Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined the effect of protein kinase (PKC) depletion in SV40-transformed Djungarian hamster fibroblasts (DM15 cells) on the level of gap junction permeability. Cx43 electrophoretic mobility, and cell sensitivity to different uncoupling stimuli. After 24 hr exposure to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the total PKC activity in DM15 cells was reduced to 20-25% in comparison with intact cells. In PKC-depleted cells the level of dye coupling was 30-40% higher than in the same untreated cultures. Western blot analysis revealed multiple forms of the gap junction protein connexin 43, which correspond to known phosphorylated and dephosphorylated forms of this protein. No decrease in the level of connexin 43 phosphorylation after PKC depletion was observed. TPA (10(-7) g/ml), mezerein (10(-7) g/ml), teleocidin (10(-8) g/ml), Ca-ionophore A23187 (10(-6) g/ml), insecticide 1,1,1-trichloro-2,2-bis-(p-chlorphenyl)-ethane (DDT) (10(-4) g/ml), and nigericin (10(-5) M in hydrolysate lactalbumin solution, pH 6.3) induced a four-to six-fold decrease in the number of recipient cells in the dye-coupling assay. PKC-depleted cells became almost completely resistant to the uncoupling effect of mezerein, teleocidin, and A23187, as well as to new exposure to TPA, and became partially resistant to the effect of DDT. Nigericin inhibited intercellular communication between PKC-depleted cells to the same extent as between control cells. Thus, in the cell system studied, PKC plays a certain role in maintaining the basal level of gap junction permeability and has an important significance as a mediator of the uncoupling effects of such substances as TPA, mezerein, teleocidin, and Ca2+.
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PMID:Role of protein kinase C in the regulation of gap junctional communication. 770 63

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.
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PMID:Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells. 814 55

In the Lewis rat model of experimental autoimmune thyroiditis (EAT), decreased immunodetectable connexin assembly into gap junctions and diminished intercellular communication are associated with the loss of thyroid function (hypothyroidism) that occurs prior to significant tissue destruction. The current study explores the hypothesis that the loss of connexin 43 (Cx43)-mediated intercellular communication in these cells is caused by upregulation of protein kinase C (pKC) activity. Thyrocytes isolated from EAT rats exhibited a 78% increase in basal pKC activity; whereas, basal protein kinase A (pKA) activity was unchanged. Increased pKC activity was a result of increased isozyme protein levels. Thyroid cells expressed pKC isozymes gamma and lambda and had elevated levels of alpha (40%), beta (30%), delta (31%), and epsilon (25%) as quantified by western blot analyses. Furthermore, modulation of pKC activity inversely altered Cx43 assembly and function in monolayer thyrocytes. For example, octoacetyl glycerol (OAG) treatment of normal thyrocyte monolayers to increase pKC activity resulted in deficient Cx43 gap junction assembly and reduced intercellular communication indistinguishable from the deficits in EAT thyrocytes. Conversely, calphostin C inhibition of pKC activity in EAT thyrocyte monolayers restored these parameters to normal. Thus, pharmacological modulations of pKC activity in cultured thyrocytes support a causal relation between the changes in pKC activity and Cx43-mediated intercellular communication. Abnormalities in autoimmune diseased thyroid tissue (eg, increased pKC) appear to contribute to reduced intercellular coordination of thyroid follicles and thereby can affect subsequent thyroid function. The persistence of target cell abnormalities in the absence of infiltrating lymphocytes and their products supports an alternative mechanism by which thyroid function can be affected that does not depend on the loss of thyroid glandular epithelium.
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PMID:Elevation of protein kinase C in thyrocytes isolated from a Lewis rat model of autoimmune thyroiditis prevents assembly of immunodetectable connexin43 gap junctions and reduces intercellular communication. 945 38

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.
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PMID:Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions. 1002 40

The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca(2+) ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53-66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.
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PMID:Identification of second messengers that induce expression of functional gap junctions in microglia cultured from newborn rats. 1210 Oct 41

Hepatocytes and other digestive epithelia exchange second messengers and coordinate their functions by communicating through gap junctions. However, little is known about intercellular communication in cholangiocytes. The aim of this study was to examine expression and regulation of gap junctions in cholangiocytes. Connexin expression was determined by confocal immunofluorescence in rat bile ducts and in normal rat cholangiocyte (NRC) cells, a polarized cholangiocyte cell line. Intercellular Ca(2+) signaling was monitored by fluorescent microscopy. Microinjection studies assessed regulation of gap junction permeability in NRC cells and in SKHep1 cells, a liver-derived cell line engineered to express connexin 43. Immunochemistry showed that cholangiocytes from normal rat liver as well as the NRC cells express connexin 43. Localization of apical, basolateral, and tight junction proteins confirmed that NRC cells are well polarized. Apical exposure to ATP induced Ca(2+) oscillations that were coordinated among neighboring NRC cells, and inhibition of gap junction conductance desynchronized the Ca(2+) oscillations. NRC cells transfected with a connexin 43 antisense were significantly less coupled. Transcellular dye spreading was inhibited by activation of protein kinase A or protein kinase C. The same was observed in transfected SKHep1 cells, which expressed only connexin 43. Rat cholangiocytes and NRC cells express connexin 43, which permits synchronization of Ca(2+) signals among cells. Permeability of connexin 43-gap junctions is negatively regulated by protein kinases A and C. In conclusion, cholangiocytes have the capacity for intercellular communication of second messenger signals via gap junctions in a fashion that is under hormonal control.
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PMID:Expression and regulation of gap junctions in rat cholangiocytes. 1219 55

Gap junction channels provide the basis for the electrical syncytial properties of the heart as a communicating electrical network. Cardiac gap junction channels are predominantly composed of connexin 40 or connexin 43. The conductance of these channels (g(j)) can be regulated pharmacologically: substances which activate protein kinase C, protein kinase A or protein kinase G may alter Cx43 gap junction conductance. However, for PKC, this seems to be subtype specific. Thus, antiarrhythmic peptides can enhance g(j) via activation of PKCepsilon, while FGF-2 reduces g(j) via PKCepsilon. Lipophilic drugs can uncouple the channels. Besides an acute regulation of g(j), the expression of the cardiac connexins can also be regulated. A decrease in Cx43 with a concomitant increase in Cx40 has been found in end-stage failing hearts, while in renovascular hypertension, an increase in Cx43 has been described. Mediators like endothelin-1, angiotensin-II, TGF-beta, VEGF, and cAMP have been shown to increase Cx43. Interestingly, endothelin-1 and angiotensin-II increased Cx43 but did not affect Cx40 expression. In contrast, in humans suffering from atrial fibrillation (AF), the content in Cx40 can be enhanced while Cx43 was unaltered, although in several other studies, other changes of the cardiac connexins were found, which might be related to the type of AF. Regarding the role of calcium, the content in both Cx40 and Cx43 was decreased in cultured neonatal rat cardiomyocytes after 24 h administration of 100 nM verapamil. Thus, gap junctional channels can be affected pharmacologically either acutely by modulating gap junction conductance or chronically by altering gap junction protein expression. Interestingly, it appears that the expression of Cx43 and Cx40 can be differentially regulated.
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PMID:Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40. 1256 16

Long-term modulation of intercellular communication via gap junctions was investigated in TM3 Leydig cells, under low and high confluence states, and upon treatment of the cells for different times with activators of protein kinase A (PKA) and protein kinase C (PKC). Cells in low confluence were readily coupled, as determined by transfer of the dye Lucifer Yellow; on reaching confluence, the cells uncoupled. Western blots and RT-PCR revealed that connexin 43 (Cx43) was abundantly expressed in TM3 Leydig cells and its expression was decreased after the cells achieved confluence. Stimulation of PKA or PKC induced a decrease in cell-cell communication. Staurosporin, an inhibitor of protein kinases, increased coupling and was able to prevent and reverse the uncoupling actions of dibutyryl cAMP and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Under modulation by confluence, Cx43 was localized to the appositional membranes when cells were coupled and was mainly in the cytoplasm when they were uncoupled. In addition, cAMP and TPA reduced the surface membrane labeling for Cx43, whereas staurosporin increased it. These data show a strong correlation between functional coupling and the membrane distribution of Cx43, implying that this connexin has an important role in intercellular communication between TM3 cells. Furthermore, increased testosterone secretion in response to luteinizing hormone was accompanied by a decrease in intercellular communication, suggesting that gap junction mediated coupling may be a modulator of hormone secretion in TM3 cells.
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PMID:Modulation of gap junction mediated intercellular communication in TM3 Leydig cells. 1274 21

Gap junction channels are essential for intercellular communication. Among the most abundant gap junction channel proteins is connexin 43 (Cx43). The goal of our study was to find out, whether Cx43 content may be regulated via adenylyl cyclase (AC)/cAMP/protein kinase A (PKA), protein kinase C (PKC) pathways or by a tyrosine kinase coupled pathway, i.e. TNF alpha-receptor dependent pathway. Therefore, we used HeLa cells transfected with Cx43 and exposed these cells for 24 h to either db-cAMP (10(-4)M), forskolin (10(-5)M), the phorbolester phorbol-12,13-didecanoate PDD (10(-7)M) (or its inactive form 4 alpha-PDD), TNF alpha (10 U/ml) with or without additional treatment with the MAP kinase inhibitors SB203580 (10(-5) M, p38 MAP-kinase inhibitor) or the MEK1-inhibitor PD98059 (10(-5)M). Cx43 content was analysed using Western blot analysis. All results were confirmed by a second series of identical experiments using Cx43 immunohistochemistry. We found significantly enhanced Cx43 content in cells treated with db-cAMP, forskolin, PDD or TNF alpha (p<0.05), while 4 alpha-PDD or the solvent DMSO exerted no effect. These increases in Cx43 content could be completely suppressed by SB203580 (p<0.05) but not by PD98059. In absence of a stimulating drug, these inhibitors (SB203580 or PD98059) did not affect Cx43 content. Additional PCR experiments revealed increases in Cx43-mRNA under the influence of db-cAMP, forskolin, PDD or TNFalpha (p<0.05), which all could be completely suppressed by SB203580. From these results we conclude that 1.Cx43 content can be regulated via AC/cAMP/PKA, PKC and TNF alpha-receptor-dependent pathways 2. Activation of p38 MAP kinase is a common pathway for regulation of Cx43 content in HeLa cells
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PMID:Chronic regulation of the expression of the gap junction protein connexin 43 in transfected HeLa cells. 1282 13

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.
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PMID:Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor. 1293 79


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