Regulation of hyaluronan secretion into rabbit synovial joints in vivo by protein kinase C

Hyaluronan (HA) is important for joint cavitation, lubrication, volume regulation and synovial fluid drainage but little is known about the regulation of joint HA synthesis/secretion in vivo. We investigated whether HA secretion into joints in vivo can be regulated by protein kinase C (PKC). Secretion into the knee joint cavity of anaesthetised rabbits was measured over 6 h by washout and chromatography. Joints received intra-articular injections of Ringer vehicle (control) or an activator of classical PKC isoforms, phorbol-12-myristate-13-acetate (PMA), at 20-2000 ng ml(-1). The effects of PKC inhibition by bisindolylmaleimide (BIM) and protein synthesis inhibition by cycloheximide (CX) on basal and stimulated HA secretion were also studied. The endogenous HA mass, 181+/-8 microg (n=26, mean +/- S.E.M.), and basal secretion rate, 4.4+/-0.4 microg h(-1), indicated a turnover time of 41 h. Secretion rate showed a dose-dependent response to PMA (n=30), rising 5-fold to 21.7+/-5.0 microg h(-1) (n=5) at 2000 ng ml(-1) PMA (P<0.0001, one-way ANOVA). PMA-induced stimulation was partially suppressed by CX (HA secretion: 5.8+/-1.7 microg h(-1), n=8, P<0.01) and totally blocked by BIM (HA secretion: 3.2+/-0.6 microg h(-1), n=9, P<0.001). Basal HA secretion was unaffected by CX over 6 h (4.2+/-0.7 microg h(-1), n=8) but was reduced by 29 % by BIM (3.1+/-0.6 microg h(-1), n=10, P=0.03). It is concluded that: (1) PKC can stimulate HA secretion into joints in vivo through mechanisms involving protein synthesis de novo as well as phosphorylation; (2) basal HA secretion is only partially PKC dependent; and (3) hyaluronan synthase turnover time is >6 h in vivo, which is slower than in vitro (<2-3 h).