| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 87 === Summary
1. Extracellular matrix (ECM) proteins such as fibronectin and laminin have been implicated in a wide variety of cellular properties which include cell adhesion, migration, differentiation and proliferation. Recent data from our laboratory showed that in the presence of TPA (0.5 mM), application of soluble fibronectin and laminin markedly increased spontaneous ACh release. The aim of the present study is to further analyze the regulatory role of fibronectin and laminin on the synaptic transmission at developing motoneuron.
2. The cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (Vh= －60~－65 mV).
3. Application of 0.5 mM TPA alone or 6mg/ml soluble fibronectin alone did not significantly affect the spontaneous ACh release (SSCs). However, sequential application of 0.5 mM TPA and 6 mg/ml fibronectin (5~10min later) markedly increased the SSC frequency. Bath application of 0.5 mM TPA plus soluble laminin (6 mg/ml) also markedly increased spontaneous ACh release.
4. Pretreatment with 50 mg/ml GRGDS for 10 min inhibited the SSC increasing effect of TPA plus fibronectin, but not that of TPA plus laminin. These results suggest that the regulatory effect of fibronectin but not that of laminin is RGD-dependent.
5. Pretreatment with 1 mM cytochalasin D for 1 hour did not significantly inhibit the SSC increasing effect of TPA plus fibronectin or laminin, indicating that cytoskeleton rearrangement is not involved in the SSC increasing action.
6. Pretreatment with genistein (50 mM), an inhibitor of tyrosine kinase, significantly inhibited the SSC increasing effect of TPA plus fibronectin or laminin. These results indicate that tyrosine kinase plays a role in the SSC increasing action.
7. We further investigated the effect of delayed application of fibronectin. The SSC frequency was not significantly affected when the cultures were pretreated with 0.5 mM TPA for 1 hour and 6 mg/ml fibronectin was then bath applied.
8. Application of high concentration of TPA (5 mM) increased the SSC frequency in a few minutes. However, subsequent application of 6 mg/ml fibronectin had no significant effect on SSC frequency, suggesting that fibronectin may enhance PKC activation, and once PKC is fully activated, the action of fibronectin is masked.
9. When cultures were plated onto fibronectin-coated or laminin-coated glasses for 1 day, application of low concentration of TPA (0.05 mM) resulted in significant enhancement of the SSC frequency.
10. Application of high concentration of soluble fibronectin (30 mg/ml) had little effect on the SSC frequency. After chronic treatment with NT-3 (50 ng/ml) or CNTF (150 ng/ml) for 1 day, the SSC frequency neither increase in response to bath application of 30 mg/ml fibronectin alone.
11. Application of an adenylate cyclase activator forskolin (20 mM) alone did not significantly affect the spontaneous ACh release. However, application of 20 mM forskolin plus 6mg/ml fibronectin (5~10min later) but not laminin markedly increased the SSC frequency.
12. For cultures plated onto fibronectin-coated glasses for 1 day, application of forskolin (20 mM) resulted in significant enhancement of the SSC frequency. But for cultures plated onto laminin-coated glass，the SSC frequency did not increase in response to bath application f 20 mM forskolin.
13. In summary, these results suggest that fibronectin and laminin may initiate acute and sustained changes in intracellular signaling such as PKC or PKA activation and play an important role in regulating synaptic transmission at developing motoneuron. .
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