Abstractα4β2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up‐regulation of α4β2 nicotinic acetylcholine receptors involves expression changes for non‐receptor genes. In a microarray analysis, 10 μM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in hα4β2 SH‐EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in 3[H]‐cytisine binding. Quantitative RT‐PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro‐inflammatory cytokines IL‐1β and IL‐6. Nicotine suppresses IL‐1β and IL‐6 expression at least in part by inhibiting NFκB activation. Antagonists dihydro‐β‐erythroidine and mecamylamine blocked these nicotine‐induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing α4β2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of α4β2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate α4β2 expression, and may explain antagonist actions in nicotine‐induced receptor up‐regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic α4β2 receptor activation promotes anti‐inflammatory effects similar to α7 receptor activation.

A bispecific enzyme-linked signal-enhanced immunoassay (BiELSIA) was developed with markedly increased sensitivity. Antimyosin, the detection antibody, was linked to the signal probespecific antibody. Probes consisted of diethylenetriamine pentaacetic acids attached to polylysine modified with up to seven or eight horseradish peroxidase (HRP) units. Each bispecific antibody bound two polymer probes, providing twice the signal. Using BiELSIA in a competitive inhibition immunoassay format with an average of 1.5, 3, 4.5, 6, and 7.5 HRP units per polymer-probe, the sensitivity of standard enzyme-linked immunosorbent assay (10(13) mole) was increased to 10(15), 10(18), 10(19), 10(20), and 10(-21) mol (< or = 1,000 molecules), respectively. BiELSIA detected cardiac myosin heavy chain fragments in sera of patients obtained at the time of emergency department admission for acute myocardial infarction, but not in normal sera. This technology should be applicable for detection of cancer, human immunodeficiency virus, prion, and other antigens that are present in concentrations too low for detection by current immunoassays.

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