Eileen M. Westphale

Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage ( G j/ V j) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The G j/ V jdata imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage (Gj/Vj) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The Gj/Vj data imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

SummaryGap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels

Characterization of the gap junction protein, connexin45

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hepl, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285–298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [35S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [32P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.

Expression of Multiple Gap Junction Proteins in Human Fetal and Infant Hearts

ABSTRACT: Mammalian cardiac myocytes express multiple gap junction channel proteins or connexins. Expression patterns of the avian homologues of the mammalian cardiac connexins change during cardiac morphogenesis in association with changes in the electrophysiologic properties of intercellular junctions in chick cardiac myocytes. To determine whether expression of cardiac connexins is developmentally regulated in humans, we characterized connexin mRNA and protein content and distribution in hearts of 11 human fetuses (74 to 122 d gestational age), seven children (0.5 mo to 3 y of age), and two adults. Northern blot analysis identified transcripts of connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45) genes in all hearts analyzed. Cx40 mRNA was approximately 5-fold more abundant in samples from fetal hearts than in hearts of children or adults. However, fetal samples used for RNA extraction included atrial as well as ventricular myocardium, whereas samples from children and adults were exclusively ventricular. Northern analysis of adult human right atrial appendages revealed abundant Cx40 mRNA, thus suggesting that the greater amount of Cx40 signal seen on Northern blots from fetal hearts could have been attributable to atrial contributions. Neither Cx43 nor Cx45 mRNA varied significantly in amount in samples from the different developmental stages analyzed. Immunofluorescence identified abundant Cx43 in the known distribution of gap junctions in myocytes in sections of all hearts. Cx45 staining was inconspicuous in fetal hearts but was readily apparent in cardiac myocytes in hearts of older subjects. In contrast, Cx40 staining in the ventricle was confined to mural coronary arteries, apparently in endothelial cells, whereas in the atrium Cx40 staining at myocyte junctions was abundant. Junctions in hypertrophied myocardium of children with congenital or acquired heart disease appeared more prominent and complex than in normal tissues. Thus, the human heart contains multiple connexins, but their distribution and developmental patterns differ from each other and from those in other species. Cx45 expression is not restricted to the fetal human heart. Cx40 expression in ventricles occurs selectively in coronary arteries.

Dexamethasone used as an antiemetic in chemotherapy protocols inhibits natural cytotoxic (NC) cell activity

Because dexamethasone is often included as an antiemetic in chemotherapy protocols that involve cisplatin and because cisplatin has been shown to increase the in vitro lysis of tumor cells by natural cytotoxic (NC) effector cells, we determined the NC activity of 27 patients who received dexamethasone in conjunction with seven different cisplatin‐based chemotherapy protocols. The results of this analysis showed that the NC activity of patients who received cisplatin‐based chemotherapy protocols that included dexamethasone was reduced significantly 24 hours after treatment compared with before treatment (P < 0.001). The addition of dexamethasone (at a concentration equivalent to the plasma level of patients treated with dexamethasone) to the in vitro assay of NC activity caused a significant decrease in NC activity compared with when dexamethasone was not added (P < 0.001). There was no cumulative effect of dexamethasone in that the reduction of NC activity by dexamethasone was not significantly different in patients who had been treated previously at least four times and in patients who were treated for the first time. When dexamethasone was not included in the chemotherapy protocol the NC activity of 19 patients was not reduced 24 hours after treatment. These results indicate that dexamethasone causes a significant reduction in NC activity. Although the tumor surveillance role of human NC cells in vivo has not been established, the effect of dexamethasone on NC cells suggests that additional research of the effect of dexamethasone in cisplatin‐based chemotherapy protocols is warranted.

Regulation of Lens Gap Junctions

Gap junctions are membrane specializations that contain groups of intercellular channels. These channels are permeable to ions and small molecules up to 1,000 Da. They communicate the cytoplasm of adjacent cells, thereby providing a direct pathway for the passage of ions, nutrients, metabolites and second messengers between coupled cells. Gap junctional channels are made of oligomeric assemblies of members of a family of related proteins called connexins (Cx). These proteins cross the plasma membrane four times leaving the amino and carboxyl termini on the cytoplasmic side (reviewed by Beyer)1.