Sphingosine 1-phosphate enhances spontaneous transmitter release at the frog neuromuscular junction

Abstract

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 micro M) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10(-5), 10(-4) or 10(-3) M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10(-5) but not 10(-8) M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10(-4) M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

Figure 1

Sample recordings of miniature endplate potentials (MEPPs) in normal Ringer solution before, during, and after superfusion of S1-P (10⁻³ M) entrapped in liposomes. Note the increase in MEPP frequency and the presence of ‘giant' MEPPs during S1-P superfusion.

Figure 2

Percent changes in MEPP frequency as a function of time. The effects of 10⁻⁵, 10⁻⁴ and 10⁻³ M S1-P delivered by liposomes (L S1-P) on MEPP frequency are superimposed for comparison. The peak effect occurs at 3 min for 10⁻⁴ and 10⁻³ M, and at 4 min for 10⁻⁵ M S1-P. MEPP frequency 100%=0.974 s⁻¹ (10⁻⁵ M S1-P), 1.12 s⁻¹ (10⁻⁴ M S1-P) and 0.983 s⁻¹ (10⁻³ M S1-P). Note that the MEPP frequency return to control level, e.g., at time=8 min for S1-P 10⁻⁵ M. Each point represents the mean from six different experiments. Asterisks denote statistically significant differences (P<0.05) from control.

Figure 3

Histogram analysis (cumulative frequency) of the changes in MEPP amplitude distribution before (control) and after S1-P delivered by liposomes (L S1-P). For each of the six single experiments, MEPP amplitudes at time=0 min (pre-exposure) and time=3 min (post-exposure) are expressed as a per cent of the median amplitude (100 samples each). Histograms reveal no change in the shape of the unimodal amplitude-frequency distribution of MEPPs (exp. 1 median=0.348 mV; exp. 2 median=0.317 mV; exp. 3 median=0.392 mV; exp. 4 median=0.329 mV; exp. 5 median=0.377 mV; exp. 6 median=0.366 mV) before and after administration of liposomes containing 10⁻³ M S1-P. Results indicate no significant changes in the median MEPP amplitude before and after S1-P treatment; however, there is an increase in the number of gMEPPs after S1-P treatment.

Figure 4

Responses of second liposomal delivery of S1-P following a low and higher concentration of liposomal delivery of S1-P on MEPP frequency. (A) Perfusion with liposomes containing S1-P 10⁻⁸ M (L S1-P 10⁻⁸ M) had no significant effect on MEPP frequency. A subsequent administration of S1-P 10⁻⁴ M-filled liposomes (L S1-P 10⁻⁴ M) increased the MEPP frequency to a degree similar to that of muscle preparations treated with S1-P 10⁻⁴ M-filled liposomes alone (P>0.05) (n=6). Control MEPP frequency (min 0)=1.17 s⁻¹. (B) Administration of S1-P 10⁻⁴ M-filled liposomes (L S1-P 10⁻⁴ M) to preparations pre-exposed to S1-P 10⁻⁵ entrapped liposomes (L S1-P 10⁻⁵ M) induced no significant changes in MEPP frequency (n=6). Control MEPP frequency (min 0)=0.88 s⁻¹. In all cases, asterisks denote statistically significant differences (P<0.05) from control.