Application of Peptides to Cells by Pressure Ejection

We found the suggestion that the slow and prolonged nature of the excitation evoked by Substance P in some studies, but not others, is due to the slow release of the peptide from the pipette by microiontophoresis (Guyenet et al, 1979) rather disquieting and turned to alternative methods for the application of peptides to single neurons. In addition, our resolve 

Although the use of pressure for the ejection of peptides is not without its drawbacks, at least it allows one to ignore the suggestion that the passage of current, or hydrogen ions (Frederickson et aL, 1971), from the peptide-containing pipette is responsible for the observed changes in neuronal excitability. 

As shown in Fig. 35 the system we adopted is almost identical to that 

The release of nine different peptides from 40 of the same pipettes was also examined by radio immunoassays, after the electrode was withdrawn from the slice and the tip immersed in 0.2 ml of 4 mM sodium acetate. Assayable amounts of peptide could be detected after about 1 hr in the absence of pressure and shown to be between 0.2 and 2.0 fmol per second. The application on average of 400-1000 kPa of pressure led to an eightfold increase in release. This would represent a flow of fluid of between 1-12 pl/sec. However, during repeated trials, the release from the same pipette was extremely variable and quite unlike the consistent results of Sakai et al. (1979). However, since in part their method depended on the direct measurement of the ejection of droplets into oil, it is possible that their electrodes were not only selected more carefully, but had bigger tips. It is also not clear how the use of siliconized glass and ethanolic solutions improved the performance of their pipettes. 

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