Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 (Cx40^–/–). In vivo, hypoxemia was more severe in Cx40^–/– mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40^–/–, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α_1G subtype Ca²⁺ channels, cytosolic phospholipase A₂, and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner.

Background— Gap junctions allow the direct exchange of ions and small molecules between cells in contact, thus coordinating physiological processes such as cell growth and differentiation. We have recently demonstrated increased expression of the gap junction protein connexin43 (Cx43) in specific subsets of cells in atherosclerotic lesions. Because the development of atherosclerosis depends critically on paracrine cell-to-cell interactions, we hypothesized that direct intercellular communication via gap junctions may be another factor controlling atherogenesis. Methods and Results— The role of Cx43 in atherogenesis was examined by use of both a genetic and a pharmacological approach. First, atherosclerosis-susceptible LDL receptor–deficient (LDLR ^−/− ) mice with normal (Cx43 ^+/+ ) or reduced (Cx43 ^+/− ) levels of Cx43 were fed a cholesterol-rich diet for 14 weeks. The progression of atherosclerosis was reduced by 50% ( P <0.01) in the thoracoabdominal aorta and in the aortic roots of Cx43 ^+/− LDLR ^−/− mice compared with Cx43 ^+/+ LDLR ^−/− controls. Atheroma in Cx43 ^+/− LDLR ^−/− mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells. Next, we observed that HMG-CoA reductase inhibitors, or “statins,” lipid-lowering drugs well known for their pleiotropic antiatherogenic effects, reduced Cx43 expression in primary human vascular cells in vitro. Atheroma of LDLR ^−/− mice treated orally with pravastatin contained fewer inflammatory cells and exhibited thicker fibrous caps than controls. This was associated with reduced Cx43 expression in lesions of statin-treated mice. Conclusions— These data indicate a critical role for Cx43-mediated gap junctional communication in atherosclerotic plaque formation.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

Background— Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. Methods and Results— Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. Conclusions— Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a “ball-and-chain” mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.