Regulation of Connexin Phosphorylation and Cell–Cell Coupling in Trabecular Meshwork Cells

Abstract

purpose. To investigate the expression of functional gap junctions and the effect of protein kinase C (PKC) on such junctions in confluent cultures of bovine trabecular meshwork (TM) cells.

methods. Expression of the gap junction protein connexin43 in TM cells was examined by immunofluorescence microscopy. Intercellular communication by gap junctions was assessed by observing the diffusion of fluorescent dye from an individual cell injected with lucifer yellow. The phosphorylation of connexin43 was evaluated by immunoblot analysis with a monoclonal antibody to this protein.

results. Immunofluorescence staining revealed that connexin43 was localized to sites of contact between adjacent TM cells. Exposure of cells to the PKC activator phorbol 12-myristate 13-acetate (PMA; 10 nM, 1 hour) had no marked effect on the pattern of connexin43 immunofluorescence. Injection of a TM cell with lucifer yellow resulted in the spread of the dye into neighboring cells. Dye coupling was inhibited by PMA in a dose- and time-dependent manner, and this inhibition was prevented by pretreatment of cells with the PKC inhibitor bisindolylmaleimide I. Immunoblot analysis of control TM cell lysates yielded connexin43 bands corresponding to the nonphosphorylated protein (43 kDa) and three phosphorylated forms (47, 48, and 49 kDa). Cells exposed to PMA (10 nM, 1 hour) yielded an additional band corresponding to a 44-kDa form of phosphorylated connexin43 and showed a decrease in the intensity of the band corresponding to the nonphosphorylated protein and an increase in the intensity of the 47-kDa band.

conclusions. TM cells communicate with each other through gap junctions, and the communication is inhibited by PKC, probably, at least in part, through phosphorylation of connexin43.

The trabecular meshwork (TM) and Schlemm’s canal are the main pathways for the outflow of aqueous humor and are important in the maintenance of intraocular pressure. The trabecular beams of the meshwork are overlaid by TM cells, ¹ the functions of which are thought to be regulated by various extracellular signals. Direct cell-to-cell communication in multicellular organs and tissues is achieved through gap junctions. ^{2 3 4} Such gap junction–mediated intercellular communication plays a predominant role in the coupling of neighboring cells and contributes to the regulation of various cellular functions, such as proliferation and differentiation, and to the development of tissues and organs. ^{5 6 7 8 9}  

Among the various junctional complexes, only gap junctions permit ions or molecules of less than 1000 Da to penetrate and to exchange between adjacent cells. ^{10 11} The freeze–fracture technique has demonstrated the presence of gap junctions in meshworks of endothelial cells in humans. ¹² Gap junctions are formed by the docking of hemichannels (connexons) located in the cell membranes of adjacent cells. ¹³ Each connexon comprises a hexameric aggregate of transmembrane proteins known as connexins. ¹³ Connexin43, with a molecular size of 43 kDa, is one subtype of connexin ¹³ and is expressed in a wide variety of tissues and organs, including the brain, ¹⁴ heart, ¹⁵ nerves, ¹⁶ and vascular smooth muscle. ¹⁷  

Protein kinase C (PKC) is an important component of intracellular signaling pathways. PKC thus participates, for example, in the regulation of ion channel activity, neurotransmitter release, and neurotransmitter receptor sensitivity. ¹⁸ Phorbol esters that activate PKC have also been shown to affect both gap junction–mediated intercellular communication and the phosphorylation of connexins in various cell types, including osteoblastic MC3T3-E1 cells, ¹⁹ Novikoff hepatoma cells, ²⁰ and liver epithelial cells. ²¹  

We have now investigated whether connexin43 is expressed in the TM, and whether the activation of PKC affects intercellular communication through gap junctions in TM cells. We examined the expression and localization of connexin43 in the bovine anterior chamber angle and cultured bovine TM cells with the use of indirect immunofluorescence microscopy. The effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on connexin phosphorylation and on the function of gap junctions in cultured TM cells were examined by immunoblot analysis and a dye-coupling technique, respectively. 

