Molecular Basis for Pacemaker Cells in Epithelia

Abstract

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca 2؉ signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V 1a vasopressin receptor (V 1a R Cells within excitable tissues such as the heart must act in a coordinated fashion to carry out organ-level functions such as muscle contraction to maintain blood flow. Excitation-contraction coupling in the heart is coordinated by anatomically defined pacemaker cells, and is mediated by cytosolic Ca 2ϩ signaling in individual myocytes. Intercellular Ca 2ϩ waves can be observed in the intact heart (1), and these Ca 2ϩ signaling patterns spread from cell to cell via gap junctions (2) and may relate to normal and abnormal cardiac function (1). In individual myocytes, intracellular Ca 2ϩ release occurs principally via the ryanodine receptor (RyR) 1 (3). Although RyR are spread throughout myocytes, focal clusters generate Ca 2ϩ sparks that can initiate cell-wide Ca 2ϩ signals (4 -6). Intra-and intercellular Ca 2ϩ signaling follows a different paradigm in non-excitable cells (3). Ca 2ϩ signaling in epithelial organs and in many other tissues instead occurs principally via inositol 1,4,5-trisphosphate (InsP 3 ) and the InsP 3 receptor (InsP 3 R) (3). As in the heart, cell-to-cell spread of Ca 2ϩ waves has been observed in epithelial organs, including the liver (7-9) and salivary gland (10). Intercellular Ca 2ϩ signaling follows a complex pattern in the liver, much as it does in the heart. For example, vasopressin induces Ca 2ϩ waves that spread from pericentral to periportal hepatocytes, opposite to the direction of blood flow (7, 9), and this may help direct canalicular motility and bile flow (11). Altered intercellular Ca 2ϩ signaling may contribute to the pathophysiology of certain disease states, since agents that block gap junction conductance also alter tissue function (12)(13)(14), and because certain cholestatic liver diseases are characterized by decreased expression of gap junctions and impaired transmission of intercellular Ca 2ϩ signals (15). Here we examined the conditions necessary to organize intercellular Ca 2ϩ signals in a cell system in which Ca 2ϩ signaling is mediated entirely via InsP 3 and the InsP 3 R. 8 ]vasopressin, tetracycline, penicillin, and streptomycin were obtained from Sigma; fluo-3/AM, fluo-4/AM, fura-2/AM, rhodamine-conjugated phalloidin, and caged compounds (NPE-caged InsP 3 and DM-nitrophen-EDTA) were obtained from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium, Liebovitz 15 (L-15) medium, and other tissue culture reagents were from Invitrogen (Basel, Switzerland). All other chemicals were of the highest quality commercially available. EXPERIMENTAL PROCEDURES Materials-[ArgAntibodies-InsP 3 receptors were labeled using an antibody from affinity-purified specific rabbit polyclonal antiserum directe...

Lipid Rafts Establish Calcium Waves in Hepatocytes

“…SKHep1 cells were used because they are a liver-derived epithelial cell line that is not polarized. Furthermore, like hepatocytes, 8 SKHep1 cells lack ryanodine receptors, 30 so that Ca 2+ signaling depends entirely on InsP3Rs in these cells. The InsP3R was diffusely distributed throughout the cytosol in SkHep1 cells, and this pattern was unchanged after treatment with mβCD ( Figure 9A).…”

“…27 Hepatocytes and SKHep1 cells were observed during perfusion with vasopressin, which causes an InsP3-mediated increase in Ca i 2+ in both of these cell types. 1,30 Similarly, pancreatic acinar cells were stimulated with ACh, which causes an InsP3-mediated increase in Ca i 2+ in these cells. 20 Apical and basal regions were identified within the cytosol of individual cells, and the Ca i 2+ signals in those regions were determined from the recorded images.…”

The Type III Inositol 1,4,5-Trisphosphate Receptor Preferentially Transmits Apoptotic Ca2+ Signals into Mitochondria

“…Immunofluorescence-Confocal immunofluorescence was performed as described previously (25,26). Cells were fixed with 4% paraformaldehyde in PBS for 10 min and then washed three times in PBS.…”

“…Immunoblotting-Standard methods were used for immunoblots (25,26). Briefly, cells grown in 35-mm dishes were washed three times with phosphate-buffered saline (PBS) and solubilized in 80 l of Nonidet P40 containing a protease inhibitor mixture (Roche Applied Science).…”

“…Detection of Cytosolic and Mitochondrial Ca 2ϩ Signals-Cytosolic and mitochondrial Ca 2ϩ were monitored in individual CHO cells by time lapse confocal microscopy as described previously (25,26). CHO cells were cultured on glass coverslips, incubated for 30 min in the presence of 6 M Fluo-4/AM to monitor cytosolic Ca 2ϩ or 6 M rhod-2/AM to monitor mitochondrial Ca 2ϩ and then transferred to a perfusion chamber on the stage of a Bio-Rad MRC-1024 confocal microscope.…”