Fig. 4. Effect of ion manipulation. (A) Elevated [Ca²⁺]. Deflection of maximally stiffened samples immersed for 2 h in normal ASW (CON), ASW containing 100 mmol l^-1 Ca²⁺ or 0.38 mol l^-1 CaCl₂ (solid bars), and of the same samples after a subsequent wash for 2 h in ASW (cross-hatched bars). (B) Effect of the same agents on partially destiffened samples. After excision, samples were left for 3-4 h in ASW then tested (solid bars). They were then left for 2 h in the stated media and retested (cross-hatched bars). (C) Ca²⁺ depletion. Deflection of maximally stiffened samples (solid bars) immersed for 2 h in normal ASW, calcium-free ASW alone (CaFASW) or CaFASW containing 5 mmol l^-1 EGTA, and of the same samples after a further wash for 4 h in ASW (cross-hatched bars). (D) Effect on maximally stiffened samples of 2 h exposure to low concentrations of EGTA (dissolved in CaFASW). (E) Effect on maximally stiffened samples of 2 h exposure to EGTA-AM (dissolved in CaFASW containing 1% DMSO). (F) Effect of EGTA on partially destiffened samples. After excision, samples were left for 3.5 h in CaFASW then tested (solid bars). They were then left for 2 h in CaFASW containing 1, 2 or 4 mmol l^-1 EGTA and retested (cross-hatched bars). (G) Inorganic Ca²⁺-channel blockers. Deflection of maximally stiffened samples (solid bars) immersed for 1-2 h in normal ASW (CON), ASW containing 20 mmol l^-1 Co²⁺ or ASW containing 20 mmol l^-1 Mn²⁺, and of the same samples after a further wash for 2 h in ASW (cross-hatched bars). (H) Effect of inorganic Ca²⁺-channel blockers on partially destiffened samples. After excision, samples were left for 3-4 h in ASW then tested (solid bars). They were then left for 2 h in the stated media and retested (cross-hatched bars). (I) Effect of verapamil (VER; 100 µmol l^-1) and nimodipine (NIM; 100 µmol l^-1) on partially destiffened samples. Control: 1% methanol in ASW. In D-F and I deflections were measure after 35 s. In A,C-F,I statistical comparison is between mean deflections of ASW control and treated samples; in B,G, statistical comparison is between mean deflections of each group of samples before (solid bars) and after (cross-hatched bars) treatment. Vertical bars represent standard deviations and asterisks indicate statistically significant differences between means: ^*P<0.05, ^**P<0.01, ^***P<0.001.