Alma Barajas-Espinosa

Two dissimilar AT1 agonists distinctively activate AT1 receptors located on the luminal membrane of coronary endothelium

Diverse intracoronary agonists cause cardiac effects while acting on coronary endothelial luminal membrane (CELM) receptor. Our data show: a) the presence of AT(1)R in isolated CELM and in all cardiac cell types and b) sustained intracoronary infusions of Ang II-POL, a large sized molecule (approximately 15,000 kDa) confined to the vessel lumen that can only act on CELMs AT(1)R or Ang II (approximately 1 kDa); both exert the same maximum positive inotropic (PIE) and coronary constriction (CPP). The effects of these two agonists are blocked by Losartan and by Sar-POL; a large size antagonist (approximately 15,000 kDa) that acts only on CELM. Ang II effects are transient due to desensitization and cause tachyphylaxis to Ang II and toward Ang II-POL suggesting that both Ang II and Ang II-POL act on the same receptor group. In contrast, Ang II-POL effects are sustained and do not cause tachyphylaxis. The results show that intravascular Ang II and Ang II-POL act differentially by an unknown mechanism on CELMs AT(1)R and suggest that intravascular Ang II and Ang II-POL cause PIE and CCP by activation limited to CELMs AT(1)R through an unknown mechanism that is space-confined to the CELMs AT(1)R.

Luminal endothelial lectins with affinity for N-acetylglucosamine determine flow-induced cardiac and vascular paracrine-dependent responses.

Coronary blood flow applied to the endothelial lumen modulates parenchymal functions via paracrine effectors, but the mechanism of flow sensation is unknown. We and others have demonstrated that coronary endothelial luminal membrane (CELM) oligosaccharides and lectins are involved in flow detection, and we proposed that cardiac effects of coronary flow result from a reversible flow-modulated lectin-oligosaccharide interaction. Recently, glycosylated and amiloride-sensitive Na(+)/Ca(++) channels (ENaCs) have been proposed to be involved in the flow-induced endothelial responses. Because N-acetylglucosamine (GlcNac) is one of the main components of glycocalyx oligosaccharides (i.e., hyaluronan [-4GlcUAbeta1-3GlcNAcbeta1-](n)), the aim of this article is to isolate and define CELM GlcNac-binding lectins and determine their role in cardiac and vascular flow-induced effects. For this purpose, we synthesized a 460-kDa GlcNac polymer (GlcNac-Pol) with high affinity toward GlcNac-recognizing lectins. In the heart, intracoronary administration of GlcNac-Pol upon binding to CELM diminishes the flow-dependent positive inotropic and dromotropic effects. Furthermore, GlcNac-Pol was used as an affinity probe to isolate CELM GlcNac-Pol-recognizing lectins and at least 35 individual lectinic peptides were identified, one of them the beta-ENaC channel. Some of these lectins could participate in flow sensing and in GlcNac-Pol-induced effects. We also adopted a flow-responsive and well-accepted model of endothelial-parenchymal paracrine interaction: isolated blood vessels perfused at controlled flow rates. We established that flow-induced vasodilatation (FIV) is blocked by endothelial luminal membrane (ELM) bound GlcNac-Pol, nitro-l-arginine methyl ester and indomethacin, amiloride, and hyaluronidase. The effect of hyaluronidase was reversed by infusion of soluble hyaluronan. These results indicate that GlcNac-Pol inhibits FIV by competing and displacing intrinsic hyaluronan bound to a lectinic structure such as the amiloride-sensitive ENaC. Nitric oxide and prostaglandins are the putative paracrine mediators of FIV.

ENZYMATIC HYDROLYSIS OF LUMINAL CORONARY GLYCOSIDIC STRUCTURES UNCOVERS THEIR ROLE IN SENSING CORONARY FLOW

Endothelial luminal glycocalyx (ELG) is a multifunctional complex structure made off of a diversity of glycosilated proteins, and glycosaminoglycans (GAG). Coronary ELG may participate as a sensor of coronary flow (CF) to induce inotropic and dromotropic effects. In isolated perfused guinea pig heart we tested the role of glycosidic groups of glycans bound to proteins and GAG of the ELG on CF-induced inotropic and dromotropic effects. To study the role of saccharide related groups of certain glycans, they were removed by selective enzyme hydrolysis or bound to a selective plant lectin. CF-induced positive inotropic and positive dromotropic control curves were obtained and the effects of intracoronary infusion of enzyme or lectin determined. The analyzed groups were as follow: 1) Fucosidase enzyme and Ulex europeasus lectin; hydrolysis and binding respectively (H&Br) to alpha-linked fucosyl related groups. 2). Endoglycanase-H and Lycopersicon esculentum (H&Br to N-linked beta-1,3GlcNAc related groups). 3) O-glycanase and Arachis hypogea (H&Br to O-linked beta-Gal1, 3GalNac related groups). 4) Sialidase and Maackia amurensis (H&Br to neuraminic acid related groups). In treatments 1-3 both. lectin and corresponding enzyme, equally depressed CF-positive dromotropic effects without affecting positive inotropic effects. In treatment 4 both lectin and enzyme equally depressed CF-positive inotropic effects without dromotropic effects. The differential role of GAG hyaluran or heparan groups on CF-positive inotropism and positive dromotropism respectively was shown. Infusing hyaluranidase removed hyaluran that solely inhibited CF- inotropism while removal of heparan with heparinase solely inhibited CF-dromotropism. Only the effects of hyaluronidase were reversed infusing hyaluronidate. Our results indicate glycans of ELG are elements of complex multimolecular sensors of coronary flow.

