Hypertonic enhancement of transmitter release from frog motor nerve terminals: Ca2+ independence and role of integrins

Abstract

Hyperosmotic solutions cause markedly enhanced spontaneous quantal release of neurotransmitter from many nerve terminals. The mechanism of this enhancement is unknown. We have investigated this phenomenon at the frog neuromuscular junction with the aim of determining the degree to which it resembles the modulation of release by stretch, which has been shown to be mediated by mechanical tension on integrins. The hypertonicity enhancement, like the stretch effect, does not require Ca2+ influx or release from internal stores, although internal release may contribute to the effect. The hypertonicity effect is sharply reduced (but not eliminated) by peptides containing the RGD sequence, which compete with native ligands for integrin bonds. There is co-variance in the magnitude of the stretch and osmotic effects; that is, individual terminals exhibiting a large stretch effect also show strong enhancement by hypertonicity, and vice versa. The stretch and osmotic enhancements also can partially occlude each other. There remain some clear-cut differences between osmotic and stretch forms of modulation: the larger range of enhancement by hypertonic solutions, the relative lack of effect of osmolarity on evoked release, and the reported higher temperature sensitivity of osmotic enhancement. Nevertheless, our data strongly implicate integrins in a significant fraction of the osmotic enhancement, possibly acting via the same mechanism as stretch modulation.

Figure 1. Lack of Ca²⁺ dependence of hypertonicity response

Addition of 25 mm sucrose to NFR solution caused a 2.45 ± 0.07-fold (n = 76) increase in the mEPP frequency (bars 1 and 2). Bathing preparations in a zero-Ca²⁺, EGTA-buffered Ringer solution, which eliminates Ca²⁺ influx, did not reduce the enhancement of mEPP frequency by addition of 25 mm sucrose. MEPP frequency increased by 3.52 ± 0.20 times (n = 12) (bars 3 and 4). Intracellular buffering with 25 μm BAPTA AM, coupled with the removal of extracellular Ca²⁺, also failed to eliminate osmotic enhancement of mEPP frequency. Addition of 25 mm sucrose caused a 3.86 ± 0.12-fold increase (n = 32) (bars 5 and 6). Hypertonicity caused a significant effect at the P < 0.01 level in all cases. Bars 7-10 show that loading of preparations with 25 μm BAPTA AM was effective in buffering intraterminal Ca²⁺. Preparations with and without BAPTA loading were slightly depolarized by elevation of K⁺ in the Ringer solution to 6 mm, increasing Ca²⁺ influx. Junctions without BAPTA showed an elevation in mEPP frequency of 2.27 ± 0.22-fold (n = 23). The same treatment of BAPTA-loaded preparations produced much smaller changes. The resting mEPP frequency after BAPTA loading was slightly reduced relative to the NFR solution condition. Addition of 6 mm K⁺ caused a much reduced increase (bars 9 and 10).

Figure 2. Effects of thapsigargin, an irreversible inhibitor of endoplasmic reticulum Ca²⁺-ATPase, on osmotic enhancement of release by Ringer solution containing 50 mm sucrose

In NFR solution the baseline frequency (0.74 ± 0.05 Hz, n = 94) increased to 4.25 ± 0.25 Hz, (n = 37), a 5.74 ± 0.06-fold change (bars 1 and 2). Other preparations were treated for 1 h with 20 μm thapsigargin, during which the mEPP frequency increased sharply but then returned to slightly below the original level. Addition of 50 mm sucrose then increased the frequency from 0.47 ± 0.03 Hz (n = 104) to 3.37 ± 0.26 Hz (n = 32), a 7.17 ± 0.08-fold increase (bars 3 and 4). Similarly, in 0 Ca²⁺, EGTA-buffered Ringer solution, thapsigargin-treated terminals still showed a prominent sucrose effect (bars 5 and 6). The effect of hypertonicity was significant at P < 0.01 in all cases.

Figure 3. Effect of 0.2 mm RGD on the enhancement of mEPP frequency by addition of 25, 50, 75 and 100 mm sucrose

Control and experimental preparations were pretreated for 90 min in low-divalent (LD) Ringer solution. At all sucrose concentrations there was a significant (P < 0.01) inhibition of the hyperosmotic enhancement with the peptide (•) compared to the LD Ringer solution controls (•). Also plotted are control values showing the effect of 75 mm sucrose on mEPP frequency in the presence of the RGE peptide (▵, n = 30) and paired junctions in RGD (▴, n = 28).

Figure 4. Effects of RGD on Mn²⁺-treated junctions

In muscles treated with zero-Ca²⁺ Ringer and 0.5 mm Mn²⁺, the resting mEPP frequency was close to normal (single traces and summary bar graph a), and addition of 25 mm sucrose caused an approximately 4-fold increase in frequency (b). Preparations treated similarly but in the presence of 0.2 mm RGD showed a slightly depressed resting mEPP frequency (c) and a severely reduced enhancement of release by 25 mm sucrose (d).

Figure 5. Covariance of effects of 15-20% stretch and 25 mm hypertonicity on mEPP frequency

Data are shown for 59 identified junctions in 5 experiments with preparations held at 12-14° C to help keep fibres healthy through both manipulations. The correlation coefficient was 0.71.

Figure 6. Partial mutual occlusion of effects of stretch and hypertonicity on mEPP frequency

a, data from 23 identified fibres in two muscles studied first in NFR solution, then stretched by approximately 15%, and finally exposed to 25 mm sucrose in NFR solution. b, data from 25 identified fibres in the two muscles paired with those above, tested first in NFR solution, then in 25 mm hypertonic solution, and finally with stretch. Occlusion was particularly evident when stretch preceded hypertonicity.