INVOLVEMENT OF THE IP3 CASCADE IN THE DAMAGE TO GUINEA-PIG VENTRICULAR MYOCYTES INDUCED BY CYTOTOXIC T-LYMPHOCYTES

We have shown previously that the interaction between cytotoxic T lymp hocytes (CTL) and ventricular myocytes, an in vitro model for heart tr ansplant rejection, results in electrophysiological and morphological alterations indicative of overload of the intracellular [Ca2+] ([Ca2+] (i)). Since these deleterious effects cannot be accounted for by incre ased L-type Ca2+ current (I-Ca,I-L), we hypothesize that [Ca2+](i) ove rload due to Ca2+ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inosito l trisphosphate (IP3) cascade. Patch-clamp and fura-2-fluorescence tec hniques were utilized to record transmembrane potentials and [Ca2+](i) from ventricular myocytes bound to peritoneal exudate CTL (PEL). In v entricular myocyte-PEL conjugates (after 60 min), resting potential wa s reduced (compared with the nonconjugated state) from -80.9 +/- 0.7 t o -59.9 +/- 2.5 mV, action potential amplitude from 139.5 +/- 1.4 to 8 0.6 +/- 1.7 mV and action potential duration to 50% repolarization (AP D(50)) from 797 +/- 97 to 52 +/- 12 ms. The ratio of fluorescence at 3 40 and 380 nm (R-340/380) increased from a control value (in nonconjug ated myocytes) of 0.71 +/- 0.02 to 2.07 +/- 0.03, 30 min after conjuga te formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 mu g/ml), an antagonist of IP3-induced Ca2+ release from S R channels, or U-73122 (2 mu M), a phospholipase C (PLC) inhibitor (dr ugs were included in the pipette solution), prevented PEL-induced morp hological and electrophysiological alterations. Accordingly, heparin a ttenuated the PEL-induced increase in [Ca2+](i); after 60 min of PEL-m yocyte interaction, R-340/380 was 1.15 +/- 0.09 (compared with approxi mately 4.0 in the absence of heparin). The results indicate that CTL-m ediated damage to ventricular myocytes is, at least partially, mediate d by PLC activation and IP-induced Ca2+ release from intracellular sto res. Pharmacological targeting of IP3 in heart transplant rejection is thus suggested.