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

Abstract

S U M M A R YThe calcium channel ␣ 1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of ␣ 1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac ␣ 1E was determined by SDS-PAGE and immunoblotting (218 Ϯ 6 kD; n ϭ 3). Compared to ␣ 1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of ␣ 1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of ␣ 1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of ␣ 1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of ␣ 1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca 2 ϩ -dependent secretion of peptide hormones such as ANP and urodilatin.

Endothelium-derived nitric oxide mediates the antiadrenergic effect of human vasostatin-1 in rat ventricular myocardium

“…Conceivably, the intracardiac concentrations of CgA and its derived fragments may be even higher under certain conditions, e.g., in response to stress and/or secretagogues, promoting their secretion by various cell types. In fact, immunodetection studies have documented the presence of CgA, colocalized with atrial natriuretic peptides, in the secretory granules of rat atrial myoendocrine cells (25), as well as in rat Purkinje cells of the conducting system and in H9c2 rat cardiomyocytes (27). These data, together with the recent biochemical identification of CgA-derived peptides containing the vasostatin motif in the rat heart (16), strongly suggest that CgA-derived peptides play an important autocrine/paracrine role in cardiovascular homeostasis in physiological and pathophysiological conditions.…”

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

“…Several previous workers have prepared polyclonal antibodies directed against the T-type Ca 2ϩ channel (30,31). The antibody was prepared as described by using a peptide corresponding to amino acids 1-22 of the NH 2 -terminal region of human ␣1G subunit of the low-voltage T-type Ca 2ϩ channel with the addition of a COOH-terminal cysteine: NH 2 -MDEEEDGAGAEESGQPRSFMRL(C)-COOH (30).…”

Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette

“…Eight additional nucleotides were linked to the 5 0 -end of each primer containing an EcoRI site for subcloning. The annealing temperature for the a1E cassettes was 57 8C during 32 cycles of amplification (21). Oligonucleotide primers were purchased from Eurogentec Bel SA (Seraing, Belgium).…”