Cardiac Ischemia and Ischemia/Reperfusion Cause Wide Proteolysis of the Coronary Endothelial Luminal Membrane: Possible Dysfunctions

Background: Ischemia and ischemia-reperfusion (I/R) are common clinical insults that disrupt the molecular structure of coronary vascular endothelial luminal membrane (VELM) that result in diverse microvasculature dysfunctions. However, the knowledge of the associated biochemical changes is meager. We hypothesized that ischemia and I/R-induced structural and functional VELM alterations result from biochemical changes. First, these changes need to be described and later the mechanisms behind be identified. Methods: During control conditions, in isolated perfused rat hearts VELM proteins were labeled with biotin. The groups of hearts were: control (C), no flow ischemia (I; 25 min), and I/R (I; 25 min, reperfusion 30 min). The biotinylated luminal endothelial membrane proteins in these three different groups were examined by 2-D electrophoresis and identified. But, it must be kept in mind the proteins were biotin-labeled during control. Results: A comparative analysis of the protein profiles under the 3 conditions following 2D gel electrophoresis showed differences in the molecular weight distribution such that MWC > MWI > MWI/R. Similar analysis for isoelectric points (pHi) showed a shift toward more acidic pHi under ischemic conditions. Of 100 % proteins identified during control 66% and 88% changed their MW-pHi during ischemia and I/R respectively. Among these lost proteins there were 9 proteins identified as adhesins and G-protein coupled receptors. General significance: I and I/R insults alter MW-pHi of most luminal glycocalyx proteins due to the activation of nonspecific hydrolizing mechanisms; suspect metalloproteases and glycanases. This makes necessary the identification of hydrolyzing enzymes reponsible of multiple microvascular dysfunctions in order to maintain the integrity of vascular endothelial membrane. VELM must become a target of future therapeutics.

Recreating Intestinal Peristalsis in the Petri Dish

Here we describe a culture technique of cells dissociated from the external muscularis of the guinea pig small intestine, which allows us to maintain all the elements involved in the intestinal peristaltic reflex. After a few days in culture, these cells reorganize to form a small group of cells that permit the generation of pacemaker activity, spontaneous contractions, and the development of inhibitory and excitatory junction potentials in the petri dish, all elements involved in the peristaltic reflex. Therefore, these co-cultures are suitable to study the cellular and molecular aspects related to the development, maintenance, and modulation of motor intestinal functions.

Physiological Concentrations of Zinc Have Dual Effects on P2X Myenteric Receptors of Guinea Pig

We, hereby, characterize the pharmacological effects of physiological concentrations of Zinc on native myenteric P2X receptors from guinea-pig small intestine and on P2X2 isoforms present in most myenteric neurons. This is the first study describing opposite effects of Zinc on these P2X receptors. It was not possible to determine whether both effects were concentration dependent, yet the inhibitory effect was mediated by competitive antagonism and was concentration dependent. The potentiating effect appears to be mediated by allosteric changes induced by Zinc on P2X myenteric channels, which is more frequently observed in myenteric neurons with low zinc concentrations. In P2X2-1 and P2X2-2 variants, the inhibitory effect is more common than in P2X myenteric channels. However, in the variants, the potentiatory effect is of equal magnitude as the inhibitory effect. Inhibitory and potentiatory effects are likely mediated by different binding sites that appear to be present on both P2X2 variants. In conclusion, in myenteric native P2X receptors, Zinc has quantitatively different pharmacological effects compared to those observed on homomeric channels: P2X2-1 and P2X2-2. Potentiatory and inhibitory Zinc effects upon these receptors are mediated by two different binding sites. All our data suggest that myenteric P2X receptors have a more complex pharmacology than those of the recombinant P2X2 receptors, which is likely related to other subunits known to be expressed in myenteric neurons. Because these dual effects occur at Zinc physiological concentrations, we suggest that they could be involved in physiological and pathological processes.