Abstract
1. Responses of isolated rat lacrimal cells to local applications of ATP were studied using tight-seal whole-cell recording and/or Fura-2-derived calcium concentration measurements. 2. In cells where variations in Ca2+ concentration were prevented by use of a strong Ca2+ buffer, ATP was found to induce an inward current response at negative holding potentials. With 10 microM-ATP, the current amplitude ranged between 20 and 200 pA. The reversal potential of this ATP-induced current was close to 0 mV with normal external solution and shifted to -19 +/- 3 mV (mean +/- S.D.) when the concentration of external monovalent cations was halved. These results indicate that the channels have a cationic selectivity. The response amplitude decreased markedly from trial to trial, indicating a desensitization process which was irreversible on the time scale of the recordings. 3. Steady state I-V curves for the ATP-induced current in normal saline showed a marked inward rectification. This rectification appeared to be linked to a time-dependent activation of the channels, as hyperpolarizing voltage jumps elicited a time-dependent current increase. This relaxation could be fitted by a double-exponential function, with time constants (at -120 mV) of 0.9 +/- 0.3 ms and 110 +/- 6.4 ms. 4. Variance analysis of the ATP-induced current gave a single-channel current value of 0.34 pA at -60 mV. The single-channel current amplitude varied linearly with potential, with a slope close to 6 pS. The relation between noise covariance and time could be fitted by a double-exponential function, with time constants (at -60 mV) of 0.8 +/- 0.4 ms and 6.8 +/- 3.4 ms (mean +/- S.D.). 5. In an isotonic Ca2+ solution, 10 microM-ATP induced an inward current at -60 mV with a calculated single-channel current amplitude obtained from noise analysis close to 0.2 pA. In an external solution containing 10 mM-calcium and no sodium, 50 microM-ATP elicited a current with a reversal potential of -19 mV. 6. Fura-2 measurements were performed in intact cells or in cells dialysed with a low concentration of Ca2+ buffer (e.g. 0.5 mM-EGTA). Under such conditions ATP induced increases of the internal Ca2+ concentration with very variable amplitudes. In some cells Ca2+ rises of 50 nM or lower were found. Minimal activation of Ca(2+)-dependent channels was then observed. In other cells large Ca2+ rises (up to 500 nM) were observed and were then correlated with marked activation of Ca(2+)-dependent channels.(ABSTRACT TRUNCATED AT 400 WORDS)