Connexins and M3 muscarinic receptors contribute to heterogeneous Ca(2+) signaling in mouse aortic endothelium.

BACKGROUND/AIMS: Smooth muscle tone is controlled by Ca(2+) signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40) and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca(2+) signaling of the mouse aorta. METHODS: Ca(2+) imaging was performed on intact aortic endothelium from both wild type (Cx40+/+) and Connexin40-deficient (Cx40 -/-) mice. RESULTS: Acetylcholine (ACh) induced early fast and high amplitude Ca(2+) transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca(2+) transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca(2+) waves, indicating that Cx40 contributes to the spreading of Ca(2+) signals. The propagation of those Ca(2+) responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca(2+) waves. CONCLUSIONS: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca(2+) signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Perinatal hypoxia enhances cyclic adenosine monophosphate-mediated BKCa channel activation in adult murine pulmonary artery.

Exposure to perinatal hypoxia results in alteration of the adult pulmonary circulation, which is linked among others to alterations in K(+) channels in pulmonary artery (PA) smooth muscle cells. In particular, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels protein expression and activity were increased in adult PA from mice born in hypoxia compared with controls. We evaluated long-term effects of perinatal hypoxia on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway-mediated activation of BK(Ca) channels, using isoproterenol, forskolin, and dibutyryl-cAMP. Whole-cell outward current was higher in pulmonary artery smooth muscle cells from mice born in hypoxia compared with controls. Spontaneous transient outward currents, representative of BK(Ca) activity, were present in a greater proportion in pulmonary artery smooth muscle cells of mice born in hypoxia than in controls. Agonists induced a greater relaxation in PA of mice born in hypoxia compared with controls, and BK(Ca) channels contributed more to the cAMP/PKA-mediated relaxation in case of perinatal hypoxia. In summary, perinatal hypoxia enhanced cAMP-mediated BK(Ca) channels activation in adult murine PA, suggesting that this pathway could be a potential target for modulating adult pulmonary vascular tone after perinatal hypoxia.

Identification and functional response of interstitial Cajal-like cells from rat mesenteric artery.

Cells with irregular shapes, numerous long thin filaments, and morphological similarities to the gastrointestinal interstitial cells of Cajal (ICCs) have been observed in the wall of some blood vessels. These ICC-like cells (ICC-LCs) do not correspond to the other cell types present in the arterial wall: smooth muscle cells (SMCs), endothelial cells, fibroblasts, inflammatory cells, or pericytes. However, no clear physiological role has as yet been determined for ICC-LCs in the vascular wall. The aim of this study has been to identify and characterize the functional response of ICC-LCs in rat mesenteric arteries. We have observed ICC-LCs and identified them morphologically and histologically in three different environments: isolated artery, freshly dispersed cells, and primary-cultured cells from the arterial wall. Like ICCs but unlike SMCs, ICC-LCs are positively stained by methylene blue. Cells morphologically resembling methylene-blue-positive cells are also positive for the ICC and ICC-LC markers α-smooth muscle actin and desmin. Furthermore, the higher expression of vimentin in ICC-LCs compared with SMCs allows a clear discrimination between these two cell types. At the functional level, the differences observed in the variations of cytosolic free calcium concentration of freshly dispersed SMCs and ICC-LCs in response to a panel of vasoactive molecules show that ICC-LCs, unlike SMCs, do not respond to exogenous ATP and [Arginine](8)-vasopressin.

Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta.

Upon agonist stimulation, endothelial cells trigger smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). Endothelial cells of mouse aorta are interconnected by gap junctions made of connexin40 (Cx40) and connexin37 (Cx37), allowing the exchange of signaling molecules to coordinate their activity. Wild-type (Cx40(+/+)) and hypertensive Cx40-deficient mice (Cx40(-/-)), which also exhibit a marked decrease of Cx37 in the endothelium, were used to investigate the link between the expression of endothelial connexins (Cx40 and Cx37) and endothelial nitric oxide synthase (eNOS) expression and function in the mouse aorta. With the use of isometric tension measurements in aortic rings precontracted with U-46619, a stable thromboxane A(2) mimetic, we first demonstrate that ACh- and ATP-induced endothelium-dependent relaxations solely depend on NO release in both Cx40(+/+) and Cx40(-/-) mice, but are markedly weaker in Cx40(-/-) mice. Consistently, both basal and ACh- or ATP-induced NO production were decreased in the aorta of Cx40(-/-) mice. Altered relaxations and NO release from aorta of Cx40(-/-) mice were associated with lower expression levels of eNOS in the aortic endothelium of Cx40(-/-) mice. Using immunoprecipitation and in situ ligation assay, we further demonstrate that eNOS, Cx40, and Cx37 tightly interact with each other at intercellular junctions in the aortic endothelium of Cx40(+/+) mice, suggesting that the absence of Cx40 in association with altered Cx37 levels in endothelial cells from Cx40(-/-) mice participate to the decreased levels of eNOS. Altogether, our data suggest that the endothelial connexins may participate in the control of eNOS expression levels and function.

