Effect of charge substitutions at residue his-142 on voltage gating of connexin43 channels

Previous studies indicate that the carboxyl terminal of connexin43 (Cx43CT) is involved in fast transjunctional voltage gating. Separate studies support the notion of an intramolecular association between Cx43CT and a region of the cytoplasmic loop (amino acids 119–144; referred to as “L2”). Structural analysis of L2 shows two α-helical domains, each with a histidine residue in its sequence (H126 and H142). Here, we determined the effect of H142 replacement by lysine, alanine, and glutamate on the voltage gating of Cx43 channels. Mutation H142E led to a significant reduction in the frequency of occurrence of the residual state and a prolongation of dwell open time. Macroscopically, there was a large reduction in the fast component of voltage gating. These results resembled those observed for a mutant lacking the carboxyl terminal (CT) domain. NMR experiments showed that mutation H142E significantly decreased the Cx43CT-L2 interaction and disrupted the secondary structure of L2. Overall, our data support the hypothesis that fast voltage gating involves an intramolecular particle-receptor interaction between CT and L2. Some of the structural constrains of fast voltage gating may be shared with those involved in the chemical gating of Cx43.     

Immunocytochemical localization of ornithine decarboxylase in cultured murine cells

Summary Antiserum elicited to ornithine decarboxylase (ODC) purified from murine RAW 264 macrophage-like cells has been employed to localize ODC in cultured murine cells. The antiserum immunoprecipitated 100% of the ODC activity from the cultured cells. The specificity of the antiserum was demonstrated by the immunoprecipitation from ³⁵S-methionine metabolically-labeled cell extracts of a single protein which migrated upon SDS-gel electrophoresis coincident with authentic ODC. Indirect immunofluorescence experiments were performed on paraformaldehyde-fixed RAW 264 cells and JB6 epidermal cells using the rabbit anti-ODC antiserum and FITC-conjugated goat anti-rabbit IgG. Little immunofluorescence was apparent in non-stimulated cells. Intense immunofluorescence was detectable in stimulated cells at times of peak cellular ODC activity. Antigenically-reactive ODC was localized diffusely in the cytoplasm and was absent in the nuclei of RAW 264 cells, whereas in the JB6 cells the immunodetectable enzyme protein was localized in a punctate pattern in both the cytoplasm and nucleoplasm and was absent in the nucleolus. The appearance and disappearance of immunoreactive ODC in both cell types after stimulation was consistent with the alterations in ODC activity.     

Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester.

Nuclear factor kappa-B (NF-kappa B) has been shown to play an important role in LPS-mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP-dependent protein kinase in the regulation of NF-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B element was transfected into the RAW264 macrophage-like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8-bromo cAMP. To further study NF-kappa B induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF-kappa B binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA-binding activity confirmed these results and indicated that LPS induced a 55 KD DNA-binding protein. Induction of this NF-kappa B binding activity was not inhibited by pretreatment with the PKC inhibitor H-7. H-7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down-regulates PKC, had no effect on LPS induction of NF-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC.     

Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.     

Kinetics of Protein-Protein Interactions of Connexins: Use of Enzyme Linked Sorbent Assays

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.     

Hemodynamic changes in apolipoprotein E-knockout mice

Apolipoprotein E-knockout (ApoE-KO) mice develop advanced atherosclerotic lesions by 1 yr of age and have been well characterized pathologically and morphologically, but little is known regarding their cardiovascular physiology and hemodynamics. We used noninvasive Doppler ultrasound to measure aortic and mitral blood velocity and aortic pulse-wave velocity in 13-mo-old ApoE-KO and wild-type (WT) mice anesthetized with isoflurane. In other mice from the same colony, we measured systolic blood pressure, body weight, heart weight, cholesterol, and hematocrit. Heart rate and blood pressure were comparable (P = not significant) between ApoE-KO and WT mice, but significant decreases (P < 0.001) were found in body weight (-22%) and hematocrit (-11%), and significant increases were found in heart weight (+23%), aortic velocity (+60%), mitral velocity (+81%) (all P < 0.001), and pulse-wave velocity (+13%, P < 0.05). We also found inflections in the aortic arch velocity signal consistent with enhanced peripheral wave reflection. Thus ApoE-KO mice have phenotypic alterations in indexes of peripheral vascular resistance and compliance and significantly elevated cardiac outflow velocities and heart weight-to-body weight ratios.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Summary Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B. This investigation was supported by PHS Grant CA32444, awarded by the National Cancer Institute. A. R. L. G. is a recipient of a USPHS fellowship, GM09226-01, and S. M. T. was supported by NIH training Grant AMO 7282.     

Lymphokine maintains macrophage activation for tumor cell killing by interfering with the negative regulatory effect of prostaglandin E2

Mouse resident peritoneal macrophages activated with bacterial lipopolysaccharide (LPS) rapidly lost their ability to kill tumor cells in vitro. Such loss of killing has previously been attributed to the effects of prostaglandin E (PGE) produced by the LPS-stimulated macrophages. Macrophages exposed in the current study to both LPS and partially purified lymphokine did not lose cytolytic activity, in spite of the fact that these cells produced undiminished amounts of PGE, compared to controls. Cytolytic activity was shown to be retained under these conditions because lymphokine decreased the sensitivity of activated macrophages to the negative regulatory effects of PGE. The mechanism responsible for the lymphokine effect is not known; however, generalized inhibition of macrophage responsiveness to the hormone does not appear to be involved because lymphokine did not reduce the cyclic AMP response of macrophages, measured on a whole cell basis, after they were exposed to PGE.     

Membrane Asymmetry in Isolated Canine Cardiac Sarcoplasmic Reticulum: Comparison with Skeletal Muscle Sarcoplasmic Reticulum

Cardiac sarcoplasmic reticulum (CSR), isolated from dog hearts, was shown to be asymmetric in the distribution of phospholipids across the CSR bilayer. Phosphatidylethanolamine was mostly resident in the outer leaflet, phosphatidylcholine was equally distributed across both monolayers and phosphatidylserine was found primarily in the inner monolayer. This distribution of headgroups is similar to that found in fast skeletal muscle sarcoplasmic reticulum (SSR); however, the asymmetry in CSR is not as striking as that in SSR. Phospholipids retained by the CSR calcium pump protein (CaATPase) after detergent ``stripping' were similar to those intimate to the SSR CaATPase, although the percentages of unsaturated phospholipids and plasmalogenic phospholipids are not as great as in the skeletal system. Lipids associated with the CSR CaATPase following DFDNB cross-linking showed a preference for retention of the aminophospholipids, again similar to the SSR CaATPase. Because the nonrandom distribution of membrane lipids modifies SSR function, it is likely these membrane lipids impact in situ the function of the CSR. Received: 19 December 1997/Revised: 3 April 1998