Search

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others. [ABSTRACT FROM AUTHOR]

Background: Ischemic heart disease (IHD) is associated with decreased cardiac conduction, providing a substrate for reentrant arrhythmia and sudden cardiac death. Connexin43 (Cx43) is the principal gap junction protein responsible for allowing current to pass through cardiomyocytes in the ventricles. Cx43 expression is known to be reduced in the left ventricle following IHD. Recently, we showed that ROS stimulates c-Src phosphorylation (p-Src), allowing p-Src binding to the scaffolding protein zonula occludens-1 (ZO-1) and destabilizing Cx43, leading to Cx43 lateralization and degradation. We tested whether Src inhibition would prevent Cx43 degradation in IHD. Methods: Coronary artery occlusion was performed on 12-week-old mice causing myocardial infarction (MI). MI mice were treated with PP1, a p-Src inhibitor, or PP3, an inactive analogue. PP1, PP3, and sham hearts were compared functionally by echocardiography, optical mapping, ECG telemetry analysis, and arrhythmia inducibility by ventricular pacing. Tissues were collected for immunohistochemistry and Western blot analysis. Results: Scar borders of PP1 groups demonstrated restored conduction velocity compared with PP3-treated mice (PP1 = 33 cm/s, PP3 = 18 cm/s). In PP1-treated mice, there was a 60 % decrease in p-Src activation and a 25% increase in Cx43 expression at the scar border compared with PP3 groups. PP1 did not change infarct size, ECG pattern, or cardiac function (ejection fraction: sham = 61 ± 1, PP1 = 39 ± 2, PP3 = 39 ± 2). Conclusions: Src inhibition improves Cx43 levels and conduction velocity after MI. Src inhibitors may represent a new class of antiarrhythmic compounds. [ABSTRACT FROM AUTHOR]

We previously found that the plasma of patients with sickle cell disease (SCD) contains large numbers of small extracellular vesicles (EVs) and that the EVs disrupt the integrity of endothelial cell monolayers (especially if obtained during episodes of acute chest syndrome, ACS). The present study was designed to test the generality of this finding to other complications of SCD, specifically to evaluate the possibility that circulating EVs isolated during a vaso-occlusive crises (VOC) also cause damage to the intercellular connections between endothelial cells. Plasma was obtained from nine pediatric subjects at baseline and during VOC episodes. EVs isolated from these samples were added to cultures of microvascular endothelial cells. Immunofluorescence microscopy was employed to assess monolayer integrity and to localize two intercellular junction proteins (VE-cadherin and connexin43). The EVs isolated during VOC caused significantly greater monolayer disruption than those isolated at baseline. The extent of disruption varied between different episodes of VOC or ACS in the same patient. The VOC EVs disrupted the integrity of both junction proteins at appositional membranes. These results suggest that circulating EVs may be involved in modulating endothelial integrity contributing to the pathogenesis of different complications of SCD.

Background: Small cell-derived extracellular vesicles (EVs) can affect endothelial function. We previously found that patients with sickle cell disease (SCD) have greater numbers of circulating EVs than subjects without the disease, and the EVs differentially disrupt endothelial integrity in vitro. Because endothelial disruption is a critical component of acute chest syndrome (ACS), we hypothesized that EVs isolated during ACS would induce greater endothelial damage than those isolated at baseline., Methods: Nine pediatric subjects had plasma isolated at baseline and during ACS from which EVs were isolated. Cultured microvascular endothelial cells were treated with EVs and then studied by immunofluorescence microscopy to localize VE-cadherin and F-actin., Results: The EVs had a diameter of 95 nm. They contained CD63 and flotillin-1, which were increased in SCD patients (5-13-fold compared to control) and further increased between baseline and ACS (24-57%). The EVs contained hemoglobin, glycophorin A, and ferritin. Treatment with baseline EVs caused modest separation of endothelial cells, while ACS EVs caused substantial disruptions of the endothelial cell monolayers. EVs from subjects with ACS also caused a 50% decrease in protein levels of VE-cadherin., Conclusions: These results suggest that circulating EVs can modulate endothelial integrity contributing to the development of ACS in SCD patients by altering cadherin-containing intercellular junctions., Impact: Sickle cell disease patients have circulating extracellular vesicles (EVs) that modulate endothelial integrity by altering cadherin-containing intercellular junctions. Disruption is more severe by EVs obtained during acute chest syndrome (ACS). These results expand our knowledge of the pathophysiology of acute chest syndrome and the vasculopathies of sickle cell disease.

