Mechanosensitive hyaluronan secretion: stimulus-response curves and role of transcription-translation-translocation in rabbit joints.
ABSTRACT Joint movement was recently shown to stimulate the secretion of the lubricant hyaluronan (HA); also, exercise therapy and intra-articular hyaluronan injections are used to treat moderate osteoarthritis. The present study quantifies the stimulus-response curves for HA secretion in vivo and reports a role of transcription-translation-translocation in the secretory response. After washing out endogenous HA from anaesthetized, cannulated rabbit knees, the joints were cycled passively at various frequencies and durations, with or without intra-articular inhibitors of protein synthesis and Golgi processing. Newly secreted HA was harvested for analysis after 5 h. Joints displayed graded, non-linear stimulus-response curves to both duration and frequency of movement; 1 min duration per 15 min or a frequency of 0.17 Hz raised HA secretion by 42-54%, while rapid (1.5 Hz) or prolonged cycling (9 min per 15 min) raised it by 110-130%. Movement-stimulated secretion and phorbol ester-stimulated secretion were partly inhibited by the translation inhibitor cycloheximide, by the transcription-translation inhibitors actinomycin D and puromycin and by the Golgi translocation inhibitor brefeldin A. There is thus a graded coupling between HA secretion and cyclic joint movement that depends partly on new protein synthesis. This is likely to be important for joint homeostasis, providing protection during repetitive cycling and potentially contributing to exercise therapy for osteoarthritis.
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ABSTRACT: In this study, we wished to determine whether angiopoietin-1 (Ang1) modified the permeability coefficients of non-inflamed, intact continuous, and fenestrated microvessels in vivo and to elucidate the underlying cellular mechanisms. Permeability coefficients were measured using the Landis-Michel technique (in frog and rat mesenteric microvessels) and an oncopressive permeability technique (in glomeruli). Ang1 decreased water permeability (L(P): hydraulic conductivity) in continuous and fenestrated microvessels and increased the retention of albumin (sigma: reflection coefficient) in continuous microvessels. Endothelial glycocalyx is common to these anatomically distinct microvascular beds, and contributes to the magnitude of both L(P) and sigma. Ang1 treatment increased the depth of endothelial glycocalyx in intact microvessels and increased the content of glycosaminoglycan of cultured microvascular endothelial cell supernatant. Ang1 also prevented the pronase-induced increase in L(P) (attributable to selective removal of endothelial glycocalyx by pronase) by restoration of glycocalyx at the endothelial cell surface. The reduction in permeability was inhibited by a cell transport inhibitor, Brefeldin. Ang1 modifies basal microvessel permeability coefficients, in keeping with previous reports demonstrating reduced solute flux in inflamed vessels. Anatomical, biochemical, and physiological evidence indicates that modification of endothelial glycocalyx is a novel mechanism of action of Ang1 that contributes to these effects.Cardiovascular research 05/2009; 83(1):24-33. · 5.80 Impact Factor
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ABSTRACT: Joint lubrication, synovial fluid conservation and many pathophysiological processes depend on hyaluronan (HA). Intra-articular HA injection and exercise, which stimulates articular HA production, ameliorate osteoarthritis. We therefore investigated the pathways regulating movement-stimulated articular HA secretion rate ( ) in vivo. Endogenous HA was removed from the knee joint cavity of anaesthetised rabbits by washout. Joints were then cycled passively or remained static for 5 h, with/without intra-articular agonist/inhibitor, after which newly secreted HA was harvested for analysis. Movement almost doubled . Similar or larger increases were elicited in static joints by the intra-articular Ca(2+) ionophore ionomycin, prostaglandin E(2), cAMP-raising agents, serine/threonine phosphatase inhibitor and activation of protein kinase C (PKC). PKC-stimulated secretion was inhibited by the PKC inhibitor bisindolylmaleimide I and inhibitors of the downstream kinases MEK-ERK (U0126, PD98059). These agents inhibited movement-stimulated secretion of HA (MSHA) only when the parallel p38 kinase path was simultaneously inhibited by SB203580 (ineffective alone). The phospholipase C inhibitor U73122 almost fully blocked MSHA (P = 0.001, n = 10), without affecting static . The ENaC channel blocker amiloride inhibited MSHA, whereas other inhibitors of stretch-activated channels (Gd(3+), ruthenium red, SKF96365) did not. It is proposed that MSHA may be mediated by PLC activation, leading to activation of parallel PKC-MEK-ERK and p38 kinase pathways.The Journal of Physiology 08/2009; 587(Pt 17):4361-76. · 4.38 Impact Factor
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ABSTRACT: Hyaluronan, a joint lubricant and regulator of synovial fluid content, is secreted by fibroblast-like synoviocytes lining the joint cavity, and secretion is greatly stimulated by Ca(2+)-dependent protein kinase C. This study aimed to define synoviocyte membrane currents and channels that may influence synoviocyte Ca(2+) dynamics. Resting membrane potential ranged from -30 mV to -66 mV (mean -45 ± 8.60 mV, n = 40). Input resistance ranged from 0.54 GΩ to 2.6 GΩ (mean 1.28 ± 0.57 GΩ; ν = 33). Cell capacitance averaged 97.97 ± 5.93 pF. Voltage clamp using C(s+) pipette solution yielded a transient inward current that disappeared in Ca(2+)-free solutions and was blocked by 1 μM nifedipine, indicating an L-type calcium current. The current was increased fourfold by the calcium channel activator FPL 64176 (300 nM). Using K(+) pipette solution, depolarizing steps positive to -40 mV evoked an outward current that showed kinetics and voltage dependence of activation and inactivation typical of the delayed rectifier potassium current. This was blocked by the nonspecific delayed rectifier blocker 4-aminopyridine. The synoviocytes expressed mRNA for four Kv1 subtypes (Kv1.1, Kv1.4, Kv1.5, and Kv1.6). Correolide (1 μM), margatoxin (100 nM), and α-dendrotoxin block these Kv1 subtypes, and all of these drugs significantly reduced synoviocyte outward current. The current was blocked most effectively by 50 nM κ-dendrotoxin, which is specific for channels containing a Kv1.1 subunit, indicating that Kv1.1 is critical, either as a homomultimeric channel or as a component of a heteromultimeric Kv1 channel. When 50 nM κ-dendrotoxin was added to current-clamped synoviocytes, the cells depolarized by >20 mV and this was accompanied by an increase in intracellular calcium concentration. Similarly, depolarization of the cells with high external potassium solution caused an increase in intracellular calcium, and this effect was greatly reduced by 1 μM nifedipine. In conclusion, fibroblast-like synoviocytes cultured from the inner synovium of the rabbit exhibit voltage-dependent inward and outward currents, including Ca(2+) currents. They thus express ion channels regulating membrane Ca(2+) permeability and electrochemical gradient. Since Ca(2+)-dependent kinases are major regulators of synovial hyaluronan secretion, the synoviocyte ion channels are likely to be important in the regulation of hyaluronan secretion.AJP Cell Physiology 11/2010; 299(5):C1180-94. · 3.71 Impact Factor