Potential detrimental effects of a phytoestrogen-rich diet on male fertility in mice.

Soy and soy-based products are widely consumed by infants and adult individuals. There has been speculation that the presence of isoflavone phytoestrogens in soybean cause adverse effects on the development and function of the male reproductive system. The purpose of this study was to examine the influence of dietary soy and phytoestrogens on testicular and reproductive functions. Male mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Although adult mice fed a soy-rich diet exhibited normal male behaviour and were fertile, we observed a reduced proportion of haploid germ cells in testes correlating with a 25% decrease in epididymal sperm counts and a 21% reduction in litter size. LH and androgens levels were not affected but transcripts coding for androgen-response genes in Sertoli cells and Gapd-s, a germ cell-specific gene involved in sperm glycolysis and mobility were significantly reduced. In addition, we found that dietary soy decreased the size of the seminal vesicle but without affecting its proteolytic activity. Taken together, these studies show that long-term exposure to dietary soy and phytoestrogens may affect male reproductive function resulting in a small decrease in sperm count and fertility.

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta.

During an agonist stimulation of endothelial cells, the sustained Ca2+ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca2+ stores depletion to the opening of such channels are still elusive. We have used Ca2+ and tension measurements in intact aortic strips to investigate the role of the Ca2+-independent isoform of phospholipase A2 (iPLA2) in endothelial store-operated Ca2+ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca2+ entry when Ca2+ stores are artificially depleted. We also show that the sustained store-operated Ca2+ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca2+ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA2 upon Ca2+ stores depletion may act as an intracellular messenger that stimulates store-operated Ca2+ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca2+ entry is a key physiological mechanism regulating arterial tone.

Intracellular cAMP: the "switch" that triggers on "spontaneous transient outward currents" generation in freshly isolated myocytes from thoracic aorta.

Spontaneous transient outward currents (STOCs) have been reported in resistance and small arteries but have not yet been found in thoracic aorta. Do thoracic aorta myocytes possess cellular machinery that generates STOCs? It was found that the majority of aortic myocytes do not generate STOCs. STOCs were generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not generate STOCs we have called "silent" myocytes and myocytes with STOCs have been called "active." STOCs recorded in active myocytes were voltage dependent and were inhibited by ryanodine, caffeine, and charybdotoxin. Forskolin was reported to increase STOCs frequency in myocytes isolated from resistance arteries. Forskolin (10 microM) triggered STOCs generation in 35.1% of silent aortic myocytes. In 36.8% percent of silent myocytes, forskolin did not trigger STOCs but increased the amplitude of charybdotoxin-sensitive outward net current to 136.1 +/- 8.5% at 0 mV. Membrane-permeable 8BrcAMP triggered STOCs generation in 38.7% of silent myocytes. Forskolin- or 8BrcAMP-triggered STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open probability of BK(Ca) channels in BAPTA-AM-pretreated cells. Our data demonstrate that, in contrast to resistance arteries, STOCs are present just in the minority of myocytes in the thoracic aorta. However, cellular machinery that generates STOCs can be "switched" on by cAMP. Such an inactive cellular mechanism could modulate the contractility of the thoracic aorta in response to physiological demand.

Effects of arterial wall stress on vasomotion.

Smooth muscle and endothelial cells in the arterial wall are exposed to mechanical stress. Indeed blood flow induces intraluminal pressure variations and shear stress. An increase in pressure may induce a vessel contraction, a phenomenon known as the myogenic response. Many muscular vessels present vasomotion, i.e., rhythmic diameter oscillations caused by synchronous cytosolic calcium oscillations of the smooth muscle cells. Vasomotion has been shown to be modulated by pressure changes. To get a better understanding of the effect of stress and in particular pressure on vasomotion, we propose a model of a blood vessel describing the calcium dynamics in a coupled population of smooth muscle cells and endothelial cells and the consequent vessel diameter variations. We show that a rise in pressure increases the calcium concentration. This may either induce or abolish vasomotion, or increase its frequency depending on the initial conditions. In our model the myogenic response is less pronounced for large arteries than for small arteries and occurs at higher values of pressure if the wall thickness is increased. Our results are in agreement with experimental observations concerning a broad range of vessels.