Methods

Materials

Eagle’s minimum essential medium and bovine serum albumin (BSA; fraction V) were obtained from Nacalai Tesque (Kyoto, Japan), fetal bovine serum and blocking agent from Dainippon Pharmaceutical (Osaka, Japan), PMA and bisindolylmaleimide I from Sigma (St. Louis, MO), 4α-phorbol 12-myristate 13-acetate (4α-PMA) from Research Biochemicals (Natick, MA), and lucifer yellow CH (Li⁺ salt) from Aldrich (Dreieich, Germany). The monoclonal antibody to rat connexin43 was from Chemicon (Temecula, CA), fluorescein isothiocyanate (FITC)-conjugated goat antibodies to mouse immunoglobulin G (IgG) from Cappel (Durham, UK), and horseradish peroxidase–conjugated goat antibodies to mouse IgG from Santa Cruz Biotechnology (Santa Cruz, CA). Rhodamine-phalloidin was from Molecular Probes (Eugene, OR), BALB/c mouse control ascites fluid from Cedarlane (Hornby, Ontario, Canada), leupeptin and pepstatin A from Peptide Institute (Osaka, Japan), and aprotinin and alkaline phosphatase from Boehringer Mannheim (Indianapolis, IN). Enhanced chemiluminescence (ECL) immunoblot detection reagents and Hyperfilm were from Amersham Pharmacia Biotech (Little Chalfont, UK). Four-chamber, polystyrene vessel glass culture slides (Falcon CultureSlide) and tissue culture dishes (6 cm in diameter, Falcon) were from Becton Dickinson (Franklin Lakes, NJ). 

Preparation of Bovine TM Cells

Fresh bovine eyes were obtained from a local abattoir, and TM cells were prepared as previously described. ²² In brief, each eye was equatorially dissected under sterile conditions. The vitreous, lens, cornea, iris, and ciliary body were removed, and the remaining anterior chamber angle tissue was cut into small pieces and placed in a plastic dish. The tissue was cultured for 2 weeks in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum, after which the pieces of tissue were removed and the explanted primary cells were further cultured in the same medium. When the cultures became confluent, the cells were removed from the dish by exposure to trypsin-EDTA (0.05% trypsin and 0.53 mM EDTA; Life Technologies, Rockville, MD) and subcultured. 

Cells were subjected to experiments during the third passage. They were removed from the dish by exposure to trypsin-EDTA, and the single cells in suspension were plated on culture slides or dishes. When the cells achieved confluence, PMA or 4α-PMA dissolved in dimethyl sulfoxide (DMSO) was added at various concentrations to the culture medium, and the cells were incubated for various times. The final concentration of DMSO in the culture medium was 0.02%. Control incubations were performed in the presence of the same final concentration of DMSO alone. 

Immunofluorescence Microscopy

The localization of connexin43 in bovine eyes was examined by indirect immunofluorescence microscopy. The tissue constituting the anterior chamber angle was dissected from three fresh bovine eyes, embedded in optimal cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan), and frozen in a bath of acetone and dry ice. Frozen sections (thickness, 6 μm) were cut with a cryostat (HM 505 N; Microm, Walldorf, Germany), mounted on silane-treated slides (Dako, Kyoto, Japan), and air dried. After they were washed with phosphate-buffered saline (PBS), the sections were incubated at room temperature first for 1 hour with PBS containing 1% (wt/vol) BSA-PBS to block nonspecific binding and then for 1 hour with a monoclonal antibody to connexin43 (1:200 dilution in BSA-PBS). The specimens were again washed with PBS, incubated for 1 hour at room temperature with FITC-conjugated goat antibodies to mouse IgG (1:500 dilution in BSA-PBS), washed with PBS, and then mounted in glycerol-PBS (2:1, vol/vol). The sections were observed under a fluorescence microscope (Axiovert 135; Zeiss, Jena, Germany) and photographed with the aid of an exposure meter (MC-80; Zeiss). The same field was also examined under bright light and again photographed. At least three sections from each bovine eye were examined. 

The localization of connexin43 and actin filaments in cultured TM cells was examined by double-label cytochemical staining. After exposure to PMA or 4α-PMA, the cells were rinsed with PBS and fixed for 20 minutes in ice-cold acetone. The fixed cells were incubated with the monoclonal antibody to connexin43 and then with FITC-conjugated goat antibodies to mouse IgG, as described, for immunostaining of eye tissue. After they were washed, the cells were incubated for 30 minutes at room temperature with rhodamine-phalloidin (1:200 dilution in BSA-PBS) and observed under a laser confocal microscope (Fluoview; Olympus, Tokyo, Japan). 

