Expression Of Gap Junction Proteins Research Articles

Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulosa cells

Tris(4-chlorophenyl)methanol (TCPM) is a by-product in the manufacture of technical grade DDT, which is known to alter properties and functions of the female reproductive system. We investigated whether in vitro TCPM has an influence on the function of gap junction-mediated intercellular communication (GJIC) and gap junction protein expression of connexin 43 (Cx43) in cultured bovine granulosa cells. GJIC was assessed by fluorescent dye microinjection (dye-coupling). After a 1-h exposure to TCPM at a concentration of 32 μ m, a significant ( P<0.05) reduction in dye coupling occurred. The same result was obtained with o,p′-DDT. At a concentration of 32 μ m both pesticides were cytotoxic as indicated by significant ( P<0.05) increased propidium iodide staining of the cell nuclei. Little or no effect on the stainable pattern of connexons occurred after 1 h incubation time, while after 3 h treatment from 16 to 64 μ m TCPM, a significant inhibition in the immunostaining resulted and the concentrations of 32 and 64 μ m TCPM were cytotoxic for the granulosa cells. The freeze-fracture electron microscopy resulted in small differences in the morphology of gap junction plaques of cell cultures treated for 3 h with 8 or 16 μ m TCPM in comparison to untreated cells. After treatment with 32 μ m TCPM, gap junction plaques were very rarely detected and the lateral intramembraneous particles (IMP) distribution of many plasma membranes was strongly altered. Estimation of the cellular parameters may lead to an enhanced understanding of the mechanism of chemically induced toxicity by TCPM, that causes a general toxic effect on granulosa cells. We can conclude that TCPM is a toxic risk in the same manner as DDT.

Extracellular Matrix Modulates Expression of Connexin Messenger RNA and Protein by Alveolar Epithelial Cells

Cell-to-cell communication through gap junction channels plays an important role in both the physiologic and pathophysiologic function of many tissues and cell types.1 Data from several laboratories have documented expression and function of gap junction proteins, connexins (Cx), in alveolar epithelial cells, both in vivo and in vitro.2,3,4,5 Nevertheless, little is known concerning the biology of gap junctions in the alveolar region of the lung. Type II alveolar epithelial cells express at least six Cxs in primary culture.

Heterocellular gap junctional communication between alveolar epithelial cells.

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.

Connexin37, not Cx40 and Cx43, is Induced in Vascular Smooth Muscle Cells During Coronary Arteriogenesis

W.-J. Cai, S. Koltai, E. Kocsis, D. Scholz, W. Schaper and J. Schaper. Connexin37, not Cx40 and Cx43, is Induced in Vascular Smooth Muscle Cells During Coronary Arteriogenesis. Journal of Molecular and Cellular Cardiology (2001) 33, 957–967. The hypothesis that an altered expression of gap junction (GJ) proteins, connexin37 (Cx37), Cx40 and Cx43 will contribute to adaptive arteriogenesis was tested in growing coronary collateral vessels (CV) of the dog heart by immunoconfocal microscopy and transmission electron microscopy (TEM). We found that: (1) in the normal coronary system Cx37 and Cx40 were only expressed in endothelial cells (EC) from artery to capillary; (2) during collateral growth Cx37 was significantly induced in smooth muscle cells (SMC) from small-large arteries to precapillary arterioles (Ø=15 μ m), while Cx40 was still only present in EC; (3) both homogeneous and heterogeneous distribution of Cx37 was observed in normal vessels (NV) and growing vessels (GV); (4) in mature vessels (MV), Cx37 was downregulated, similar to NV; (5) dual immunostaining revealed an inverse correlation between expression of Cx37 and desmin in GV occurring prior to downregulation of α -smooth actin and calponin; (6) Cx43 was undetectable in any vascular cells, both in NV and GV; (7) GJ were not found in SMC by TEM. Our data for the first time show the profile of connexin expression in the coronary system and provide evidence for existence of GJ proteins in capillaries. It is a novel finding that an altered expression of Cx37 is characteristic of adaptive arteriogenesis in the dog heart and may be used as a marker of vascular growth. Induced Cx37 may be an early signal indicating that SMC are responding to haemodynamic changes, i.e. increased shear stress.

Differential expression of major gap junction proteins, connexins 26 and 32, in rat mammary glands during pregnancy and lactation.

