Pertussis Toxin Treatment of Whole Blood

This study was designed to assess G protein function in mononuclear leukocytes (MNL) of patients with congestive heart failure (CHF). MNL membranes were ADP-ribosylated in vitro in the presence of pertussis or cholera toxin. The amount of pertussis toxin substrates did not differ significantly between CHF patients (6,100±224 fmol/mg, n =23) and age-matched healthy control subjects (5,812±972 fmol/mg protein, n= 19). Among the CHF patients, no differences were observed between those with idiopathic and ischemic CHF. The amount of cholera toxin substrates also did not differ significantly between CHF patients (7,522±1,405 fmol/mg protein, n=11) and control subjects (5,654±707 fmol/mg protein, n=14). Moreover, basal and isoproterenoland prostaglandin El-stimulated cyclic AMP (cAMP) accumulation in MNL was similar in control subjects and patients. To detect more subtle alterations of the cAMPgenerating system, we incubated anticoagulated blood with 250-400 ng/ml pertussis toxin for 4 hours at 370 C. This treatment completely ADP-ribosylated the MNL pertussis toxin substrates. Incubation with pertussis toxin did not change basal or prostaglandin E1stimulated cAMP generation in MNL of control subjects, but it significantly enhanced stimulated generation (443±44 vs. 643±93 pmol/107 cells, p<0.025) in MNL of CHF patients. This enhancement was most pronounced in the most severely ill patients (New York Heart Association class lY) and correlated with plasma norepinephrine levels, another marker of CHF severity (r=0.798, n= 11, p <0.01). Isoproterenol-stimulated cAMP generation, which was much weaker than that produced by prostaglandin E1, was enhanced only in patients in class IV. We conclude that despite apparently unaltered amounts of subunits identifiable by ADP ribosylation with pertussis toxin, MNL of CHF patients appear to have enhanced functional activity (i.e., inhibition of cAMP formation) by these substrates. Thus, incubation ofMNL with pertussis toxin reveals a tonic inhibition of cAMP generation in cells of CHF patients. The results suggest that incubation of whole blood with pertussis toxin and subsequent assessment of cAMP accumulation in MNL provide a useful way to examine G protein function in patients with cardiovascular disease. (Circulation 1990;81:1198-1204)