriloquism). Recent studies have suggested that pitch–size synesthetic congruency (i.e., a natural association between the relative pitch of a sound and the relative size of a visual stimulus) might affect this phenomenon. To reexamine this, participants made temporal order judgements about small- and large-sized visual stimuli while high- or low-pitched tones were presented before the first and after the second light. We replicated a previous study showing that, at large sound–light intervals, sensitivity for visual temporal order was better for synesthetically congruent than for incongruent pairs. However, this congruency effect could not be attributed to temporal ventriloquism, since it disappeared at short sound–light intervals, if compared with a synchronous audiovisual baseline condition that excluded response biases. In addition, synesthetic congruency did not affect temporal ventriloquism even if participants were made explicitly aware of congruency before testing. Our results thus challenge the view that synesthetic congruency affects temporal ventriloquism

Sphingosine-1-phosphate reduces rat renal and mesenteric blood flow in vivo in a pertussis toxin-sensitive manner

1. Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their effects on the cardiovascular system in vivo in anaesthetized rats. 2. The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1–100 μg kg(−1)) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood flows (RBF, MBF) using a pressure transducer and electromagnetic flow probes, respectively. 3. Intravenous injection of SPP rapidly (within 30 s), transiently and dose-dependently reduced RBF (maximally −4.0±0.3 ml min(−1)) and MBF (maximally −1.4±0.2 ml min(−1)), without affecting mean arterial pressure or heart rate. Other sphingolipids had no significant effect. 4. Intrarenal arterial SPP administration caused greater blood flow reductions (maximally −6.4±0.3 ml min(−1)) than systemic administration. Upon intrarenal administration, sphingosylphos- phorylcholine also lowered RBF (maximally −2.8±0.6 ml min(−1)), while the other sphingolipids remained without effect. 5. Pretreatment with pertussis toxin (PTX, 10 μg kg(−1)) 3 days before the acute experiment abolished the SPP-induced reductions of RBF and MBF. 6. These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any effects. The PTX-sensitivity strongly suggests that the effects of SPP on renal and mesenteric blood flow are mediated by receptors coupled to G(i)-type G-proteins

Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro

1. Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G-protein-coupled receptors, some of which were cloned and designated as Edg-receptors. 2. Sphingolipid-induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid-induced elevations in intracellular Ca(2+) concentration were measured in cultured rat aortic smooth muscle cells. 3. SPPC and SPP (0.1–100 μmol l(−1)) caused concentration-dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC(50) 5.63±0.17 and E(max) 49±3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC(50) D-erythro-SPPC 5.69±0.08, L-threo-SPPC 5.31±0.06) and inhibited by pretreatment with pertussis toxin (E(max) from 44±5 to 19±4%), by chelation of extracellular Ca(2+) with EGTA and by nitrendipine (E(max) from 40±6 to 6±1 and 29±6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (E(max) from 87±3 to 70±4%). 4. SPP and SPPC caused transient increases in intracellular Ca(2+) concentrations in rat aortic smooth muscle cells in a pertussis toxin-sensitive manner. 5. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca(2+) concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin-sensitive G-proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone