Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell–cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with 32Pi yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43−/− mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of ∼50-pS channel events and a concomitant loss of ∼100-pS channel events. This change to ∼50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

Establishment of a functional vascular network is rate-limiting in embryonic development, tissue repair and engineering. During blood vessel formation, newly generated endothelial cells rapidly expand into primitive plexi that undergo vascular remodeling into circulatory networks, requiring coordinated growth inhibition and arterial-venous specification. Whether the mechanisms controlling endothelial cell cycle arrest and acquisition of specialized phenotypes are interdependent is unknown. Here we demonstrate that fluid shear stress, at arterial flow magnitudes, maximally activates NOTCH signaling, which upregulates GJA4 (commonly, Cx37) and downstream cell cycle inhibitor CDKN1B (p27). Blockade of any of these steps causes hyperproliferation and loss of arterial specification. Re-expression of GJA4 or CDKN1B, or chemical cell cycle inhibition, restores endothelial growth control and arterial gene expression. Thus, we elucidate a mechanochemical pathway in which arterial shear activates a NOTCH-GJA4-CDKN1B axis that promotes endothelial cell cycle arrest to enable arterial gene expression. These insights will guide vascular regeneration and engineering.

Abstract-Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55-to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia. Key Words: gap junction Ⅲ connexin 43 Ⅲ phosphorylation Ⅲ selectivity Ⅲ ischemia G ap junctions are clusters of intercellular channels that mediate electrical and chemical signaling throughout the cardiovascular system. 1,2 Gap junction channels are formed when hemichannels (connexons) in the membranes of neighboring cells dock. Each hemichannel is a hexamer of connexin subunits; in cells of the cardiovascular system, 4 members of the connexin gene family are commonly expressed: Cx45, Cx43, Cx40, and Cx37. The predominant connexin expressed in ventricular cells is Cx43, the focus of the current study. In the normally functioning ventricle, Cx43 is localized to intercalated disks where it supports the longitudinal and transverse (zigzag) spread of the action potential, such that coordinated contractile activation of the heart occurs. The contractile failure and arrhythmias occurring during ischemia reflect, in addition to compromised metabolism, altered excitability, and reduced electrical coupling. 3 Exposure of the heart to a brief period of ischemia and reperfusion (termed ischemic preconditioning) before a prolonged ischemic period protects the heart against necrosis and fatal arrhythmias. 4 During the ischemic preconditioning period, receptor-mediated activation of protein kinase C (PKC) occurs and appears to be necessary for protection against injury during the subsequent prolonged ischemic pe...

Activation of the Ras

The functional consequences of Connexin43 (Cx43) phosphorylation remain largely unexplored. Using an antibody that specifically recognizes Cx43 phosphorylated at serine residues 325, 328 and/or 330 (pS325/328/330-Cx43), we show that labeling of this form of Cx43 as well as of total Cx43 is restricted to the intercalated disk region of normal ventricular tissue. In ischemic heart, significant relocalization of total Cx43 to the lateral edges of myocytes was evident; however pS325/328/330-Cx43 remained predominately at the intercalated disk. Western blots indicated a eightfold decrease in pS325/328/330-Cx43 in ischemic tissue. Peptide-binding- and competition-experiments indicated that our antibody mainly detected Cx43 phosphorylated at S328 and/or S330 in heart tissue. To evaluate how this change in Cx43 phosphorylation contributes to ischemia-induced downregulation of intercellular communication, we stably transfected Cx43-/- cells with a Cx43 construct in which serine residues 325, 328 and 330 had been mutated to alanine (Cx43-TM). Cx43-TM was not efficiently processed to isoforms that have been correlated with gap junction assembly. Nevertheless, Cx43-TM cells were electrically coupled, although development of coupling was delayed. Fully opened channels were only rarely observed in Cx43-TM cells, and Lucifer-Yellow-dye-coupling was significantly reduced compared with wild-type cells. These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly.