A delayed ATP-elicited K+ current in freshly isolated smooth muscle cells from mouse aorta.

Adenosine 5'-triphosphate (ATP) activated two sequential responses in freshly isolated mouse aortic smooth muscle cells. In the first phase, ATP activated Ca(2+)-dependent K(+) or Cl(-) currents and the second phase was the activation of a delayed outward current with a reversal potential of -75.9 +/- 1.4 mV. A high concentration of extracellular K(+) (130 mM) shifted the reversal potential of the delayed ATP-elicited current to -3.5 +/- 1.3 mV. The known K(+)-channel blockers, iberiotoxin, charybdotoxin, glibenclamide, apamin, 4-aminopyridine, Ba(2+) and tetraethylammonium chloride all failed to inhibit the delayed ATP-elicited K(+) current. Removal of ATP did not decrease the amplitude of the ATP-elicited current back to the control values. The simultaneous recording of cytosolic free Ca(2+) and membrane currents revealed that the first phase of the ATP-elicited response is associated with an increase in intracellular Ca(2+), while the second delayed phase develops after the return of cytosolic free Ca(2+) to control levels.ATP did not activate Ca(2+)-dependent K(+) currents, but did elicit Ca(2+)-independent K(+) currents, in cells dialyzed with ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The delay of activation of Ca(2+)-independent currents decreased from 10.5 + 3.4 to 1.27 +/- 0.33 min in the cells dialyzed with 2 mM EGTA. Adenosine alone failed to elicit a Ca(2+)-independent K(+) current but simultaneous application of ATP and adenosine activated the delayed K(+) current. Intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) transformed the Ca(2+)-independent ATP-elicited response from a sustained to a transient one. A phospholipase C inhibitor, U73122 (1 microM), was shown to abolish the delayed ATP-elicited response. These results indicate that the second phase of the ATP-elicited response was a delayed Ca(2+)-independent K(+) current activated by exogenous ATP. This phase might represent a new vasoregulatory pathway in vascular smooth muscle cells.

Characterization of purine receptors in mouse thoracic aorta.

The contracting and relaxing effects of purines and UTP were investigated on rings of mouse thoracic aorta in vitro. UTP, ATP gamma S, and alpha-beta-Methyleneadenosine 5'triphosphate contracted rings with and without endothelium. On the contrary, adenosine, AMP, ADP, ATP, and 2-(methylthio)adenosine 5'-diphosphate had no effect on relaxed rings. When rings were tonically contracted by U46619 a thromboxane A2 analogue, ATP, ADP, ATP gamma S, 2-(methylthio)adenosine 5'-diphosphate, and UTP caused endothelium-dependent but not independent relaxations.I conclude that ATP acts on P2Y2 and P2Y1 receptors on the endothelial cells to cause endothelium-dependent relaxation. In this tissue, the relaxing effect of ATP dominates by endothelium-dependent ways when aorta rings are contracted by a stable thromboxane A2 analog. However receptors mediating contraction in response to purines and pyrimidines are present on smooth muscle cells. Indeed, the stimulation of P2Y receptors by UTP as well as the activation of P2X family receptors by ATP gamma S causes a contraction. The potential contractile effect of ATP seems masked by its hydrolysis by ectonucleotidases.

Calcium imaging of murine thoracic aorta endothelium by confocal microscopy reveals inhomogeneous distribution of endothelial cells responding to vasodilator agents.

The aim of the study was to assess, in intact murine thoracic aorta in vitro, the distribution of endothelial cells responsive to endothelium-dependent vasodilators ACh, ATP, bradykinin and substance P, using laser line confocal microscopy in combination with two Ca2+ fluorescent dyes, Fluo-4 and Fura-red. We observed that 82 +/- 3% of endothelial cells responded to ATP, 33 +/- 5% to Ach, whereas less than 3% of them responded to bradykinin or substance P. In order to determine whether the findings of pharmacological tests agree with confocal microscopy data, endothelium-dependent vasodilators induced relaxation was evaluated using isometric tension measurement. We show a marked correlation between a higher number of activated endothelial cells, using confocal microscopy, and a greater degree of endothelium-dependent relaxation using isometric tension measurement (p = 0.00286). Our results suggest that endothelial cells responding to endothelium-dependent vasodilators are not homogeneously distributed in intact murine thoracic aorta. This could be due to nonhomogeneous distribution of surface receptors or to differences in post-receptor coupling mechanisms.