α7 nicotinic receptors play a role in regulation of cardiac hemodynamics.

The α7 nicotinic receptors (NR) have been confirmed in the heart but their role in cardiac functions has been contradictory. To address these contradictory findings, we analyzed cardiac functions in α7 NR knockout mice (α7-/-) in vivo and ex vivo in isolated hearts. A standard limb leads electrocardiogram was used, and the pressure curves were recorded in vivo, in Arteria carotis and in the left ventricle, or ex vivo, in the left ventricle of the spontaneously beating isolated hearts perfused following Langedorff's method. Experiments were performed under basic conditions, hypercholinergic conditions, and adrenergic stress. The relative expression levels of α and ß NR subunits, muscarinic receptors, ß1 adrenergic receptors, and acetylcholine life cycle markers were determined using RT-qPCR. Our results revealed a prolonged QT interval in α7-/- mice. All in vivo hemodynamic parameters were preserved under all studied conditions. The only difference in ex vivo heart rate between genotypes was the loss of bradycardia in prolonged incubation of isoproterenol-pretreated hearts with high doses of acetylcholine. In contrast, left ventricular systolic pressure was lower under basal conditions and showed a significantly higher increase during adrenergic stimulation. No changes in mRNA expression were observed. In conclusion, α7 NR has no major effect on heart rate, except when stressed hearts are exposed to a prolonged hypercholinergic state, suggesting a role in acetylcholine spillover control. In the absence of extracardiac regulatory mechanisms, left ventricular systolic impairment is revealed.

Cardiac nicotinic receptors show ß-subunit-dependent compensatory changes.

Nicotinic receptors (NRs) play an important role in the cholinergic regulation of heart functions, and converging evidence suggests a diverse repertoire of NR subunits in the heart. A recent hypothesis about the plasticity of ß NR subunits suggests that ß2-subunits and ß4-subunits may substitute for each other. In our study, we assessed the hypothetical ß-subunit interchangeability in the heart at the level of mRNA. Using two mutant mice strains lacking ß2 or ß4 NR subunits, we examined the relative expression of NR subunits and other key cholinergic molecules. We investigated the physiology of isolated hearts perfused by Langendorff's method at basal conditions and after cholinergic and/or adrenergic stimulation. Lack of ß2 NR subunit was accompanied with decreased relative expression of ß4-subunits and α3-subunits. No other cholinergic changes were observed at the level of mRNA, except for increased M3 and decreased M4 muscarinic receptors. Isolated hearts lacking ß2 NR subunit showed different dynamics in heart rate response to indirect cholinergic stimulation. In hearts lacking ß4 NR subunit, increased levels of ß2-subunits were observed together with decreased mRNA for acetylcholine-synthetizing enzyme and M1 and M4 muscarinic receptors. Changes in the expression levels in ß4-/- hearts were associated with increased basal heart rate and impaired response to a high dose of acetylcholine upon adrenergic stimulation. In support of the proposed plasticity of cardiac NRs, our results confirmed subunit-dependent compensatory changes to missing cardiac NRs subunits with consequences on isolated heart physiology.NEW & NOTEWORTHY In the present study, we observed an increase in mRNA levels of the ß2 NR subunit in ß4-/- hearts but not vice versa, thus supporting the hypothesis of ß NR subunit plasticity that depends on the specific type of missing ß-subunit. This was accompanied with specific cholinergic adaptations. Nevertheless, isolated hearts of ß4-/- mice showed increased basal heart rate and a higher sensitivity to a high dose of acetylcholine upon adrenergic stimulation.

Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences.

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.