We examined the expression pattern of two major gap junction proteins, connexin 26 (Cx26) and connexin 32 (Cx32), in rat mammary glands during pregnancy and lactation. Immunohistochemically the two different Cxs were coexpressed in acinar cells and were independently modulated according to the physiological cell activity. Western blot analysis demonstrated that Cx26 gradually increased from early pregnancy, while Cx32 rapidly and dramatically increased at 16 h after parturition, and that both Cxs reached a maximum early in lactation. Increased expression of both Cxs was confirmed by Northern blot analysis showing that their mRNA transcripts were significantly induced on the day of parturition. We also analyzed double-immunofluorescent staining for Cx26 and Cx32 on a confocal laser scanning microscope, in order to examine colocalization of these Cxs in situ. Cx26 immunoreactivity mostly overlapped with Cx32-positive sites in acinar cells of lactating mammary glands, indicating that both Cxs were colocalized together in the same gap junctional plaques in lactation. These results suggest that upregulation of Cx26 and Cx32 in acinar cells at lactating stages, with colocalization in the same gap junctional plaques, may be important for control of secretion by acinar cells in rat mammary glands.

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells.

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.

Perineurium inflammation and altered connexin isoform expression in a rat model of diabetes related peripheral neuropathy

Diabetes related peripheral neuropathy involves both somatic and autonomic nerves and leads to an array of debilitating abnormalities. Mechanisms may include decreased neuronal conductance, reactive oxygen species, and decreased performance of the perineurium blood–nerve barrier. Here we studied the perineurium characteristics of the dorsal penile nerve in a rat model of diabetes related peripheral neuropathy. Immunohistochemistry showed extensive and perineurial cell-specific nitric oxide synthase2 staining in diabetic animals as compared to age matched controls ( P<0.05); however no apparent difference in immunostaining pattern was observed for 3-nitrotyrosine (a stable biomarker of peroxynitrite formation). Significant reductions in connexins 32 and 26 were seen in the diabetic perineurium with no detectable levels of connexin 43 in either control or diabetic dorsal nerve. These data provide new evidence of perineurial cell inflammatory responses and altered gap junction protein expression during diabetes related neuropathies and suggests that strategies to protect this cell type may have therapeutic value.

Connexin expression by alveolar epithelial cells is regulated by extracellular matrix.

Extracellular matrix (ECM) proteins promote attachment, spreading, and differentiation of cultured alveolar type II epithelial cells. The present studies address the hypothesis that the ECM also regulates expression and function of gap junction proteins, connexins, in this cell population. Expression of cellular fibronectin and connexin (Cx) 43 increase in parallel during early type II cell culture as Cx26 expression declines. Gap junction intercellular communication is established over the same interval. Cells plated on a preformed, type II cell-derived, fibronectin-rich ECM demonstrate accelerated formation of gap junction plaques and elevated gap junction intercellular communication. These effects are blocked by antibodies against fibronectin, which cause redistribution of Cx43 protein from the plasma membrane to the cytoplasm. Conversely, cells cultured on a laminin-rich ECM, Matrigel, express low levels of Cx43 but high levels of Cx26, reflecting both transcriptional and translational regulation. Cx26 and Cx43 thus demonstrate reciprocal regulation by ECM constituents.

Gap junction proteins in inhibitory neurons of the adult barrel neocortex

Recent studies indicate that electrical coupling among cortical neurons may persist throughout development; electrophysiological recordings made in cortical slices from young rats reveal that numerous GABAergic neurons are electrically coupled. To determine whether these in vitro findings reflect an inhibitory neural circuit that could be functionally relevant in vivo in adult rodents, we sought to identify whether inhibitory, parvalbumin-containing neurons of the mature cortex express gap junction proteins. Immunohistochemistry was used to examine the laminar distribution of the gap junction-forming proteins connexin 32 (Cx32), connexin 36 (Cx36) and connexin 43 (Cx43) in the somatosensory cortex of the adult mouse. Double labeling immunofluorescence identified Cx32, Cx36 and Cx43 in cortical neurons that were immunoreactive (-ir) for the neuronal markers neurofilament 145 kDa and neuronal nuclei (NeuN). Parvalbumin-ir neurons throughout the cortical laminae were labeled with Cx32-ir, Cx36-ir and Cx43-ir. Stereological methods were used to quantify the extent of parvalbumin colocalization with connexins. Analysis indicated that approximately 40% of parvalbumin-ir neurons were double labeled with either Cx32-ir or Cx43-ir, and approximately 50% of parvalbumin-ir neurons were double labeled with Cx36. These findings establish an anatomical substrate for widespread electrical coupling of neurons in somatosensory cortex and suggest that gap junctions among inhibitory interneurons may persist into adulthood, providing an important mechanism for neuronal communication.

The sinoatrial node, a heterogeneous pacemaker structure.