Intercellular junctions maintain the integrity of the endothelium. We previously found that the adherens and tight junctions between endothelial cells are disrupted by plasma extracellular vesicles from patients with sickle cell disease (especially those with Acute Chest Syndrome). In the current study, we evaluated the effects of these vesicles on endothelial gap junctions. The vesicles from sickle cell patients (isolated during episodes of Acute Chest Syndrome) disrupted gap junction structures earlier and more severely than the other classes of intercellular junctions (as detected by immunofluorescence). These vesicles were much more potent than those isolated at baseline from the same subject. The treatment of endothelial cells with these vesicles led to reduced levels of connexin43 mRNA and protein. These vesicles severely reduced intercellular communication (transfer of microinjected Neurobiotin). Our data suggest a hierarchy of progressive disruption of different intercellular connections between endothelial cells by circulating extracellular vesicles that may contribute to the pathophysiology of the endothelial disturbances in sickle cell disease.

Endothelial damage is central to the pathogenesis of many of the complications of sickle cell disease. Circulating extracellular vesicles (EVs) have been implicated in modulating endothelial behavior in a variety of different, diseases with vascular pathologies. As seen in other hemolytic diseases, the plasma of sickle cell patients contains EVs of different sizes and cellular sources. The medium-sized vesicles (microparticles) primarily derive from mature red blood cells and platelets; some of these EVs have procoagulant properties, while others stimulate inflammation or endothelial adhesiveness. Most of the small EVs (including exosomes) derive from erythrocytes and erythrocyte precursors, but some also originate from platelets, white blood cells, and endothelial cells. These small EVs may alter the behavior of target cells by delivering cargo including proteins and nucleic acids. Studies in model systems implicate small EVs in promoting vaso-occlusion and disruption of endothelial integrity. Thus, both medium and small EVs may contribute to the increased endothelial damage in sickle cell disease. Development of a detailed understanding of the composition and roles of circulating EVs represents a promising approach toward novel predictive diagnostics and therapeutic approaches in sickle cell disease., (Copyright © 2020 Lapping-Carr, Gemel, Mao and Beyer.)

Rationale: ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 ( TJP1 ) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined., Objective: We aim to determine the role of ZO-1 in cardiac function., Methods and Results: Inducible cardiomyocyte-specific Tjp1 deletion mice ( Tjp1 fl/fl ; Myh6 Cre/Esr1* ) were generated by crossing the Tjp1 floxed mice and Myh6 Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 ( Tjp1 fl/fl ; Hcn4 CreERt2 ) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node ( Tjp1 fl/fl ; Kcne1 CreERt2 ) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction., Conclusions: ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.

Intercellular communication is accomplished by passage of ions and small molecules through gap junction channels in directly contacting cells or by secretion and response to transmitters, hormones and extracellular vesicles in cells that are distant from each other. Recent studies have suggested that there may be overlap of these processes; specifically, small extracellular vesicles may contain subunit gap junction proteins, connexins. We isolated and analyzed extracellular vesicles secreted by cultured microvascular endothelial cells. These vesicles had a diameter of ~120 nm. They contained four exosomal proteins (flotillin-1, CD63, CD81 and Alix) and the gap junction protein, connexin43. They did not contain an endoplasmic reticulum protein (Grp94) or an adherens junction protein (VE-cadherin). Secretion of vesicles was increased by treatment of the cells with staurosporine. Our data confirm that the gap junction protein, connexin43, can be secreted in vesicles with the properties of exosomes. Although the role of vesicular connexin is not clearly known, we speculate that it might participate in docking/fusion of the exosomes with the recipient cell, transmission of vesicular contents, or cellular signaling.

