Role of beta-1 and beta-2 adrenoceptors in isoproterenol-induced renin release in conscious dogs.

The present experiments were designed to classify the ß-adrenoceptors pertaining to the renin release induced by isoproterenol in conscious dog. Atenolol (ß-1 adrenoceptor antagonist), in oral dose of 6 mg/kg, produced a significant inhibition of renin release caused by isoproterenol. This dose of atenolol suppressed effectively the tachycardia of isoproterenol. On the other hand, the renin release produced by isoproterenol was not modified significantly by a ß-2 adrenoceptor antagonist, IPS-339, at a oral dose of 3 mg/kg which fully antagonized hypotensive response to isoproterenol. These results strongly suggest that the renin release induced by isoproterenol is largely due to stimulation of ß-1 type adrenoceptors.     

Enteric alpha-2 adrenoceptors: pathophysiological implications in functional and inflammatory bowel disorders

The gastrointestinal tract is innervated by extrinsic noradrenergic nerves which regulate various digestive functions, including mucosal secretions, bowel propulsion and gut sensations, via activation of alpha2-adrenoceptors. These receptors are mostly involved in the prejunctional modulation of enteric neurotransmission, but they act also at extra-neural postjunctional sites. Alpha2-adrenoceptor population consists of distinct subtypes, designated as alpha2A, alpha2B and alpha2C, endowed with different physiological and pharmacological properties, and the attempts to classify alpha2-adrenoceptors at gastrointestinal level have indicated a large predominance of alpha2A subtypes. Studies in humans have shown a favourable influence of alpha2-adrenoceptor activation on colonic tone and sensation, and there is clinical evidence indicating that alpha2-agonists can improve intestinal functions and induce a satisfactory relief of symptoms in patients with irritable bowel syndrome. In addition, genetic investigations have highlighted significant associations of alpha2-adrenoceptor gene polymorphisms with constipation and somatic symptoms in functional disorders of lower digestive tract. Post-operative ileus is a common surgical complication characterized by severe alteration of gut motility, resulting mainly from neurogenic and inflammatory mechanisms. Experiments in models of post-operative ileus have demonstrated an intense expression of alpha2-adrenoceptors in monocytes recruited into the intestinal muscularis, and provided consistent evidence that these receptors promote post-operative gut dysfunctions by hampering enteric neurotransmission and contributing to local inflammatory reaction. Changes in the enteric nervous system are being increasingly recognized also as major determinants of digestive symptoms associated with bowel inflammation. In this regard, studies based on functional and molecular approaches concur in suggesting that the expression of enteric alpha2-adrenoceptors is up-regulated in the presence of intestinal inflammation, and that alpha2-mediated mechanisms are responsible for gut motor alterations occurring at both inflamed and non-inflamed sites. The present review discusses pathophysiological implications of enteric alpha2-adrenoceptors, in the attempt to highlight potential therapeutic applications for drugs targeted on these receptors.     

Ethnological significance of hemoglobin alpha-2 beta-2 121 LYS

It has been tacitly inferred that Hb α₂β₂ 121 LYS, sometimes called Hb O Arab, is associated with the Arabs. By analysis of published literature, however, it appears that such a linkage may not be justified.     

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.     

Implication of ENaC in salt-sensitive hypertension

Arterial blood pressure is critically dependent on sodium balance. The kidney is the key player in maintaining sodium homeostasis. Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the highly selective, amiloride-sensitive epithelial sodium channel (ENaC). Direct evidence that dysfunction of ENaC participates in blood pressure regulation has come from the molecular analysis of two human genetic diseases, Liddle’s syndrome and pseudohypoaldosteronism type 1 (PHA-1). Both, increased sodium reabsorption despite low aldosterone levels in Liddle’s patients and decreased sodium reabsorption despite high aldosterone levels in PHA-1 patients, demonstrated that ENaC is an effector for aldosterone action. Gene-targeting and classical transgenic technology enable the generation of mouse models for these diseases and the analysis of the involvement of the epithelial sodium channel (ENaC) in the progress of these diseases. A first mouse model using ENaC transgenic knockout mice [ENaC(−/−)Tg] mimicked several clinical features of PHA-1, like salt-wasting, metabolic acidosis, high aldosterone levels, growth retardation and increased early mortality. Such mouse models will be necessary in testing the involvement of genetic and/or environmental factors like salt-intake in hypertension.     