This article focuses on the regional heterogeneity of the mammalian sinoatrial (SA) node in terms of cell morphology, pacemaker activity, action potential configuration and conduction, densities of ionic currents (i(Na), i(Ca,L), i(to), i(K,r), i(K,s) and i(f)), expression of gap junction proteins (Cx40, Cx43 and Cx45), autonomic regulation, and ageing. Experimental studies on the single SA node cell to the whole animal are reviewed. The heterogeneity is considered in terms of the gradient model of the SA node, in which there is gradual change in the intrinsic properties of SA node cells from periphery to centre, and the alternative mosaic model, in which there is a variable mix of atrial and SA node cells from periphery to centre. The heterogeneity is important for the dependable functioning of the SA node as the pacemaker for the heart, because (i) via multiple mechanisms, it allows the SA node to drive the surrounding atrial muscle without being suppressed electrotonically; (ii) via an action potential duration gradient and a conduction block zone, it promotes antegrade propagation of excitation from the SA node to the right atrium and prevents reentry of excitation; and (iii) via pacemaker shift, it allows pacemaking to continue under diverse pathophysiological circumstances.

Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat.

It has been postulated that high atrial rate induced changes at the level of the gap junctions ('gap junctional remodeling', i.e. changes in distribution, intercellular orientation and expression of gap junction proteins), could be part of the vicious circle of electrophysiologic and structural changes leading to sustained atrial fibrillation (AF). To obtain experimental evidence in favour of such a postulate the timing of this remodeling process was studied in relation to the development of sustained AF in a goat model. Thin sections from the left (LAA) and right atrial appendage (RAA) from goats in sinus rhythm (SR) or AF, induced through programmed endocardial burst pacing for time periods between 0 and 16 weeks, were immunolabeled with antibodies against connexin(Cx)40 and Cx43 and analysed by immunofluorescence and confocal laser scanning microscopy. During SR the distribution pattern for Cx43 was completely homogeneous (LAA and RAA) and for Cx40 mostly homogeneous (LAA: all five goats, RAA: three out of five goats). The distribution pattern for Cx43 remained stable during AF, while the Cx40 distribution pattern became increasingly heterogeneous, both in the LAA and RAA, with increasing duration of pacing. This increase in heterogeneity in Cx40 distribution correlated (Spearman rank order) with an increase in stability of AF and the occurrence of structural changes (myolysis) in atrial myocytes. The Cx40/Cx43 immunofluorescence signal ratio in both the LAA and RAA appeared to be significantly lower in AF (1-16 weeks) as compared to SR (0 weeks); going from 0 to 16 weeks average ratios decreased 54.5% (n=5; P=0.026) in the LAA and 35.8 (n=5; P=0.034) in the RAA. Western blot analyses revealed similar decreases in the total Cx40/Cx43 protein ratio, on average 50.0% (n=5; P=0.008) and 47.8% (n=5; P=0.02) in the LAA and RAA, respectively. No changes were measured in the levels of Cx40 or Cx43 mRNA, as was assessed through RT-PCR. The time course of changes in the distribution and content of Cx40 gap junctions as observed during endocardial burst pacing of the goat atrium suggests that Cx40 gap junctional remodeling might be involved in the pathogenesis of sustained atrial fibrillation.

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium.

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.

ATP-Mediated Glia Signaling

Glia calcium signaling has recently been identified as a potent modulator of synaptic transmission. We show here that the spatial expansion of calcium waves is mediated by ATP and subsequent activation of purinergic receptors. Ectopic expression of gap junction proteins, connexins (Cxs), leads to an increase in both ATP release and the radius of calcium wave propagation. Cx expression was also associated with a phenotypic transformation, and cortical neurons extended longer neurites when co-cultured with Cx-expressing than with Cx-deficient cells. Purinergic receptor activation mediated both these effects, because treatment with receptor antagonists restored the glia phenotype and slowed neurite outgrowth. These results identify a key role of ATP in both short-term calcium signaling events and in long-term differentiation regulated by glia.

Connexin43 in porcine myocardium and non-pregnant myometrium

It is generally accepted that connexin43 (Cx43) is a major constituent of heart and myometrial gap junctions. However, the presence of Cx43 gap junctions in non-pregnant myometrium is still poorly documented. Tissue sections of porcine heart and non-pregnant uterus and myometrial smooth muscle cell cultures were immunostained with monoclonal antibody against Cx43. In the heart, intensive immunostaining was confined to the intercalated discs as previously reported. In the non-pregnant uterus, punctuate immunostaining of Cx43 was seen throughout the myometrium along cell interfaces between myocytes. The expression of Cx43 was sustained in cultured smooth muscle cells isolated from non-pregnant myometrium. Western blotting has detected single isoform of Cx43 in both, cardiac and myometrial tissues. The electrophoretic mobility of porcine heart Cx43 was similar to that of myometrial isoform but different from the pattern of mobility of Cx43 of the rat heart. Hence, porcine myometrium may provide attractive model for studying cellular mechanisms triggering expression of gap junction protein in normal (non-pregnant) uterus.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins.

