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sn-1,2-Diacylglycerol (DAG) mass and translocation of protein kinase C a and ,B to a membrane fraction increased -7 min after insemination of Xenopus laevis eggs. The DAG mass increase of 48 pmol (from 62 to 110 pmol/cell) was greater than that for inositol 1,4,5-trisphosphate (1P3; an increase of -170 fmol or -280-fold smaller than the DAG increase), and DAG peaks -5 min after IP3. Choline mass (a measure of phosphatidylcholine-specific phospholipase D) also peaked before DAG and the choline increase (134 pmol/cell) was greater than that of DAG. There was no detectable change in phosphocholine mass (a measure of phosphatidylcholine-specific phospholipase C). During first cleavage, DAG decreased, PKC translocation was low, and choline increased and peaked (whereas published work shows an increase in IP3 mass). Artificial elevation of intracellular calcium ([Ca2+]

Embryonic quail neural crest cells migrate towards the negative pole of an imposed dc electric field as small as 7 mV/mm (0.4 mV per average cell length). The involvement of protein kinases in the mechanism utilized by these cells to detect and respond to such imposed fields was tested through the use of several kinase inhibitors. Evidence for the involvement of protein kinase C (PKC) included: (1) inhibition of the directed motility by 1 microM sphingosine that was reversed by the addition of the phorbol ester, PMA; (2) stimulation of a faster response to the imposed field by PMA; and (3) inhibition of the directed translocation by 5 microM H-7. However, another PKC inhibitor, staurosporine, did not inhibit the directed translocation (1 nM-1 microM). We also found evidence for the involvement of either cAMP- or cGMP-dependent protein kinase. The galvanotactic response was partially inhibited by the addition of 10 microM H-9 and the response was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. However, the adenylate cyclase stimulant, forskolin, had no significant influence on the directed motility, although it reduced the average cell velocity. While these experiments suggest that cAMP- or cGMP-dependent protein kinase or PKC may be involved in the galvanotaxis response, two other protein kinases appeared not to be required. The myosin light chain kinase inhibitor, ML-7, had no effect on the directed motility in an imposed field, so myosin light chain kinase may not be required for galvanotaxis. Similarly, 5 microM W-7 had no significant effect on the directed translocation, suggesting that calmodulin-dependent protein kinase is not involved. Interestingly, the continuous activity of a protein kinase is apparently not required for the directed translocation response. The addition of the PKC and cAMP-dependent protein kinase inhibitor, H-7, after the cells had been exposed to the field for 1 hour, had no effect on the subsequent directed translocation. Thus, for these inhibitors to block the directed translocation, they must be present at the same time as the initial field application. This implies that an integral step in the cellular response mechanism for galvanotaxis involves the stimulation of a protein kinase whose effect is long lasting.

We have been studying the response of neural crest cells from 56-h-old quail embryos to small, imposed d.c. electrical fields. These cells exhibit directed translocation or galvanotaxis towards the negative pole of fields as low as 7 mV/mm. With an average cell length of 60 µm, these cells respond to fields as low as 0.4 mV along their length. A significant change in the average cosine of the cellular translocation distribution can be detected as early as 7 min after field application for all field strengths greater than 10 mV/mm. Extracellular Ca is not required for the motility of these cells as long as extracellular Mg is increased to 10 mM. However, the galvanotaxis response does appear to require Ca influx since it is completely blocked by the addition of either 10 mM Mg or 100 µM Gd. Moreover, the galvanotaxis response is partially reversed by the complete removal of extracellular Ca.