Abnormal magnesium metabolism in etiology of salt-sensitive hypertension and type 2 diabetes mellitus

A previously unknown genetic defect in magnesium metabolism (i.e., the magnesium-binding defect [MgBD]) was found to be associated with the cause of “salt-sensitive” essential hypertension in humans and rats. It inhibits the entrance of Mg²⁺ into the cell so that the intracellular concentrations of Mg²⁺ and MgATP²⁻ are decreased. Consequently, the 300 enzyme reactions in the cell, especially the 100 that either use or produce MgATP²⁻, are inhibited. Thus, because the extrusion of intracellular Na⁺ requires MgATP²⁻, hypertension results when the involved MgATP²⁻ requiring enzyme is inhibited. The MgBD is corrected by the tachykinin substance P, which occurs in normal blood plasma, and by the pentapeptide and its contained tetrapeptide, which are released from the C-terminal region of substance P by plasma aminopeptidases. In vivo, the intravenous administration of the tetrapeptide corrects the hypertension and the MgBD as well. The MgBD also occurs in type 2 diabetes mellitus and, thus, the decreased intracellular concentrations of Mg²⁺ and MgATP²⁻ ions appear to be involved also in the cause of this disease, which is reputed to be the fifth most deadly disease in the world.     

Effect of glucocorticoids on alpha-2 adrenoceptors in vas deferens of reserpinized rat in organ culture

The changes in alpha adrenoceptors in vasa deferentia of reserpinized rats in organ culture were examined by binding studies with 3H-clonidine, 3H-yohimbine and 3H-prazosin. On organ culture for 1 day, the 3H-clonidine binding sites decreased by 1.07 pmol/g tissue and after 2 days no binding sites were detectable. 3H-Yohimbine binding sites also decreased during culture, but in smaller extent than 3H-clonidine binding sites. On the other hand, 3H-prazosin binding sites showed no apparent change in amount during culture. Addition of 0.1 mM hydrocortisone or dexamethasone to the culture medium inhibited the decrease in 3H-clonidine binding sites (1.7 pmol/g tissue), and the effect of glucocorticoid was blocked by the inhibitors of protein synthesis, cycloheximide and puromycin. However, hydrocortisone showed no effect on the 3H-yohimbine and the 3H-prazosin binding sites. An anti-endocytotic agent, an anti-microtubular agent and protease inhibitors had no effects on the decrease of 3H-clonidine binding sites during culture. These results suggest that the amount of alpha-2 adrenoceptors can change rapidly while alpha-1 adrenoceptors are stable and that glucocorticoids are important in regulation of conformation of alpha-2 adrenoceptor through synthesis of certain protein(s).     

alpha- and betaCaMKII. Inverse regulation by neuronal activity and opposing effects on synaptic strength

We show that alpha and betaCaMKII are inversely regulated by activity in hippocampal neurons in culture: the alpha/beta ratio shifts toward alpha during increased activity and beta during decreased activity. The swing in ratio is approximately 5-fold and may help tune the CaMKII holoenzyme to changing intensities of Ca(2+) signaling. The regulation of CaMKII levels uses distinguishable pathways, one responsive to NMDA receptor blockade that controls alphaCaMKII alone, the other responsive to AMPA receptor blockade and involving betaCaMKII and possibly further downstream effects of betaCaMKII on alphaCaMKII. Overexpression of alphaCaMKII or betaCaMKII resulted in opposing effects on unitary synaptic strength as well as mEPSC frequency that could account in part for activity-dependent effects observed with chronic blockade of AMPA receptors. Regulation of CaMKII subunit composition may be important for both activity-dependent synaptic homeostasis and plasticity.     

