Effects of calcium on gap junctions between osteoblast-like cells in culture.

The present study investigated the effects of elevated cytoplasmic free calcium concentrations ([Ca2+]i) on the permeability of gap junctions between cultured osteoblast-like (OB) cells derived from calvarial and periosteal fragments of newborn rats. This was studied using the double whole cell patch clamp technique and intracellular dye injections. To increase [Ca2+]i, patch pipette solutions contained 100 micromol/liter Ca2+. About 1-2 minutes after whole cell modes had been attained, the total number of gap junction channels was reduced from an average of 400 in normal Ca2+ to 20 in high Ca2+. Thereafter, remaining gap junction channels were active for up to 8 minutes. In normal rat kidney (NRK) cells, gap junction channels were closed by high Ca2+ within 1 minute, pointing to a similar sensitivity of Connexin43 gap junction channels in OB and NRK cells. To study the effects of elevated [Ca2+]i on the dye permeability of gap junctions between extended OB cells, the spread of Lucifer Yellow to neighboring cells was evaluated. [Ca2+]i was gradually increased from 1.5- to 14-fold the normal value by application of either ouabain, Na+-free/ouabain, or A23187. Reduced dye spread correlated with the increase of [Ca2+]i measured by analyzing the fluorescence of fura-2. These data show that gap junctions in OB cells are sensitive to Ca2+.

Cell-to-cell communication in osteoblastic networks: cell line-dependent hormonal regulation of gap junction function.

We have characterized the distribution, expression, and hormonal regulation of gap junctions in primary cultures of rat osteoblast-like cells (ROBs), and three osteosarcoma cell lines, ROS 17/2.8, UMR-106, and SAOS-2, and a continuous osteoblastic cell line, MC3T3-E1. All cell lines we examined were functionally coupled. ROS 17/2.8 were the more strongly coupled, while ROB and MC3T3-E1 were moderately coupled and UMR-106 and SAOS-2 were weakly coupled. Exposure to parathyroid hormone (PTH) for 1 h increased functional coupling in ROB cells in a concentration-dependent manner. Furthermore, PTH(3-34), an analog of PTH with binds to the PTH receptor and thus attenuates PTH-stimulated cAMP accumulation, also attenuated PTH-stimulated functional coupling in ROB. This suggests that PTH increases functional coupling partly through a cAMP-dependent mechanism. A 1 h exposure to PTH did not affect coupling in ROS 17/2.8, UMR-106, MC3T3-E1, or SAOS-2. To examine whether connexin43 (Cx43), a specific gap junction protein, is present in functionally coupled osteoblastic cells, we characterized Cx43 distribution and expression. Indirect immunofluorescence with antibodies to Cx43 revealed that ROS 17/2.8, ROB, and to a lesser extent MC3T3-E1 and UMR-106, expressed Cx43 immunoreactivity. SAOS-2 showed little if any Cx43 immunoreactivity. Cx43 mRNA and Cx43 protein were detected by Northern blot analysis and immunoblot analysis, respectively, in all cell lines examined, including SAOS-2. Our findings suggest that acute exposure to PTH regulates gap junction coupling, in a cell-line dependent manner, in osteoblastic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic modulation of immunoglobulin secretion from avian plasma cells: the role of calcium.

The existence of a functional connection between the nervous and immune systems has long been argued. To determine if such a link exists in the secretory immune system, we have examined the avian lacrimal gland (Harderian gland) which contains large numbers of plasma cells. We have shown that these plasma cells bind an antibody to muscarinic acetylcholine receptor and that carbachol, an acetylcholine agonist, increases the secretion rate of IgG by these cells above a constitutive baseline level. This neurotransmitter-dependent increase of immunoglobulin secretion requires an influx of Ca2+, whereas the constitutive baseline secretion is apparently less dependent on such a flux. Furthermore, the Ca2+ flux appears to be mediated by voltage-dependent calcium channels. These data support the hypothesis that plasma cells can respond to neurotransmitters and, in the case of acetylcholine, increase immunoglobulin secretion.

Expression of connexin-43 in human myometrium and leiomyoma.

OBJECTIVES: The expression of connexin-43 was examined in human leiomyomas and in autologous myometrium. STUDY DESIGN: Indirect immunofluorescence was used to detect connexin-43 gap junctions in myometrial and leiomyoma tissues and in primary cultures. Immunoblot and Northern analyses were used to examine the levels of connexin-43 protein and cx43 messenger ribonucleic acid in primary cultures. RESULTS: Connexin-43 gap junctions were detected in leiomyoma tissue from nonpregnant premenopausal women but not in autologous myometrial tissue. Connexin-43 gap junctions form spontaneously in primary cultures of myometrium and leiomyoma in the presence of 17 beta-estradiol. Fluorescent dye injection confirmed that the gap junctions were coupled in the primary cells. Connexin-43 protein and messenger ribonucleic acid levels generally were higher in leiomyoma cells compared with those of autologous myometrial cells. Steady-state levels of cx43 messenger ribonucleic acid decreased with addition of medroxyprogesterone acetate to the cultures. CONCLUSIONS: Differences in the levels of cx43 expression in leiomyoma and myometrial cells may reflect differences in responses to steroid hormones.