Norepinephrine Pretreatment Attenuates Ca2+Overloading in Rat Trabeculae During Subsequent Metabolic Inhibition: Improved Contractile Recovery Via an α1-adrenergic, PKC-dependent Signaling Mechanism

The present study was designed in order to investigate more precisely the role of calcium homeostasis maintenance in protein kinase C (PKC) mediated preconditioning. We used a 15 min pre-incubation period, with 1μmol/l exogenous norepinephrine (NE) to pharmacologically precondition isolated, superfused rat trabeculae against contractile dysfunctioning following 120 min of metabolic inhibition (MI, in 2 mmol/l CN−containing Tyrode without glucose at 1 Hz stimulation frequency). Contractile recovery was studied during a subsequent 60 min recovery period (RP, in glucose containing Tyrode at 0.2 Hz). Tyrode was gassed with 95% O2/5% CO2and kept at a constant temperature of 24°C. Force and intracellular free calcium ([Ca2+]i) were monitored throughout the experimental protocol; [Ca2+]iwas measured using fura-2. Pretreatment with NE (group NE-I) significantly increased the fraction of trabeculae that resumed to contract during RP, from 36±13% (mean± ) in controls to 82±10% (P<0.05). In correspondence with this, NE-pretreatment increased the proportion of trabeculae in which the Ca2+rise from the onset of rigor development during MI was attenuated. After 40 min of MI [Ca2+]iin the failing control, as well as failing group NE-I, trabeculae (1.08±0.20 and 1.51±0.26μmol/l, respectively) was increased significantly compared to the mean value registered in the recovering preparations of these groups (0.34±0.04μmol/l;P<0.05). Specific inhibition of PKC with 2μmol/l chelerythrine (group NE-IV) almost completely blocked the protection induced by NE-pretreatment, including its protective action against Ca2+overload, i.e. the fraction of trabeculae that resumed to contract during RP returned to untreated control level (46±11%;P<0.05vgroup NE-I). Also in this case [Ca2+]iin the failing group NE-IV trabeculae after 40 min of MI was increased substantially, compared to the value measured in the recovering preparations (4.75±1.00 and 0.60±0.08μmol/l, respectively). The relative importance of bothα-adrenergic andβ-adrenergic receptor pathways in this preconditioning-like effect of NE-pretreatment, was investigated using specific blockers. The results point to anα1-adrenergic receptor mediated signaling mechanism, which enhances PKC-dependent control of [Ca2+]ifrom the onset of rigor development during MI.