Immunohistochemical Detection of α1E Voltage-gated Ca²⁺ Channel Isoforms in Cerebellum, INS-1 Cells, and Neuroendocrine Cells of the Digestive System

Abstract

SUMMARY Polyclonal antibodies were raised against a common and a specific epitope present only in longer ␣ 1E isoforms of voltage-gated Ca 2 ϩ channels, yielding an "anti-Ecom" and an "anti-E-spec" serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an ␣ 1E splice variant with a longer carboxy terminus, the so-called ␣ 1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter ␣ 1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer ␣ 1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, ␣ 1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. ␣ 1E therefore appears to be a common voltagegated Ca 2 ϩ channel linked to neuroendocrine and related systems of the body.

Immunodetection of α1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney

“…Peptide Nast-197 contains one additional cysteine residue for coupling to a matrix, as shown in brackets. Immunization of the rabbits with the hemocyanin-coupled peptides was described previously Grabsch et al 1999).…”

“…Tissue sections from human kidney were obtained from surgically resected specimens. All tissues were fixed in 10% PBS-buffered formalin at room temperature overnight and embedded in paraffin (Grabsch et al 1999). Immunohistochemistry was performed in 4 m deparaffinized serial tissue sections.…”

“…Endogenous peroxidase activity was quenched by putting the slides in 0.3% hydrogen peroxide in methanol for 20 min at 37C. After washing with PBS, sections were immunostained by the streptavidin-biotin-horseradish peroxidase technique (Grabsch et al 1999) according to the instructions of the manufacturer (Super Sensitive detection system from BioGenex; Hamburg, Germany). The polyclonal antibodies were used at a dilution of 1:100-1:300 in heart, 1:300 in H9c2 cells, and 1:20 in kidney.…”

“…The reactivity and the specificity of the polyclonal anti-␣1E-spec serum had been evaluated recently by immunocytochemistry (Grabsch et al 1999), which indicates that the anti-1E-spec serum is useful for immunocytochemical detection of longer ␣1E splice variants in cell lines and tissues.…”

“…In serial tissue sections, anti-chromogranin A/B ( Figure 6A) and anti-ANP antibodies ( Figure 6C) were used as markers for endocrine cells, first because we had described ␣1E in neuroendocrine cells of the gastrointestinal system (Grabsch et al 1999) and, second, because endocrine functions in the rat heart are assigned to Purkinje fibers (Hansson and Forsgren 1993;Benvenuti et al 1997). In the atrium, the anti-chromogranin ( Figure 6A) and anti-ANP ( Figure 6C) sera stained the same muscle cells as detected by anti-␣1E-spec ( Figure 6B).…”