John R. Boot

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the alpha1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same alpha1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert-Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K+-stimulated [Ca2+]i increase in the alpha1A cell line but not in the alpha1B, alpha1C, alpha1D, and alpha1E cell lines, establishing the alpha1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the alpha1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs.

1. Basolateral to apical albumin flux has been measured in sheets of bovine bronchial and tracheal mucosa mounted in vitro. 2. Addition of guinea-pig peritoneal eosinophils or neutrophils to the basolateral side of such tissues had no significant influence on the transmucosal flux of albumin in either the bronchial or tracheal mucosa. 3. Stimulation of eosinophils or neutrophils by the calcium ionophore A23187, or by their presentation to an opsonized airways mucosa, resulted in a significant increase in the transbronchial flux of albumin. This effect was seen after only 60 min incubation of the leucocytes with the bronchial mucosa, and was no greater when the contact time was extended to 180 min. Incubation of bronchial mucosal tissues with 1 mg ml-1 polyarginine for 3 h produced a significant increase in albumin flux, but was ineffective at 0.5 mg ml-1. 4. In contrast to the bronchial mucosa, the tracheal mucosa appeared resistant to the effects of stimulated eosinophils and neutrophils. 5. The lipoxygenase inhibitor AA-861 failed to influence the ability of eosinophils to augment the transmembrane flux of albumin. However, insertion of a Millipore filter mask between the eosinophils and the bronchial mucosa significantly inhibited the eosinophil-dependent enhancement of mucosal permeability. 6. The broad spectrum antiproteinase alpha 2-macroglobulin achieved almost total ablation of the action of stimulated eosinophils in the bronchial mucosa. These results suggest that proteinases may make a significant contribution to the genesis of epithelial injury, whereas leukotrienes do not.