Dye Coupling

Gap junction–mediated intercellular communication was measured by dye coupling with the fluorescent dye lucifer yellow according to a modified version of a previously described method. ^{23 24} TM cells were cultured in four-chamber culture slides until they achieved confluence, exposed to test agents for various times at 37°C, and then washed with PBS. A single cell was then injected with lucifer yellow CH (10% wt/vol in 0.33 M LiCl) with the use of a microinjector (Micromanipulator and Transjector; Eppendorf, Hamburg, Germany). One minute after injection, the cells were washed with PBS, observed under a fluorescence inverted microscope (Axioscope; Zeiss), and photographed. Given that the lucifer yellow clearly stained the nuclei, the number of stained nuclei was counted from photographs and was assumed to reflect the number of cells that had taken up the dye. We injected dye into two cells in each chamber and repeated each experiment in at least four chambers. Gap junctional intercellular communication activity was expressed as the mean (± SE) number of cells that contained lucifer yellow after each injection. 

Immunoblot Analysis

The phosphorylation state of connexin43 in cultured TM cells was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. ^{19 25} The cells were cultured in tissue culture dishes (6 cm in diameter) until confluence, after which PMA or 4α-PMA was added to the culture medium, and the cells were incubated for various times at 37°C. Cells were then rinsed with PBS and lysed by scraping and sonication in 0.2 ml of a solution containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% (vol/vol) Triton X-100, 3% (wt/vol) SDS, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, leupeptin (1 μg/ml), pepstatin A (1μ g/ml), and aprotinin (10 μg/ml). The lysate was centrifuged at 15,000g for 5 minutes, and a portion of the resultant supernatant was assayed for protein concentration with a protein assay (Bio-Rad, Hercules, CA). Lysate supernatant or biotinylated protein markers (broad range; New England Biolabs, Beverly, MA) were mixed with 0.5 volume of a solution containing 187.5 mM Tris-HCl (pH 6.8), 6% SDS, 30% (vol/vol) glycerol, 0.03% bromophenol blue, and 0.125 M dithiothreitol; boiled for 5 minutes; and subjected to SDS-PAGE (4 μg of lysate protein per lane) on a 12.5% gel with a bisacrylamide–acrylamide ratio of 0.15:29.2. The separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), which was then treated with Block Ace to block nonspecific sites before incubation for 2 hours at room temperature with the monoclonal antibody to connexin43 (1:1000 dilution) in washing buffer (20 mM Tris-HCl [ pH 7.4]), 2.5% vol/vol Block Ace, and 0.1% vol/vol Tween 20). The membrane was washed in washing buffer, incubated for 1 hour at room temperature with horseradish peroxidase–conjugated goat antibodies to mouse IgG (1:2000 dilution in washing buffer), washed again, immersed in ECL detection reagents for 1 minute, and exposed to film (Hyperfilm; Amersham). 

The effect of alkaline phosphatase on the electrophoretic mobility of connexin43 was examined by preparing the cell lysates in lysis buffer without Na₃VO₄ (an inhibitor of alkaline phosphatase). The lysate supernatants were incubated for 1 hour at 37°C with alkaline phosphatase (2.5 IU/ml) in the absence or presence of 10 mM Na₃VO₄. Samples were then subjected to SDS-PAGE and immunoblot analysis, as described. 

The relative amounts of nonphosphorylated and phosphorylated connexin43 were determined with a densitometer (Arcus II; PDI, New York, NY) equipped with analysis software (Quantity One, PDI). All immunoblot experiments were performed three times with similar results. 

Statistical Analysis

Quantitative data are expressed as means ± SE and were analyzed with the unpaired Student’s t-test. P < 0.05 was considered statistically significant. 

Results

Localization of Connexin43 in Bovine Anterior Chamber Angle

We first investigated whether connexin43 is expressed in the anterior chamber angle of the bovine eye. Indirect immunofluorescence microscopy revealed a punctate pattern of specific connexin43 immunoreactivity in the TM region (Figs. 1A 1B 1C 1D) . No fluorescence was observed when the primary antibody was replaced with control ascites fluid (Figs. 1E 1F) . As a positive control, we examined the cornea. Connexin43-specific fluorescence was detected in the basal cell layer of the cornea (Figs. 1G 1H) , consistent with previous observations. ²⁶ These results thus demonstrated the expression of connexin43 in the TM as well as in the basal layer of the cornea in the bovine eye. 