Background: Obstructive sleep apnea has been linked to the development of heart disease and arrhythmias, including atrial fibrillation. Since altered conduction through gap junction channels can contribute to the pathogenesis of such arrhythmias, we examined the abundance and distributions of the major cardiac gap junction proteins, connexin40 (Cx40) and connexin43 (Cx43) in mice treated with sleep fragmentation or intermittent hypoxia (IH) as animal models of the components of obstructive sleep apnea., Results: Wild type C57BL/6 mice or mice lacking NADPH 2 (NOX2) oxidase activity (gp91phox(-/Y)) were exposed to room air or to SF or IH for 6 weeks. Then, the mice were sacrificed, and atria and ventricles were immediately dissected. The abundances of Cx40 or Cx43 in atria and ventricles were unaffected by SF. In contrast, immunoblots showed that the abundance of atrial Cx40 and Cx43 and ventricular Cx43 were reduced in mice exposed to IH. qRT-PCR demonstrated significant reductions of atrial Cx40 and Cx43 mRNAs. Immunofluorescence microscopy revealed that the abundance and size of gap junctions containing Cx40 or Cx43 were reduced in atria by IH treatment of mice. However, no changes of connexin abundance or gap junction size/abundance were observed in IH-treated NOX2-null mice., Conclusions: These results demonstrate that intermittent hypoxia (but not sleep fragmentation) causes reductions and remodeling of atrial Cx40 and Cx43. These alterations may contribute to the substrate for atrial fibrillation that develops in response to obstructive sleep apnea. Moreover, these connexin changes are likely generated in response to reactive oxygen species generated by NOX2.

Normal atrial conduction requires similar abundances and homogeneous/overlapping distributions of two connexins (Cx40 and Cx43). The remodeling of myocyte connections and altered electrical conduction associated with atrial fibrillation (AF) likely involves perturbations of these connexins. We conducted a comprehensive series of experiments to examine the abundances and distributions of Cx40 and Cx43 in the atria of AF patients. Atrial appendage tissues were obtained from patients with lone AF (paroxysmal or chronic) or normal controls. Connexins were localized by double label immunofluorescence confocal microscopy, and their overlap was quantified. Connexin proteins and mRNAs were quantified by immunoblotting and qRT-PCR. PCR amplified genomic DNA was sequenced to screen for connexin gene mutations or polymorphisms. Immunoblotting showed reductions of Cx40 protein (to 77% or 49% of control values in samples from patients with paroxysmal and chronic AF, respectively), but no significant changes of Cx43 protein levels in samples from AF patients. The extent of Cx43 immunostaining and its distribution relative to N-cadherin were preserved in the AF patient samples. Although there was variability of Cx40 staining among paroxysmal AF patients, all had some fields with substantial Cx40 heterogeneity and reduced overlap with Cx43. Cx40 immunostaining was severely reduced in all chronic AF patients. qRT-PCR showed no change in Cx43 mRNA levels, but reductions in total Cx40 mRNA (to <50%) and Cx40 transcripts A (to ~50%) and B (to <25%) as compared to controls. No Cx40 coding region mutations were identified. The frequency of promoter polymorphisms did not differ between AF patient samples and controls. Our data suggest that reduced Cx40 levels and heterogeneity of its distribution (relative to Cx43) are common in AF. Multiple mechanisms likely lead to reductions of functional Cx40 in atrial gap junctions and contribute to the pathogenesis of AF in different patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several of these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by the transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20-31% of wild type levels. After the inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (Vj) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of Vj gating properties and increased the gap junction channel conductance (γj) by 20-30 pS. While co-expression of the two mutations had similar effects on Vj gating, it synergistically increased γj by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel., (Copyright © 2014 Federation of European Biochemical Societies. All rights reserved.)