Renal NF-kappaB activation and TNF-alpha upregulation correlate with salt-sensitive hypertension in Dahl salt-sensitive rats

Molecular mechanisms of salt-sensitive (SS) hypertension related to renal inflammation have not been defined. We seek to determine whether a high-salt (HS) diet induces renal activation of NF-kappaB and upregulation of TNF-alpha related to the development of hypertension in Dahl SS rats. Six 8-wk-old male Dahl SS rats received a HS diet (4%), and six Dahl SS rats received a low-sodium diet (LS, 0.3%) for 5 wk. In the end, mean arterial pressure was determined in conscious rats by continuous monitoring through a catheter placed in the carotid artery. Mean arterial pressure was significantly higher in the HS than the LS group (177.9 +/- 3.7 vs. 109.4 +/- 2.9 mmHg, P < 0.001). There was a significant increase in urinary albumin secretion in the HS group compared with the LS group (22.3 +/- 2.6 vs. 6.1 +/- 0.7 mg/day; P < 0.001). Electrophoretic mobility shift assay demonstrated that the binding activity of NF-kappaB p65 proteins in the kidneys of Dahl SS rats was significantly increased by 53% in the HS group compared with the LS group (P = 0.007). ELISA indicated that renal protein levels of TNF-alpha, but not IL-6, interferon-gamma, and CCL28, were significantly higher in the HS than the LS group (2.3 +/- 0.8 vs. 0.7 +/- 0.2 pg/mg; P = 0.036). We demonstrated that plasma levels of TNF-alpha were significantly increased by fivefold in Dahl SS rats on a HS diet compared with a LS diet. Also, we found that increased physiologically relevant sodium concentration (10 mmol/l) directly stimulated NF-kappaB activation in cultured human renal proximal tubular epithelial cells. These findings support the hypothesis that activation of NF-kappaB and upregulation of TNF-alpha are the important renal mechanisms linking proinflammatory response to SS hypertension.     

Hemoglobin variant found in Koreans, Chinese, and North American Indians: alpha-2 beta-2 22 Glu Ala

Hemoglobin G Taegu, an electrophoretically slow hemoglobin variant found in four among 6700 apparently normal Korean subjects, has been shown to have a structural anomaly at position 22 of the beta-chain where an alanyl residue occurs in place of the glutamyl group normally found at that position in Hemoglobin A. The same structural anomaly initially was established by other workers in slow hemoglobin variants occurring in North American Indians and more recently has been reported in a Northern Chinese subject. The identical hemoglobins in the three ethnic groups are Hemoglobins G Coushatta, found in several Alabama-Coushatta Indians in Tex.; G Saskatoon, seen in a few descendants of Santee Indians currently living in Canada; G Hsin-Chu, in a Chinese from the northern province of Liaoning and currently living in Taiwan; and G Taegu in Koreans. It is assumed that the Chinese and Korean subjects have the same hemoglobin variant because of gene flow. No similar assumption connecting these two groups with the North American Indian subjects is considered warranted with the presently limited available information.     

Interactions between oxidative stress and inflammation in salt-sensitive hypertension

The goal of this study was to test the hypothesis that increases in oxidative stress in Dahl S rats on a high-salt diet help to stimulate renal nuclear factor-kappaB (NF-kappaB), renal proinflammatory cytokines, and chemokines, thus contributing to hypertension, renal damage, and dysfunction. We specifically studied whether antioxidant treatment of Dahl S rats on high Na intake would decrease renal inflammation and thus attenuate the hypertensive and adverse renal responses. Sixty-four 7- to 8-wk-old Dahl S or R/Rapp strain rats were maintained for 5 wk on high Na (8%) or high Na + vitamins C (1 g/l in drinking water) and E (5,000 IU/kg in food). Arterial and venous catheters were implanted at day 21. By day 35 in the high-Na S rats, antioxidant treatment significantly increased the renal reduced-to-oxidized glutathione ratio and decreased renal cortical H(2)O(2) and O(2)(*-) release and renal NF-kappaB. Antioxidant treatment with vitamins C and E in high-Na S rats also decreased renal monocytes/macrophages in the glomeruli, cortex, and medulla, decreased tumor necrosis factor-alpha by 39%, and decreased monocyte chemoattractant protein-1 by 38%. Vitamin-treated, high-Na S rats also experienced decreases in arterial pressure, urinary protein excretion, renal tubulointerstitial damage, and glomerular necrosis and increases in glomerular filtration rate and renal plasma flow. In conclusion, antioxidant treatment of high-Na Dahl S rats decreased renal inflammatory cytokines and chemokines, renal immune cells, NF-kappaB, and arterial pressure and improved renal function and damage.     

Synthesis of alpha-, beta- and cyclic spaglumic acids.