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.

Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression

A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.

Nerve Injury Induces Gap Junctional Coupling among Axotomized Adult Motor Neurons

Neonatal spinal motor neurons are electrically and dye-coupled by gap junctions, but coupling is transient and disappears rapidly after birth. Here we report that adult motor neurons become recoupled by gap junctions after peripheral nerve injury. One and 4-6 weeks after nerve cut, clusters of dye-coupled motor neurons were observed among axotomized, but not control, lumbar spinal motor neurons in adult cats. Electrical coupling was not apparent, probably because of the electrotonic distance between dendrodendritic gap junctions and the somatic recording location. Analyses of gap junction protein expression in cat and rat showed that the repertoire of connexins expressed by normal adult motor neurons, Cx36, Cx37, Cx40, Cx43, and Cx45, was unchanged after axotomy. Our results suggest that the reestablishment of gap junctional coupling among axotomized adult motor neurons may occur by modulation of existing gap junction proteins that are constitutively expressed by motor neurons. After injury, interneuronal gap junctional coupling may mediate signaling that maintains the viability of axotomized motor neurons until synaptic connections are reestablished within their targets.

Gap Junctional Coupling and Patterns of Connexin Expression among Neonatal Rat Lumbar Spinal Motor Neurons

Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)-P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state.

Induction of gap junctional intercellular communication, connexin43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway

Expression of gap junction proteins and cell–cell communication was studied in the human neural-glial cell line, SVG, as a first step in defining whether the SVG cells could be used as a model system to study the role of gap junctions in neuronal precursor cells. SVG cells were found to express connexin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-loaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional intercellular communication in the SVG cultures. A 24–48 h treatment of SVG cells with 5 μM forskolin or 5 μM forskolin+200 μM 3-isobutyl-1-methylxanthine increased the percentage of dye-coupled cells from 5–65%, using the fluorescent recovery after photobleaching method. The increase in dye coupling induced by forskolin or forskolin+3-isobutyl-1-methylxanthine was inhibited by octanol, which is known to block gap junction-mediated cell communication. Western blot analysis of total protein extracts revealed the appearance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin+3-isobutyl-1-methylxanthine, that was not observed in vehicle-treated controls. Alkaline phosphatase treatment of total protein extracts from forskolin or forskolin+3-isobutyl-1-methylxanthine-treated cells reduced the higher molecular weight band to ≈41,000 the same as observed in the control extracts. The alkaline phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskolin+3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin+3-isobutyl-1-methylxanthine stimulated outgrowth of neurite-like processes from the cell body which immunostained positive for the connexin43 protein as well as protein markers for neurons and oligodendrocytes. We hypothesize that the SVG cells may represent a neuronal progenitor cell population that has the ability to differentiate when exposed to the appropriate signals.

Increased acetylcholine-induced vasodilation in pregnant rats: A role for gap junctional communication.

We have tested the hypothesis that increased gap junctional communication contributes to the augmented endothelium-dependent vasodilation in pregnancy. Contractile force and connexin43 expression were measured in aortic rings from nonpregnant and pregnant rats. Norepinephrine-constricted aortas from pregnant rats were more sensitive to acetylcholine, but not to sodium nitroprusside, compared with those from nonpregnant rats. Vessels from pregnant rats, constricted either with 45 mmol/L KCl or with norepinephrine + 10(-4) mol/L N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase, also exhibited greater relaxation to acetylcholine. Heptanol, an uncoupler of gap junctional communication, inhibited acetylcholine responses in norepinephrine-constricted aortas from nonpregnant rats but greatly impaired acetylcholine relaxation in aortas from pregnant rats. Heptanol also inhibited in both groups acetylcholine responses in vessels constricted with KCl, only minimally affected acetylcholine relaxation in arteries constricted with norepinephrine + L-NMMA, and did not change sodium nitroprusside-induced relaxation. Tetraethylammonium chloride induced greater contractions in control aortas compared with aortas from pregnant rats. Increased connexin43 mRNA levels were found in the uterus and in the mesenteric, uterine, and thoracic aortic arteries, but not in the heart and brain, from pregnant rats. These results suggest that increased gap junctional communication, possibly due to increased gap junction protein expression, may facilitate the effects of endothelium-derived relaxing factors, contributing to the augmented endothelium-dependent relaxation in arteries from pregnant rats.