A LIGHT-EMITTING DIODE MICROSPECTROPHOTOMETER - INTRACELLULAR CA2-RECEPTOR( MEASUREMENTS IN ISOLATED STRETCH)

A microspectrophotometer was designed to measure absorbance changes in single cells. The device utilizes sequentially activated light emitti ng diodes (LED) to provide different wave lengths of light. The instru ment has the advantage of relative simplicity and less cost compared t o other devices. The spectrophotometer was tested by measuring absorba nce changes of the metallochromic Ca2+ indicator Arsenazo III (AIII) i njected into the crayfish (Astacus astacus) stretch receptor. Under th e conditions described the detection limit of the concentration of AII I was 0.05 mM and absorbance changes of 0.0005 can be reliably determi ned which correspond to a detection limit of 10-20 nM for free Ca2+ ch anges assuming a light path length of 0.003 cm and an apparent dissoci ation constant (K(D)) of 2 muM for the Ca2+-AIII complex. The upper fr equency limit of the device is 3000 Hz. The absorbance measurements of AIII injected into the crayfish stretch receptor neurons revealed a C a(i) of 375 +/- 177 nM (mean +/- SD: 14 cells). LiCl substituted for N aCl increased Ca(i) 45-100 nM in different cells, suggesting that a Na + gradient is necessary for Ca2+ homeostasis in this receptor.