elevate intracellular calcium and drugs that activate or suppress the enzyme PKC was examined.
Calcium ionophore A21387: Addition of the calcium ionophore A21387 enhances the effect o f cAMP elevation on G A L -C expression. If 5xlO“ ^M calcium ionophore is present when cAMP analogues are used to induce the myelin phenotype, the number of cells expressing G A L -C after 72h is 45 ± 5.3% (n = 4), compared to 19 ± 4.3% (n = 4) when cells are treated with cAMP analogues alone (p <0.05, Students t-test, indicating a significant change). The level of P^ expression was not increased in these cultures, 77 ± 2.5% (n = 4) of the cells expressed Pq when A21387 was present compared to 77 ± 8.3% (n = 4) when the cells were treated with cAMP analogues alone. In these experiments, however, the Pq induction was already high,
nevertheless it was clear when looking at the cells that they were more strongly labelled with anti Pq than those without calcium ionophore. Calcium ionophore alone
at 5 -7 .5 X 10~^M did not induce Pq protein (0 ± 0.3%, n = 5 ) or G A L -C (0 ± 0, n = 3).
It will be interesting to quantify this increased Pq expression in individual cells, this could be done by photometric analysis of individual cells. Alternatively, using larger numbers of cells, cell extracts of treated cells could be analysed on a W estern blot, an increase in total Pq levels would be indicated by a heavier band of Pq immunoreactivity produced by cells treated with A21387.
Pilot studies monitoring levels of Pq mRNA by in situ hybridization indicate that the level of Pq mRNA is enormously increased when cAMP is raised in the presence of with A21387 compared to the effect of cAMP elevation alone, see Figure 3.8.
Agents that enhance or inhibit the action of PK C: In some systems, agents that activate PKC will enhance the amount of cAMP released in response to other drugs, whilst having no cAMP elevating effects themselves (Shenolikar et al. 1986). If such a cooperative mechanism were to exist in Schwann cells, then it is possible that agents that activate PKC may enhance the effects of cAMP elevating drugs. Phorbol esters are tumour promoters which mimic DAG and activate PKC (see Harper 1988 for review).
J. cAnP on Schwann c e l l s
The phorbol ester phorbol dibutyrate (PDB) caused no elevation in Pq levels when the drug was applied constantly at 10“ ^M (Pq = 0 ± 0%, n = 3) or pulsed on to the cells for Ih each day (Pq = 0 ± 0%, n = 3). The cells showed an immediate change in morphology with cell processes becoming distorted and wavy. When PDB was added at 10“ ^M, pulsed on to the cells for Ih each day as above, in combination w ith the calcium ionophore A21387, again no Pq was induced ( Pq = 0 ± 0%, n = 3).
When cells were treated with the cAMP analogues 8b-cA M P with db-cA M P in the presence o f PDB 10“ ^M for 72h, elevation of immunodetectable Pq protein was completely abolished (reduced from 40 ± 9.7 to 0 ± 0.3%, n = 7) (p <0.05, Students t-test, indicating a significant change). The inactive phorbol ester Aa phorbol had no effect on cAMP analogue-induced Pq expression. Pq expression was 67 ± 16.1% when cAMP analogues were added alone, and 61 ± 12.1% in the presence o f 4 a phorbol 10” ^M (n = 3) (p >0.5, Students t-test, indicating no significant change).
When PDB was applied for only Ih each day, then the induction of Pq by cAMP analogues was not blocked, occasionally more labelled cells were seen, but this was not reproducible. Only 3 times out of 10 was the number of cells expressing Pq doubled when compared to cells induced without PDB treatment.
Phorbol esters initially activate PKC, but the enzyme can be inactivated after longer exposure to these drugs (Shenolikar et al. 1986, or Harper 1988 for review). It was hoped that long term activation of PKC might have been achieved by carefully limiting the time for which the cells are exposed to PDB. However, it was not possible to use this screening method to detect an enhancement of Pq induction in response to cAMP analogues in the presence of activated PKC. If such an effect exists, then it might be demonstrated by running extracts of the treated cells on W estern blots, to show an increased level of Pq in each cell, rather than looking for increased numbers of cells expressing Pq, as discussed above for calcium ionophore.
Although it is clear that long term exposure to PDB prevents induction of the myelin phenotype, the reason for this is not clear. W hether this is the result of reduced or increased PKC activity could only be determined by measuring the activity of this enzyme. When another PK C -inhibiting drug was used in an attempt to block
3. cAMP on Schwann cells
PKC activity, no reduction in induction was seen. The lysosphingolipid sphingosine is competitive with DAG, and inhibits PKC (for review see Harper 1988). W hen 10” ^M sphingosine was present as cAMP analogues were added to cultured Schwann cells, no inhibition in Pq protein induction was seen. 69 ± 11.0% of the cells became Pq'*' when cAMP analogues added alone, compared to 59 ± 11.0% Pq'*’ when sphingosine was present (n = 4) (p >0.5, Students t-test, indicating no significant change).
DMSO induces differentiation in several transformed cell lines, but suppresses differentiation of glial cells from embryonal tetracarcinoma cells (see Sutrina et al. 1987 for discussion). DMSO inhibits the synthesis of glactoceramide and sulphatide in transformed rat Schwann cells (Sutrina et al. 1987). As DMSO was used to dissolve the phorbol esters in my experiments, it was important to determine that the levels used did not affect differentiation. The addition of 0.5% DMSO induced half maximal suppression of sulphatide synthesis (Sutrina et al. 1987), when DMSO was used to dissolve PDB at 10“ ^M, the final concentration of DMSO when PDB was used at 10“ ^M was 0.0001% and thus well below the levels expected to suppress glial differentiation.
Cvcloheximide. The protein synthesis inhibitor cycloheximide blocks the elevation of Pq mRNA seen when cAMP mimicking drugs are added to cultured Schwann cells. Pilot studies (repeated twice) indicate a complete block of Pq mRNA induction when 25-100/^g.ml” ^ cycloheximide was added for 24h with 8b-cA M P and db-cA M P each at 5xlO“ ^M, see Figure 3.9d. Cycloheximide reduces the level of Pq mRNA in Schwann cells to below that seen in control cells that have not been exposed to cAMP, even in the presence of cAMP elevating agents (compare Figures 3.9b and 3.9d).
The role of ^ tissue culture substrate. Lam inin-coated substrates were not obligatory for induction of the myelin phenotype, since cells cultured on PLL coated coverslips or tissue culture plastic could be induced to make Pq in response to elevation of cAMP. In a representative experiment, 63% of cells plated on laminin and 42% of cells plated on PLL expressed Pq after a 3d exposure to cAMP analogues. Extracellular matrix from bovine comeal endothelial cells was also effective although