Objectives: The aim of this study was to evaluate the role of tyrosine kinase cellular-Src (c-Src) inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI)., Background: MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation., Methods: MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry electrocardiographic monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative polymerase chain reaction, and immunohistochemistry., Results: Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p = 0.003) and distal ventricle (346%, p = 0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p = 0.95) and distal ventricle (94%, p = 1.0). PP1 raised Cx43 expression by 69% in the scar border (p = 0.048) and by 73% in the distal ventricle (p = 0.043) compared with PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, electrocardiographic pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1., Conclusions: c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ∼1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 μm Ca(2+) showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mm Ca(2+), > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mm EDTA showed large (5.6- to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mm Ca(2+) showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and

Since most cells in the heart co-express multiple connexins, we studied the possible heteromeric interactions between connexin30.2 and connexin40, connexin43 or connexin45 in transfected cells. Double-label immunofluorescence microscopy showed that connexin30.2 extensively co-localized with each co-expressed connexin at appositional membranes. When Triton X-100 solubilized connexons were affinity purified from co-expressing cells, connexin30.2 was isolated together with connexin40, connexin43, or connexin45. Co-expression of connexin30.2 with connexin40, connexin43, or connexin45 did not significantly reduce total junctional conductance. Gap junction channels in cells co-expressing connexin30.2 with connexin43 or connexin45 exhibited voltage-dependent gating intermediate between that of either connexin alone. In contrast, connexin30.2 dominated the voltage-dependence when co-expressed with connexin40. Our data suggest that connexin30.2 can form heteromers with the other cardiac connexins and that mixed channel formation will influence the gating properties of gap junctions in cardiac regions that co-express these connexins.

The cytoplasmic N-terminal domain in the connexins (Cx) has been implicated in determining several properties including connexin hetero-oligomerization, channel gating and regulation by polyamines. To elucidate the roles of potentially crucial amino acids, we produced site-directed mutants of connexins Cx40 and Cx43 (Cx40E12S,E13G and Cx43D12S,K13G) in which the charged amino acids at positions 12 and 13 were replaced with serine and glycine as found in Cx32. HeLa, N2a and HEK293 cells were transfected and studied by immunochemistry and double whole-cell patch clamping. Immunoblotting confirmed production of the mutant proteins, and immuno-fluorescence localized them to punctuate distributions along appositional membranes. Cx40E12S,E13G and Cx43D12S,K13G formed homotypic gap junction channels that allowed intercellular passage of Lucifer Yellow and electrical current, but these channels exhibited negligible voltage-dependent gating properties. Unlike wild-type Cx40, Cx40E12S,E13G channels were insensitive to block by 2 mM spermine. Affinity purification of material solubilized by Triton X-100 from cells co-expressing mutant Cx43 or mutant Cx40 with wild-type Cx40, Cx43 or Cx26 showed that introducing the mutations did not affect the compatibility or incompatibility of these proteins for heteromeric mixing. Co-expression of Cx40E12S,E13G with wild-type Cx40 or Cx43 dramatically reduced voltage-dependent gating. Thus, whereas the charged amino acids at positions 12 and 13 of Cx40 or Cx43 are not required for gap junction assembly or the compatibility of oligomerization with each other or with Cx26, they strongly influence several physiological properties including those of heteromeric channels.

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.

The ventricular action potential was applied to paired neonatal murine ventricular myocytes in the dual whole cell configuration. During peak action potential voltages >100 mV, junctional conductance (g(j)) declined by 50%. This transjunctional voltage (V(j))-dependent inactivation exhibited two time constants that became progressively faster with increasing V(j). G(j) returned to initial peak values during action potential repolarization and even exceeded peak g(j) values during the final 5% of repolarization. This facilitation of g(j) was observed <30 mV during linearly decreasing V(j) ramps. The same behavior was observed in ensemble averages of individual gap junction channels with unitary conductances of 100 pS or lower. Immunohistochemical fluorescent micrographs and immunoblots detect prominent amounts of connexin (Cx)43 and lesser amounts of Cx40 and Cx45 proteins in cultured ventricular myocytes. The time dependence of the g(j) curves and channel conductances are consistent with the properties of predominantly homomeric Cx43 gap junction channels. A mathematical model depicting two inactivation and two recovery phases accurately predicts the ventricular g(j) curves at different rates of stimulation and repolarization. Functional differences are apparent between ventricular myocytes and Cx43-transfected N2a cell gap junctions that may result from posttranslational modification. These observations suggest that gap junctions may play a role in the development of conduction block and the genesis and propagation of triggered arrhythmias under conditions of slowed conduction (<10 cm/s).