A short, one-pot synthesis of alpha- and beta-spaglumic acids (N-acetyl-L-aspartyl-L-glutamic acids, NAAGA) has been developed based on ultrasound-promoted acetylation of aspartic acid, followed by dehydration, condensation with glutamic acid dibenzyl ester and hydrogenolysis. The alpha- and beta-peptides were separated by anion-exchange chromatography. The alpha-peptide shows a remarkable tendency to cyclize during methylation with diazomethane and yields cyclic N-acetylaspartylglutamic acid dimethyl ester, which could be hydrolysed to the hitherto unreported diketopiperazine dicarboxylic acid, cyclic spaglumic acid (cyclic NAAGA).     

Factor XIII binds to the A alpha- and B beta- chains in the D-domain of fibrinogen: an immunoblotting study

The binding sites in fibrinogen for Factor XIII were localized using an immunoblotting technique. Platelet Factor XIII bound to fibrinogen and to plasmin degradation products of fibrin(ogen) including Fragments: X, D1-D3, and D-dimer, but did not bind to Fragment E. Binding of Platelet Factor XIII was independent of calcium ions but could be inhibited by the presence of 0.5 M NaCl. Binding could also be inhibited by preincubating Factor XIII with a 100-fold molar excess of fibrinogen but not by 100-fold molar excess of Fragment E. Binding of Factor XIII to fibrinogen was specific, since several other proteins tested (ovalbumin, bovine serum albumin, alpha 2-macroglobulin, beta-galactosidase, fructose kinase, lactic dehydrogenase, triose phosphate isomerase, fumarase and pyruvate kinase) did not bind Factor XIII. Furthermore, binding was not observed either when Factor XIII was left out or when antiFactor XIII antiserum was substituted with nonimmune serum. When fibrinogen was reduced prior to electrophoresis, Factor XIII bound to the A alpha and B beta chains of fibrinogen and des A,B fibrinogen, the B beta-chain of Fragment X, but not the gamma-chains. Localization of the Factor XIII binding sites to the carboxy terminal segments of the A alpha and B beta chains in the Fragment D-domain of fibrinogen could have important physiological consequences.     

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin?phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha₁ adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ?prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha₂ adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha₁ and alpha₂ adrenoceptors but about 90% of the binding sites were alpha₂. These data support the hypothesis that alpha₂ effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha₁ adrenoceptor activation.     

Genes involved in vasoconstriction and vasodilation system affect salt-sensitive hypertension

The importance of excess salt intake in the pathogenesis of hypertension is widely recognized. Blood pressure is controlled primarily by salt and water balance because of the infinite gain property of the kidney to rapidly eliminate excess fluid and salt. Up to fifty percent of patients with essential hypertension are salt-sensitive, as manifested by a rise in blood pressure with salt loading. We conducted a two-stage genetic analysis in hypertensive patients very accurately phenotyped for their salt-sensitivity. All newly discovered never treated before, essential hypertensives underwent an acute salt load to monitor the simultaneous changes in blood pressure and renal sodium excretion. The first stage consisted in an association analysis of genotyping data derived from genome-wide array on 329 subjects. Principal Component Analysis demonstrated that this population was homogenous. Among the strongest results, we detected a cluster of SNPs located in the first introns of PRKG1 gene (rs7897633, p = 2.34E-05) associated with variation in diastolic blood pressure after acute salt load. We further focused on two genetic loci, SLC24A3 and SLC8A1 (plasma membrane sodium/calcium exchange proteins, NCKX3 and NCX1, respectively) with a functional relationship with the previous gene and associated to variations in systolic blood pressure (the imputed rs3790261, p = 4.55E-06; and rs434082, p = 4.7E-03). In stage 2, we characterized 159 more patients for the SNPs in PRKG1, SLC24A3 and SLC8A1. Combined analysis showed an epistatic interaction of SNPs in SLC24A3 and SLC8A1 on the pressure-natriuresis (p interaction = 1.55E-04, p model = 3.35E-05), supporting their pathophysiological link in cellular calcium homeostasis. In conclusions, these findings point to a clear association between body sodium-blood pressure relations and molecules modulating the contractile state of vascular cells through an increase in cytoplasmic calcium concentration.