The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture.

In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.     

B6 vitamer content of rat tissues measured by isotope tracer and chromatographic methods

Feeding [14C]pyridoxine to growing rats for 146 days produced uniform labelling of the total vitamin B6 pool, thus permitting the radioactivity to be used as an absolute standard for evaluating the accuracy of vitamin B6 analyses. The results demonstrated that trichloroacetic acid extraction followed by cation exchange chromatography accurately measures the B6 vitamers. It is essential to homogenize tissues in a protein-denaturing agent in order to avoid shifts in the vitamer content, particularly in liver. In rats approximately 80% of the radioactivity was found in carcass and 8-9% each in liver and skin. Pyridoxamine phosphate equalled or exceeded the concentration of pyridoxal phosphate in heart, brain and kidney. The total vitamin B6 pool in weanling and adult rats averaged about 16 nmol/g body wt. Meta-phosphoric acid extraction followed by reverse phase chromatography gave good agreement with the cation exchange method in rat liver but with cat plasma yielded pyridoxal phosphate values below those of the cation exchange or enzymatic methods. The discrepancies encountered between different homogenization techniques and chromatographic methods emphasize the need for constant vigilance and continual verification of results by independent methods.