Effects of PMA on Connexin43 Expression and Dye Coupling in Cultured TM Cells

We next determined whether cultured bovine TM cells express connexin43 and communicate with each other through gap junctions. Indirect immunofluorescence microscopy revealed a punctate pattern of specific connexin43 fluorescence at sites of contact between adjacent cells (Fig. 2A ). No fluorescence was observed when the monoclonal antibody to connexin43 was replaced by control ascites fluid (data not shown). Double labeling of cells with rhodamine-conjugated phalloidin to detect actin filaments revealed no direct association of connexin43 with these filaments (Fig. 2A) . Microinjection of lucifer yellow into individual TM cells revealed that the fluorescent dye was transferred to neighboring cells (Figs. 3A 3B ). 

The effect of PMA on the expression of connexin43 in cultured bovine TM cells was also investigated by double-label immunofluorescence analysis with the monoclonal antibody to connexin43 and rhodamine-phalloidin. Whereas incubation of cells with 10 nM PMA for 1 hour had no effect on the pattern of connexin43 immunofluorescence (Fig. 2B) , treatment of cells with this agent for 6 hours resulted in a marked decrease in connexin43 immunoreactivity (Fig. 2C) . In contrast, incubation of cells for 6 hours with 10 nM 4α-PMA, which does not activate PKC, had no substantial effect on the pattern of connexin43 staining (Fig. 2D) . Exposure of cells to PMA or 4a-PMA for 1 or 6 hours had no consistent marked effect on cell shape as revealed by the pattern of actin staining. 

Fluorescence microscopy also revealed that incubation of cultured bovine TM cells with PMA for 1 hour resulted in a concentration-dependent decrease in the number of dye-coupled cells (Figs. 3C 3D 3E 3F 3G 3H 3I 3J) ; at the highest concentration of PMA (200 nM) examined, the number of cells coupled to the injected cell was reduced to almost zero (Figs. 3I and 3J) . The effect of PMA on gap junctional intercellular communication was quantitated by counting the number of dye-coupled cells. The PMA-induced decrease in the number of coupled cells was statistically significant at concentrations of 10, 100, and 200 nM (Fig. 4) . Exposure of cells to 200 nM 4α-PMA for 1 hour had no significant effect on the number of dye-coupled cells (Fig. 4) . Examination of the time course of inhibition of dye coupling by PMA (10 nM) revealed that the effect was significant as early as 30 minutes after exposure to the drug; although the extent of coupling had increased somewhat by 6 hours, it was still significantly reduced compared with that apparent in control cells (Fig. 5) . 

We further investigated the role of PKC in the PMA-induced inhibition of gap junction–mediated intercellular communication in cultured TM cells by examining the effect of the PKC inhibitor bisindolylmaleimide I. This agent reversed the inhibitory effect of PMA (10 nM) on dye coupling in a concentration-dependent manner (Fig. 6) . Bisindolylmaleimide I by itself had no effect on dye coupling. 

Together, these results thus demonstrated that cultured bovine TM cells expressed connexin43 and that they communicated with each other through gap junctions. Furthermore, PMA, apparently acting through PKC, reduced the extent of gap junction–mediated communication between cells. 

Immunoblot Analysis of Connexin43 in Bovine TM Cells

We next investigated the effects of PMA on the phosphorylation of connexin43 in cultured bovine TM cells. Cells were incubated in the absence or presence of 10 nM PMA for 1 hour, lysed, and subjected to immunoblot analysis with the monoclonal antibody to connexin43. Cells incubated in the absence of PMA yielded four immunoreactive bands, corresponding to molecular masses of 43, 47, 48, and 49 kDa; cells incubated in the presence of PMA yielded the same four bands plus an additional band at 44 kDa (Fig. 7) . On the basis of their molecular sizes, these bands appeared to correspond to nonphosphorylated connexin43 (Cx43-NP, 43 kDa) and four types of phosphorylated connexin43 (Cx43-P₁ at 47 kDa, Cx43-P₂ at 48 kDa, Cx43-P₃ at 49 kDa, and Cx43-P′ at 44 kDa). ²⁷ To confirm which bands corresponded to phosphorylated connexin43, we incubated cell lysates with alkaline phosphatase (2.5 IU/ml) for 1 hour before immunoblot analysis. The bands presumably corresponding to Cx43-P₁, Cx43-P₂, Cx-P₃, and Cx43-P′ disappeared, and the intensity of the band corresponding to Cx43-NP increased. Furthermore, inclusion of the alkaline phosphatase inhibitor Na₃VO₄ in the incubations with alkaline phosphatase prevented the effects of the latter on the pattern of connexin43 staining. These results show that the band at 43 kDa corresponded to nonphosphorylated connexin43; that the four bands at 44, 47, 48, and 49 kDa corresponded to phosphorylated forms of connexin43; and that exposure of cells to PMA resulted in the appearance of the band at 44 kDa. 

