Connexin 43 (Cx43) is a protein expressed in a variety of mammalian tissues. However, the lack of specific blockers and the absence of known genetic mutants have hampered the investigation of the function of this protein. Cx43-null mice die shortly after birth, thus preventing functional studies in vivo. Here, we report the generation and characterization of a vascular endothelial cell-specific deletion of the Cx43 gene (VEC Cx43 KO) in mice by using the loxP͞Cre system. Using homologous recombination, a mouse line was created carrying loxP sites flanking exon 2 of the Cx43 gene (''floxed'' mice). To produce cell specific deletion of the Cx43 gene, these mice were crossed with animals from a line carrying the Tie 2-Cre transgene. The homozygous VEC Cx43 KO mice survived to maturity. However, they were hypotensive and bradycardic when compared with heterozygous VEC Cx43 KO mice, or to the floxed Cx43 gene mice. The hypotension was associated with marked elevation of plasma nitric oxide (NO) levels as well as elevated plasma angiotensin (Ang) I and II. We hypothesize that endothelial cell Cx43 plays a key role in the formation and͞or action of NO, and that the elevation of Ang II is a secondary event. The specific cellular basis for the hypotension remains to be established, but our findings support the idea that endothelial Cx43 gap junctions are involved in maintaining normal vascular function; moreover, these animals provide the opportunity to determine more clearly the role of endothelial Cx43 in vascular development and homeostasis.
Abstract-When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40 Ϫ/Ϫ ) mice to study vasomotor responses induced by 10-second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 m upstream). In wild-type (Cx40 ϩ/ϩ ) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40 Ϫ/Ϫ mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 mol/L) had no effect on the local vasoconstriction in either Cx40 ϩ/ϩ or Cx40 Ϫ/Ϫ mice, but enhanced the conducted vasodilation in Cx40 ϩ/ϩ animals. In Cx40 Ϫ/Ϫ mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40 Ϫ/Ϫ mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40 ϩ/ϩ mice and complete elimination of this connexin in the Cx40
Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.
In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.Abstract. Volunteers make an important contribution to Canadian society. What motivates people to volunteer, and do they respond to government expenditure decisions? We address this question with the use of a 1987 microdata set and find that while the level of government spending influences the decision to volunteer, it has no effect on the number of hours donated. Furthermore, the nature of this relationship depends upon the particular type of government expenditures. For instance, the number of volunteers contracts when expenditures on certain economy-related activities fall, while reductions in health care spending bring about an increased number of volunteers.
In the vessel wall, endothelial cells are metabolically and electrically coupled to each other and to the adjacent smooth muscle cells by gap junctions composed of connexins. Gap junctions may be formed from combinations of several different connexin proteins, and deletion of one connexin can lead to modification of the expression of another. To reveal a possible interaction between connexin40 (Cx40) and connexin43 (Cx43) in endothelium, we studied their distribution in vessels from C57Bl/6 and Cx40 knockout mice (Cx40-/-) using immunoblots and immunocytochemistry on aortic cross sections and en face whole mounts. En face preparations from C57Bl/6 mice revealed two distinct pools of Cx43, one pericellular and the other intracellular. Cx40 was largely restricted to the periphery of the cells, and in Cx40-/- mice it was, as expected, undetectable. In the Cx40-/- mice, total Cx43 protein was also modestly reduced (immunoblots), but there was a major redistribution of the protein within the cell. The pericellular component of Cx43 was rendered virtually undetectable, and the intracellular compartments were normal or even slightly elevated. Smooth muscle Cx43 was also reduced in the Cx40-/- animals. These findings indicate that the cellular distribution of Cx43 is dependent on the presence of Cx40, and in view of the profound effects on the pericellular pool of the Cx43, the findings suggest that interactions between Cx40 and Cx43 regulate communication between endothelial cells and perhaps between smooth muscle and endothelial cells as well.
Adipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in STZ-treated NOD-SCID mice. Co-culture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with pro-inflammatory cytokines. Analysis of hASC-derived factors revealed VEGF and TIMP-1 to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro pro-survival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with IL-1β, IFN-γ and TNF-α. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against β cell death in rodent and in vitro models of Type 1 diabetes through a combination of local paracrine as well as systemic effects.
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