ABSTRACTbility arises mainly from the limitations of the discrete data model and from the polygon-based mapping prac- A geographical information system (GIS) or expert knowledge-tice employed in conventional soil surveys.based fuzzy soil inference scheme (soil-land inference model, SoLIM) is described. The scheme consists of three major components: (i) a Zhu (1997a,b), Zhu and Band (1994), Zhu et al.model employing a similarity representation of soils, (ii) a set of Zhu et al. (1997) survey approach are first discussed to provide a context for the SoLIM, which is followed by an overview of the SoLIM. The assessment of the SoLIM for soil survey D etailed soil spatial and attribute information is through two case studies is described in the third part required for many environmental modeling and of this paper. land management applications

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric͞homotypic channels with distinct permeability and gating properties but do not form functional homomeric͞heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric͞homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.Gap junction channels connect the cytoplasms of adjacent cells and provide a pathway for intercellular diffusion of ions, second messenger molecules, and small metabolites. Functional gap junction channels are formed by connexins (Cxs), a gene family with at least 14 mammalian members that are distinguished from one another by their predicted molecular weights expressed in kilodaltons (e.g., Cx43, the 43-kDa connexin). Cxs oligomerize to form connexons (hemichannels), which are defined as homomeric when the six comprising Cxs are identical or heteromeric when two or more Cxs comprise the connexon. Connexons in adjacent cells join in the extracellular space to form the functional intercellular channel, which is defined as homotypic when the Cx composition of the contributing connexons is identical or heterotypic when different.The ability of Cxs to form homomeric͞heterotypic channels has been examined in the Xenopus oocyte and HeLa cell expression systems as well as in other settings (for review, see ref. 1). Homomeric Cx43 connexons successfully dock with homomeric connexons comprised of Cxs 30.3, 37, and 45 but not with Cxs 50, 40,33,32, 31.3,31,or 26. Homomeric Cx40 connexons successfully dock with and form functional homomeric͞heterotypic channels with Cxs 37 and 45 but not with Cxs 50, 46, 43,32, 31.1,31,or 26. Clearly, there are far more incompatible than compatible combinations.The capacity of Cxs to form functional heteromeric connexons and channels has only recently received attention. Biochemical and structural tools have demonstrated the existence of heteromeric connexons comprised of Cxs 46 and 50 (2) and Cxs 32 and 26 (3). That these Cx pairs form functional hetero...

Abstract-During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-␤ (TGF-␤) activation. We investigated whether gap junction channels are formed between endothelial cells and recruited mesenchymal progenitors and whether intercellular communication is necessary for endothelial-induced mural cell differentiation. Key Words: blood vessel formation Ⅲ gap junctions Ⅲ endothelial cells Ⅲ mural cell differentiation Ⅲ transforming growth factor-␤ E mbryonic blood vessel formation begins with the coalescence of mesodermal progenitors and their differentiation into endothelial and blood cells, forming blood islands 1 that fuse and define a primitive circulatory plexus. 2 During branching and remodeling of the initial plexus that leads to a well-defined vascular network, 3 endothelial tubes acquire a surrounding vessel wall of mural cells (pericytes or smooth muscle cells) via the secretion of platelet-derived growth factor-B that acts as a chemoattractant and mitogen for mural cell precursors. 4 -6 On contact with endothelial cells, the recruited progenitors are induced toward a mural cell fate, 7 in a process mediated by the activation of transforming growth factor-beta (TGF-␤).Although TGF-␤ activation is not completely understood, 8,9 it is clear that activated TGF-␤ plays a critical role in the induction of mural cell differentiation. Via TGF-␤ control elements 10 or upregulation of serum response factor (SRF), 11 TGF-␤ promotes coordinated transcriptional activation of cytoskeletal and contractile genes, including smooth muscle (SM)-␣-actin, SM-␥-actin, SM22␣, calponin, and SM-myosin heavy chain, needed for differentiated function. 12 Mural cells not only modulate blood flow but also stabilize vessels 6,13 and may sustain endothelial survival through the production of Ang-1 14 or VEGF-A. 15 The exact interactions between endothelial and mesenchymal cells during vessel assembly that lead to TGF-␤ activation and mural cell differentiation are not well defined but are presumably complex given the number of genes implicated by mutation studies to regulate these processes during vascular development. Our studies aim to elucidate the role of one component of endothelial-mesenchymal cell interactions in vessel assembly-heterocellular communication via gap junctions.Gap junctions are aggregates of intercellular channels 16 that connect the cytoplasms of adjoining cells and allow the passage of second messengers, ions, and metabolites. 17 Gap junction channels are composed of connexin (Cx) proteins, of which there are at least 20. 18,19 The presence of gap junctions has been documented among and between vascular cells. 20 -22 Conflicting reports of their exact Cx composition suggest that Cx distribution varies among vascular beds and vessel types and is differentially regulated in response to injury and growth.In adults, quiescent endothelial cells predominantly express Cx37 and Cx40 [23][24][25][26][2...