ANÁLISIS Y DISCUSIÓN DE RESULTADOS

Experiments described in Section 4.2.2 suggest that the protective effect o f inositol treatment on closure o f the PNP in curly tail embryos is mediated via activation of PKC. In order to test this hypothesis further, embryos were treated with a water-soluble form o f the specific PKC inhibitor GF109203X, which was predicted to reduce the effects of inositol and TPA if these are mediated through PKC.

In cellular models o f phosphorylation, PKC activity is inhibited with IC50 values (half- maximal inhibitory concentration) of 0.2 to 2.5 pM . For example, in an assay of vimentin phosphorylation in Swiss 3T3 cells maximal inhibition of PKC was obtained at a concentration of 2.5-5 pM GF109203X with no inhibition o f cAMP-dependent PKA even at the highest concentration (Toullec et a l, 1991). In another study GF109203X was added at a concentration of 10 pM to COST cells in order to investigate the role of PKC in RA-induced transcription (Tahayato e ta l, 1993). Based on these data, the

embryos. The higher concentration was selected with the aim o f overcoming any decreased uptake of the inhibitor by embryos with intact yolk sacs, as compared with cultured cells which are likely to be more accessible to the cell-permeable inhibitor. The influence of PKC inhibitor on closure of the PNP in curly tail embryos was

examined following culture in whole serum, in serum containing 50 pg/ml inositol and in serum containing 500 nM TPA for one hour after 17 hours o f culture (Figure 4.1). In a preliminary set o f cultures, GF109203X was added at 2 or 10 pM for the entire 24 hour culture period, but the caudal region of a number o f the embryos exhibited a "crinkly" neural tube, which is indicative of a teratogenic effect (Table 2.2). Therefore, the period of treatment with inhibitor was reduced to one hour both in order to avoid the problem o f growth retardation or teratogenicity and, in the case o f TPA treatment, to reduce the possibility of the inhibitor being degraded before the addition of the PKC agonist. GF109203X was added to the culture medium to 10 pM final concentration 15 minutes prior to the addition of TPA after 17 hours of culture. Following one hour exposure to TPA, embryos were transferred to fresh serum and cultured for a further 6 hours, as before (Figure 4.1). Embryos cultured in whole serum or in serum containing 50 pg/ml inositol were also treated with PKC inhibitor at the equivalent time points.

Inhibition of protein kinase C reverses the protective effect of TPA

Embryos treated simultaneously with 10 pM GF109203X and 500 nM TPA exhibited a mean PNP length at the 30-31 somite stage which was significantly increased in

comparison with embryos cultured with TPA alone (Table 4.3, Figure 4.4). This increase suggests that the protective effect of TPA is reversed by inhibition o f PKC. There was no apparent effect of GFl 09203X on the closure o f the PNP in embryos cultured in rat serum alone since mean PNP length at the 30-31 somite stage was unaffected (Table 4.3, Figure 4.4). Addition of PKC inhibitor for one hour to cultures with 50 pg/ml inositol appeared to cause an increase in the mean PNP length at the30-31

somite stage although this difference is not statistically significant (Table 4.3, Figure 4.4). However, unlike embryos treated with 50 |ig/ml inositol (Table 4.2a), the PNP length of embryos treated with both 10 |iM GF109203X and 50 |ig/ml inositol is no longer significantly reduced compared to embryos cultured in serum alone.

Short-term inhibition of protein kinase C does not cause growth retardation

The addition of PKC inhibitor for 1 hour 15 minutes had no apparent effect on growth at this stage of development since there was no alteration in crown-rump length of embryos following culture to the 30-31 somite stage (Table 4.3). In addition, no teratogenic effects were observed; embryos exhibited vigorous yolk sac circulation and appeared normal as judged by external morphology.

Table 4,3 Development of curly tail embryos cultured fo r 24 hours in r a t serum , in the presence of 50 pg/ml inositol or with TPA treatm ent after 17 hours and with GF109203X (mean values ± SEM)

Treatment Number o f embryos Crown-rump length (mm) Somites P N P length (mm) R at serum f 32 4.00 ±0.05 i 30.44 ±0.09 0.61 ±0.04 Serum + lOpM GF 18 4.04 ±0.06 1 30.50 ±0.12 0.55 ±0.04

Statistically significant - Noa 1 Nob Nob

(P<0.05) P= 0.6233 1 P= 0.7231 P= 0.2711 t= -0.494 I T= 477.0 T= 404.0 50 pg/ml inos.f 30 4.06 ±0.06 I 30.37 ±0.09 0.45 ±0.03

50 pg/ml inos. + GF 19 3.91 ±0.05 j 30.26 ±0.10 0.56 ±0.05

Statistically significant - Noa I Nob Nob* (P<0.05) P= 0.0685 1 P= 0.1931 P= 0.1238

t= 1.86 1 T= 705.0 T= 550.5 500 nM TPA (17 hrs) t 41 4.02 ±0.04 1 30.29 ±0.07 0.42 ±0.03

500 nM TPA + GF 20 3.98 ±0.08 I 30.50 ±0.11 0.62 ±0.05

Statistically significant - Nob Nob Yesb

(P<0.05) P= 0.7645 1 P= 0.1931 P= 0.0022 T= 600.0 ! T= 705.0 T= 819.5. a= t-test, b= Mann-Whitney Rank Sum Test

GFl 09203X (GF) was used at 10 |iM in the culture medium.

t = The data for embryos cultured with are repeated from Table 4.2a for comparison. * = There is no significant difference between the mean PNP length for the inositol + GF treatment group compared with the inositol alone or rat serum groups.

Figure 4.4 Mean PNP length of embryos at the 30-31 somite stage following culture in serum supplemented with GF109203X and inositol or TPA. Embryos were cultured with 10 pM GF109203X in serum alone, serum supplemented with 50 pg/ml inositol for the entire 24 hours or serum with 500 nM TPA added for one hour after 17 hours (see Figure 4.1 for experimental strategy). Data for culture in the absence of G Fl09203X are reproduced from Figure 4.3 for comparison.

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