Connexin40 (Cx40) contains a specific binding site for spermine (affinity approximately 100 microm) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 mm spermine, reduced the transjunctional voltage (V(j)) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state (G(min)) and V(j) gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild-type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced V(j) sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore-forming domain that serves as the receptor for V(j)-dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.

Many cells contain two (or more) gap junction proteins that are able to oligomerize with each other to form heteromeric gap junction channels and influence the properties of intercellular communication. Cx26 and Cx43 are found together in a number of cell types, but previous data have suggested that they might not form heteromeric connexons. We studied the possible interactions of these connexins by co-expression in three different cell lines. Analysis of N2aCx26/Cx43 cell pairs by double whole-cell patch-clamp methods showed that these cells were coupled, but contained only a small number of sizes of single channels consistent with those formed by homomeric Cx26 or Cx43 channels. Immunofluorescence studies showed that both connexins localized to appositional membranes, but in largely distinct domains. Analysis of Triton X-100-solubilized connexons from co-expressing cells by centrifugation through sucrose gradients or by affinity purification using a Ni-NTA column showed no evidence of mixing of Cx26 and Cx43. These results contrast with our observations in cells co-expressing other connexins with Cx43 and suggest that Cx26 and Cx43 do not form heteromeric hemichannels. Moreover, the incorporation of Cx26 and Cx43 into oligomers and into the membrane were similarly affected by treatment of co-expressing cells with brefeldin A or nocodazole, suggesting that the lack of mixing is due to incompatibility of these connexins, not to differences in biosynthetic trafficking.

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.

In order to better understand control of the mitochondrial pyruvate dehydrogenase complex (PDC), total catalytic activity was determined during development of the primary leaves of pea (Pisum sativum L.) seedlings, as well as in each leaf pair of 21-day-old plants. Activity of the PDC in clarified homogenates was highest in the youngest organs and then dropped dramatically as the leaves matured and became photosynthetically competent. As leaves began to senesce, total PDC activity dropped to zero. Steady-state mRNA levels were determined using E1 and E3 cDNA probes. The overall pattern of transcript abundance matched the pattern observed for total PDC activity; transcript levels for E1alpha and E1beta approached zero during senescence. Levels of the E1alpha, E1beta, E2 and E3 subunits of the PDC were analyzed in the same samples, using specific antibodies. Quantitation of the immunoblotting results throughout this developmental series showed a pattern in parallel with that of catalytic activity and mRNA levels, although the relative changes in subunit protein levels were not as extreme as the changes in activity. The exception to the global pattern was that of the E3 subunit: lipoamide dehydrogenase. Expression of this enzyme was highest in mature, fully expanded leaves, which were active in photosynthesis and photorespiration, reflecting the additional role of E3 as a component of glycine decarboxylase.

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.

Fibroblast growth factor-8 (FGF8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. We now report on the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 was shown to contain promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 was found to enhance human growth hormone expressed from the Fgf8 promoter 3-4-fold in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments documented the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrated that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicated that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment.

Recent evidence indicates that Fgf8 is expressed during vertebrate development in multiple locations involved in the patterning and outgrowth of important embryo structures. Cloning and analysis of the murine gene revealed at least eight potential protein isoforms that share a common carboxyl region, encoded by exons 2 and 3, but possess different amino termini, generated by alternative splicing of RNA encoded by multiple 5' exons (exons 1A, 1B, 1C, and 1D). We now report the cloning and sequence of the human FGF8 gene. Human FGF-8 isoforms are identical to their murine counterparts in the common carboxyl region. Four of the human isoforms are identical to, or very similar to, the murine isoforms in the amino termini. However, four of the potential murine isoforms do not have corresponding human isoforms due to marked sequence divergence, leading to a blocked reading frame in exon 1B of FGF8. The lack of the four murine isoforms in humans raises the question of their function in murine development.

