Na(+)-Ca2+ exchanger in arteries: identification by immunoblotting and immunofluorescence microscopy

Abstract

Antibodies raised against dog cardiac Na(+)-Ca2+ exchanger were employed to determine the presence and distribution of the exchanger in arterial smooth muscle (ASM) cells. The antiserum cross-reacted with protein bands of approximately 70, 120, and 150-160 kDa from the membranes of ASM cells, as well as heart sarcolemma. A cardiac Na(+)-Ca2+ exchanger cDNA probe hybridized to 7-kilobase (kb) mRNA from myocytes of the mesenteric artery. Thus ASM cells possess a "cardiac type" Na(+)-Ca2+ exchanger. The relative amounts of 7-kb mRNA and antigen detected on Northern and Western blots, respectively, however, indicate that vascular myocytes contain much less of this transporter than do cardiac myocytes. Immunofluorescence studies on cultured arterial myocytes suggest that the exchanger molecules are organized in reticular patterns over the cell surfaces. A similar pattern is observed when cells are stained for sarcoplasmic reticulum (SR) Ca(2+)-ATPase. This raises the possibility that the exchanger in the plasmalemma of arterial myocytes may be associated, perhaps functionally as well as structurally, with the underlying SR. The antiserum also cross-reacted with endothelial cell membranes, but labeling was lighter and more diffuse than in the myocytes.

Na + pump low and high ouabain affinity α subunit isoforms are differently distributed in cells

“…The location of the high affinity isoforms appears to provide a clue because the SR and ER sequester Ca 2ϩ , and the Ca 2ϩ released from these organelles controls numerous cell activities. A critical link between the Na ϩ pump and cell Ca 2ϩ is the PM Na ϩ ͞Ca 2ϩ exchanger, which is also localized to PM regions that overlie junctional SR and ER (11,14,15,31). The energy required to control cytosolic Ca 2ϩ via the Na ϩ ͞Ca 2ϩ exchanger is derived from the Na ϩ electrochemical gradient generated by the Na ϩ pump (12).…”

“…Myocytes were dissociated and cultured from adult rat mesenteric artery (MA) (14). Virtually all cultured MA cells crossreacted with antibodies raised against smooth muscle ␣-actin.…”

“…Membranes were prepared from cultured myocytes, astrocytes and neurons, and fresh rat soleus muscle, as described (14). Membrane proteins were separated by SDS͞PAGE and immunolabeled with antibodies raised against specific ␣ subunit isoforms (17).…”

“…Cells on coverslips were fixed (45 min, 22ЊC) with 0.45% (wt͞vol) formaldehyde in: 75 mM cyclohexylamine (free base), 75 mM NaCl, 10 mM EGTA, 10 mM MgCl 2 , and 10 mM piperazine-N,-NЈbis (2-ethane sulfonic acid) adjusted to pH 6.8 with HCl. The cells were then immunolabeled (14). Nonspecific crossreactivity was blocked (4 hr, 22ЊC) with normal donkey or goat serum diluted 1:6 with ''antibody buffer'' (500 mM NaCl͞10 mM MgCl 2 ͞20 mM NaN 3 ͞20 Tris⅐HCl, pH 7.4).…”

Na+/Ca2+ Exchange Facilitates Ca2+-dependent Activation of Endothelial Nitric-oxide Synthase

“…During depolarization, the exchanger operates in reversed mode (Ca 2ϩ entry/Na ϩ extrusion) and triggers Ca 2ϩ -induced Ca 2ϩ release during cardiac excitation (18,19). In endothelial cells, the presence of a Na ϩ /Ca 2ϩ exchanger (NCX) has recently been demonstrated by immunoblotting and immunofluorescence microscopy (20), and these results have been verified by the detection of cDNA coding for NCX1 (21). However, the role of this protein in endothelial Ca 2ϩ homeostasis is still a matter of debate.…”