Finally, we examined the time course of the effects of PMA on the phosphorylation of connexin43. Densitometric analysis of immunoblots for cells incubated with 10 nM PMA revealed that the intensity of the Cx43-NP band was decreased at 1 hour but had returned to control values by 6 hours, the intensity of the Cx43-P′ band was increased at both 1 and 6 hours, the intensity of the Cx43-P₁ band was increased at 1 hour but had returned to control values by 6 hours, the intensity of the Cx43-P₂ band was decreased at both 1 and 6 hours, and the intensity of the Cx43-P₃ band was unaffected at 1 hour but decreased at 6 hours (Fig. 8) . Similar treatment of cells with 10 nM 4α-PMA had no marked effects on the intensity of the various connexin43 bands at either 1 or 6 hours. These results showed that exposure of cells to 10 nM PMA for 1 hour increased the relative amounts of the Cx43-P′ and Cx43-P₁ phosphorylated forms of connexin43. 

Discussion

We have shown that connexin43 is expressed in the anterior chamber angle of the bovine eye as well as in cultured bovine TM cells at the junctional regions of neighboring cells. Furthermore, our results demonstrate that cultured TM cells are coupled by gap junctions and that PMA inhibits intercellular communication by these junctions, presumably by inducing the activation of PKC and the subsequent phosphorylation of connexin43. Such a junctional localization of connexins suggests that the attached cells communicate with each other by exchanging small molecules and that their activities are coupled. ^{2 3 4} Our data thus indicate that TM cells coupled with their neighbors by gap junctions may function as one unit. 

PKC is an important regulator of cellular functions in response to extracellular stimuli. PMA has previously been shown to downregulate gap junctional intercellular communication in various cell types. ^{19 20 21} In the present study, incubation of cultured bovine TM cells with PMA for 1 hour reduced the extent of dye coupling among cells in a dose-dependent manner. Exposure of the cells to 10 nM PMA for 1 hour thus significantly inhibited gap junctional intercellular communication. Although such treatment had no apparent effect on the pattern of connexin43 immunoreactivity revealed by indirect immunofluorescence microscopy, it induced an apparent increase in the phosphorylation of this protein. The inhibitory effect of PMA on dye coupling was prevented by the PKC inhibitor bisindolylmaleimide I, indicating that it was indeed mediated through activation of PKC and probably, at least in part, through the consequent phosphorylation of connexin43. 

Immunoblot analysis with a monoclonal antibody to connexin43 revealed the presence of various immunoreactive proteins in cultured bovine TM cells. On the basis of their mobility and the effects of alkaline phosphatase, these protein bands were assumed to correspond to a nonphosphorylated form and various phosphorylated forms of connexin43. These results are consistent with those of previous studies showing that monoclonal antibodies to connexin43 recognize various forms of the protein that differ in apparent molecular size. ^{19 20 21 27 28 29} Incubation of cultured bovine TM cells with PMA (10 nM) for 1 hour induced a decrease in the amount of nonphosphorylated connexin43 and increases in the amounts of the phosphorylated forms Cx43-P′ and Cx43-P₁. PMA-induced phosphorylation of connexin43 mediated by PKC (directly or indirectly) may thus underlie the inhibitory effect of PMA on gap junctional intercellular communication in these cells. 

The amounts of Cx43-P′ and Cx43-P₁ in cultured bovine TM cells exposed to PMA (10 nM) for 6 hours appeared reduced compared with those detected at 1 hour, consistent with the recovery of dye coupling observed in cells incubated with this drug for 6 hours. The inhibition of gap junctional intercellular communication by PMA in other cell types is also transient, with recovery apparent within 4 to 6 hours. ^{21 29} These observations may be attributable to the downregulation of PKC induced by prolonged exposure to PMA that has been described in other cell types. ^{30 31 32} However, the recovery of dye coupling in cultured bovine TM cells exposed to PMA (10 nM) for 6 hours appears inconsistent with the apparent downregulation of connexin43 expression revealed at this time by immunofluorescence staining. Further investigations are required to resolve this apparent inconsistency. 

 Supported in part by a Grant-in-Aid 11771057 for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture of Japan (TS).