The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.

The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.

Light-dependent inactivation of mitochondrial pyruvate dehydrogenase complex (mtPDC) in pea (Pisum sativum L.) leaves was further characterized, and this phenomenon was extended to several monocot and dicot species. The light-dependent inactivation of mtPDC in vivo was rapidly reversed in the dark, even after prolonged illumination. The mtPDC can be efficiently cycled through the inactivated-reactivated status by rapid light-dark cycling. Light-dependent inactivation of mtPDC was shown to be suppressed by inhibitors of photorespiratory carbon metabolism, including 2-pyridylhydroxymethane sulfonate, isonicotinic acid hydrazide, and aminoacetonitrile, and by an inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Glycine fed to pea leaf strips in the dark yielded partially inactivated leaf mtPDC, and this inactivation was blocked by inhibitors of glycine oxidation. It is concluded that the photorespiratory glycine to serine conversion that occurs in C(3) leaf mitochondria can provide the NADH to drive oxidative phosphorylation and subsequent inactivation of mtPDC. Glycine oxidation also produces ammonium ion, which has been shown to enhance the inactivation of mtPDC in vitro by stimulating the pyruvate dehydrogenase kinase that catalyzes the phosphorylation (inactivation) of the mtPDC. Thus, light-dependent, photorespiration-stimulated inactivation of the mtPDC can regulate carbon entry into the Krebs cycle during C(3) photosynthesis.

Galactolipase activity in chloroplasts of several chill-resistant plants was found to be very low [0.02-0.13 mumol free fatty acid (FFA) liberated min-1 mg protein-1] or not detected. The same phenomenon was observed for soybean and members of the Cucurbitaceae such as cucumber, pumpkin, melon and squash. Since, following cold storage of cucumber leaves, the levels of monogalactosyl-diacylglycerol and digalactosyl-diacylglycerol in chloroplasts decrease while those of FFA accumulate it seems likely that in these typical chill-sensitive plants galactolipase is present but inactivated during isolation procedure. The low galactolipase activity in chloroplasts was accompanied by a relatively low FFA content ranging from 0.05 mumol to 0.30 mumol FFA mg chlorophyll (Chl)-1. However, both pea and horse bean chloroplasts (with low galactolipase activity) exhibit about 0.45 mumol FFA mg Chl-1. Elevated galactolipase activity was observed in chloroplasts of most chill-sensitive species (ranging from 0.31 mumol to 1.32 mumol FFA liberated min-1 mg protein-1) as well as in chloroplasts from broad bean, a member of a chill-resistant species (1.26 mumol FFA liberated min-1 mg protein-1). In addition in this latter group of plants FFA level in chloroplasts often did not fit the galactolipase activity. The results suggest that there exists a tendency for chilling tolerance of plants to decrease both galactolipase activity and FFA level. However, in some plant species with elevated galactolipase activity the chloroplast FFA level does not correlate with enzyme activity.

The effect of octanoate, palmitate and oleate on glucose formation was studied with lactate, pyruvate or malate as substrate in kidney tubules isolated from fasted rabbits. All fatty acids studied inhibited the rate of glucose production by about 30-50% depending on the glucose precursor and fatty acid used, stimulated the oxygen uptake by about 50% and increased the mitochondrial NADH/NAD+ ratio, as manifested by a marked rise of 3-hydroxybutyrate/acetoacetate ratio. Octanoate was twice as quickly utilized for ketone body production than palmitate and oleate were. As concluded from the 'crossover' plot the inhibitory effect of fatty acids on gluconeogenesis in rabbit kidney tubules may be due to: (i) a decrease of mitochondrial generation of phosphoenolpyruvate and (ii) an inhibition of flux through fructose-1,6-bisphosphatase.