 Submitted for publication June 16, 1999; revised February 2, 2000; accepted February 15, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Immunofluorescence microscopic analysis of the expression of connexin43 in the anterior chamber angle and cornea of the bovine eye. Bright-field micrographs (A, C) and immunofluorescence micrographs (B, D) show the expression of connexin43 in the anterior chamber angle of a bovine eye at low magnification (A, B) and high magnification (C, D). (E) Bright-field and (F) immunofluorescence micrographs for control staining of the anterior chamber angle with nonimmune ascites fluid in place of the monoclonal antibody to connexin43. (G) Bright-field and (H) immunofluorescence micrographs show the expression of connexin43 in the cornea. TM, trabecular meshwork; AP, aqueous plexus; AC, anterior chamber; S, sclera; CE, corneal epithelium; CS, corneal stroma. Bars, 100 μm.

Figure 2.

 

Effect of PMA on the pattern of connexin43 immunofluorescence in cultured bovine TM cells. Cells were incubated in the absence (A) or presence of 10 nM PMA for 1 hour (B) or 6 hours (C) or 10 nM 4α-PMA for 6 hours (D), after which connexin43 was detected by indirect immunofluorescence staining with a monoclonal antibody to connexin43, and actin filaments were visualized with rhodamine-phalloidin. Bar, 50 μm.

Figure 3.

 

Concentration-dependent effect of PMA on dye coupling in cultured bovine TM cells. Cells were incubated for 1 hour in the absence (A, B) or presence of PMA at concentrations of 1 nM (C, D), 10 nM (E, F), 100 nM (G, H), or 200 nM (I, J). An individual cell (∗) was then microinjected with lucifer yellow, and, after incubation for 1 minute, the cells were washed and examined under a fluorescence microscope (B, D, F, H, and J) as well as under phase-contrast optics (A, C, E, G, and I). Bars, 100 μm.

Figure 4.

 

Quantitation of the inhibitory effect of PMA on dye coupling in cultured bovine TM cells. Cells were incubated for 1 hour in the absence (control) or presence of the indicated concentrations of PMA or 4α-PMA. A single cell was then microinjected with lucifer yellow, and the dye was allowed to spread for 1 minute. The extent of gap-junctional intercellular communication (GJIC) was assessed by counting the number of coupled cells. Data are means ± SE (n = 30). *P < 0.0001 versus control.

Figure 5.

 

Time course of the inhibitory effect of PMA on gap junction–mediated intercellular communication in cultured bovine TM cells. Cells were incubated in the presence of 10 nM PMA for the indicated times, after which an individual cell was microinjected with lucifer yellow, and the dye was allowed to spread for 1 minute. Data are means ± SE (n = 16). *P = 0.001,** P < 0.0001 versus zero time.

Figure 6.

 

Effect of bisindolylmaleimide I on PMA-induced inhibition of gap-junctional intercellular communication in cultured bovine TM cells. Cells were incubated first for 1 hour with the indicated concentrations of bisindolylmaleimide I and then for an additional 1 hour in the absence or presence of 10 nM PMA in the continued presence of the respective concentration of bisindolylmaleimide I. A single cell was microinjected with lucifer yellow, and the dye was allowed to spread for 1 minute. Data are means ± SE for the indicated number (n) of injected cells. *P < 0.005,** P < 0.0001.

Figure 7.

 

Immunoblot analysis of the effects of PMA on the phosphorylation of connexin43 in cultured bovine TM cells. Cells were incubated for 1 hour in the absence or presence of 10 nM PMA, after which cell lysates were prepared and incubated for 1 hour in the absence or presence of alkaline phosphatase (2.5 IU/ml; AP) or 10 mM Na₃VO₄, as indicated. Protein samples were then subjected to immunoblot analysis with a monoclonal antibody to connexin43. The positions of a 46.5-kDa molecular size marker and of nonphosphorylated and various phosphorylated forms of connexin43 are indicated.

Figure 8.

 

Time course for the effects of PMA and 4α-PMA on connexin43 phosphorylation in cultured bovine TM cells. (A) Cells were incubated for the indicated times with 10 nM PMA or 10 nM 4α-PMA, after which cell lysates were prepared and subjected to immunoblot analysis with a monoclonal antibody to connexin43. The position of a 46.5-kDa molecular size standard is shown. (B) Immunoblots similar to that shown in (A) were subjected to densitometric analysis for determination of the intensity of the bands corresponding to the indicated nonphosphorylated and phosphorylated forms of connexin43. Data are expressed in absorbance units and are means ± SE of three different experiments.