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Background and Purpose: To analyse the relative contribution of β1 -, β2 - and β3 -adrenoceptors (Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/cGMP signalling pathways.Experimental Approach: Rat mesenteric resistance artery (MRA) and aorta were used to analyse the Adrb expression by real-time-PCR and immunohistochemistry, and for the pharmacological characterization of Adrb-mediated activity by wire myography and tissue nucleotide accumulation.Key Results: The mRNAs and protein for all Adrb were identified in endothelium and/or smooth muscle cells (SMCs) in both vessels. In MRA, Adrb1 signalled through cAMP, Adrb3 through both cAMP and cGMP, but Adrb2, did not activate nucleotide formation; isoprenaline relaxation was inhibited by propranolol (β1 , β2 ), CGP20712A (β1 ), and SQ22536 (adenylyl cyclase inhibitor), but not by ICI118,551 (β2 ), SR59230A (β3 ), ODQ (soluble guanylyl cyclase inhibitor), L-NAME or endothelium removal. In aorta, Adrb1 signalled through cAMP, while β2 - and β3 -subtypes through cGMP; isoprenaline relaxation was inhibited by propranolol, ICI118,551, ODQ, L-NAME, and to a lesser extent, by endothelium removal. CL316243 (β3 -agonist) relaxed aorta, but not MRA.Conclusion and Implication: Despite all three Adrb subtypes being found in both vessels, Adrb1, located in SMCs and acting through the adenylyl cyclase/cAMP pathway, are primarily responsible for vasodilatation in MRA. However, Adrb-mediated vasodilatation in aorta is driven by endothelial Adrb2 and Adrb3, but also by the Adrb2 present in SMCs, and is coupled to the NO/cGMP pathway. These results could help to understand the different physiological roles played by Adrb signalling in regulating conductance and resistance vessels. [ABSTRACT FROM AUTHOR]

Background and Purpose To analyse the relative contribution of β1-, β2- and β3-adrenoceptors ( Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/ cGMP signalling pathways. Experimental Approach Rat mesenteric resistance artery ( MRA) and aorta were used to analyse the Adrb expression by real-time -PCR and immunohistochemistry, and for the pharmacological characterization of Adrb-mediated activity by wire myography and tissue nucleotide accumulation. Key Results The mRNAs and protein for all Adrb were identified in endothelium and/or smooth muscle cells ( SMCs) in both vessels. In MRA, Adrb1 signalled through cAMP, Adrb3 through both cAMP and cGMP, but Adrb2, did not activate nucleotide formation; isoprenaline relaxation was inhibited by propranolol (β1, β2), CGP20712A (β1), and SQ22536 (adenylyl cyclase inhibitor), but not by ICI118,551 (β2), SR59230A (β3), ODQ (soluble guanylyl cyclase inhibitor), L- NAME or endothelium removal. In aorta, Adrb1 signalled through cAMP, while β2- and β3-subtypes through cGMP; isoprenaline relaxation was inhibited by propranolol, ICI118,551, ODQ, L- NAME, and to a lesser extent, by endothelium removal. CL316243 (β3-agonist) relaxed aorta, but not MRA. Conclusion and Implication Despite all three Adrb subtypes being found in both vessels, Adrb1, located in SMCs and acting through the adenylyl cyclase/ cAMP pathway, are primarily responsible for vasodilatation in MRA. However, Adrb-mediated vasodilatation in aorta is driven by endothelial Adrb2 and Adrb3, but also by the Adrb2 present in SMCs, and is coupled to the NO/ cGMP pathway. These results could help to understand the different physiological roles played by Adrb signalling in regulating conductance and resistance vessels. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1. EXPERIMENTAL APPROACH SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O2•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively. KEY RESULTS I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ETB mRNA expression, and down-regulated ETA mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ETB receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ETB receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ETB receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, Nω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O2•− production by increasing NADPH oxidase 1 (NOX-1) and p47phox mRNA. CONCLUSIONS AND IMPLICATIONS Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ETB receptors and decrease in NO bioavailability. [ABSTRACT FROM AUTHOR]

Ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM) produced a slow, sustained contractile response in rat aorta that only can be observed in Ca2+-containing solution. In Ca2+-free medium, no response to the drugs was obtained, which suggests that the contraction elicited in presence of Ca2+ is mainly due to the contribution of extracellular influx. This Ca2+ entry does not depend on the opening of dihydropyridine-dependent Ca2+-channels for nimodipine does not affect this. Noradrenaline (1 μM) induced a biphasic response in Ca2+-free medium that was mediated by two different Ca2+ compartments, one of which is common to caffeine (10 mM), and is also depleted by ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM). This compartment loses its Ca2+ content after long exposure (65 min) to Ca2+-free EDTA-containing solution and its refilling was also affected by the three agents tested. The other compartment depleted by noradrenaline, but not by caffeine, was also insensitive to ryanodine, thapsigargin and cyclopiazonic acid, and did not lose its Ca2+ after 65 min in Ca2+-free medium. Contractions induced by noradrenaline (1 μM) or caffeine (10 mM) in Ca2+-free medium were not affected by ryanodine, thapsigargin and cyclopiazonic acid when these agents were added 1 min before or during the response to each agonist. After depletion of internal Ca2+ stores sensitive to noradrenaline, an increase in the resting tone (IRT) of rat aorta was observed when Ca2+ was added again in absence of the agonist. This IRT was not affected by treatment with ryanodine, thapsigargin and cyclopiazonic acid, and represents a Ca2+ entry pathway dependent on the depletion of the noradrenaline-sensitive Ca2+ compartment. In conclusion, we can differentiate two Ca2+ entry pathways in rat aorta that depend on the previous depletion of two internal Ca2+ compartments: One corresponds to the classic capacitative Ca2+ entry model and is promoted by depletion of the internal pool sensitive to noradreanline, caffeine, ryanodine, thapsigargin and cyclopiazonic acid, the other is dependent only on depletion of an α1-adrenoceptor-sensitive Ca2+ pool. [ABSTRACT FROM AUTHOR]

In rat aorta, depletion of internal Ca2+ stores by addition of noradrenaline (1μM) induces a biphasic response (an initial phasic response and a tonic one) mediated by two different intracellular Ca2+ pools. This response cannot be repeated, suggesting a depletion of internal Ca2+ stores sensitive to noradrenaline. In absence of the agonist, this depletion is the signal for the entry of extracellular Ca2+, not only to refill the stores but also, under our experimental conditions, to activate the contractile proteins thus inducing an increase in the resting tone (IRT) that constitutes functional evidence of this Ca2+ entry. The ionic channels involved in the mechanism of the IRT have been studied in the present work. The fact that the addition of nimodipine (10–15– 10–11M) selectively inhibits the IRT suggests that this mechanical response is mediated by Ca2+ influx through dihydropyridine-sensitive Ca2+ channels. Moreover, the inhibitory action of nimodipine is attenuated by glibenclamide (10μM). Cromakalim (10–10–10–6M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10μM). These results relate the ATP-dependent K+ channels to the mechanism of the IRT. The refilling of the two internal Ca2+ compartments sensitive to noradrenaline was, like the IRT, altered in presence of the compounds tested, since the subsequent contractile response to noradrenaline was decreased. The present results suggest that nimodipine treatment inhibits the refilling of the Ca2+ compartment responsible for the tonic contraction induced by noradrenaline in Ca2+-free medium, whereas the refilling of the Ca2+ pool responsible for the phasic response to noradrenaline remained unaltered. Both the phasic and tonic responses to noradrenaline in Ca2+-free medium decreased after treatment with cromakalim. We can therefore assume that the refilling of both Ca2+ compartments sensitive to noradrenaline was inhibited. In conclusion, these results are consistent with the contraction of the rat aorta in response to noradrenaline in Ca2+-free medium consisting of an initial phasic response and a tonic one. The former is due to the release of internal Ca2+ from a compartment refilled through a special channel that is cromakalim but not dihydropyridine sensitive. The tonic response is due to Ca2+ release from another compartment refilled through a cromakalim- and dihydropyridine-sensitive Ca2+ channel. The Ca2+ entry through this latter channel intervenes in the IRT observed during the refilling of these stores previously depleted by noradrenaline, and the opening state of this channel is also modulated by ATP-dependent K2+ channels. [ABSTRACT FROM AUTHOR]

As a contribution to the understanding of the thermodynamic state of single salts in living systems, the activity coefficients of KCl were determined in concentrated bovine serum albumin (BSA) solutions. The concentration range studied was 0.01 to 0.5 M KCl and zero to 18% wt BSA, thus amply covering physiological conditions. The activity coefficients of the salt were measured using the EMF method with ion exchange membrane electrodes. Keeping the salt concentration constant, the activity coefficients of the salt decrease linearly with protein concentration, the effect being more pronounced for low salt content. The maximal deviations of the activity coefficients with respect to those in pure salt solution amount to ca. 40% for 0.01 M KCl and 18% wt BSA. The results were interpreted on the assumption of the superposition of three effects i.e. water bound to BSA molecules as 'non-solvent' water, specific Cl ion binding and the electrostatic interactions of the polyions with the salt ions. In view of the results it can be concluded that only a small portion of simple intracellular ions are bound, based on the assumption that the cytoplasm of living cells may be regarded as a concentrated protein-salt solution. [ABSTRACT FROM AUTHOR]

Here we describe a new HLA class II null allele at the DRB5 gene. Serologic HLA typing of a Spanish gypsy family rendered the following paternal haplotype: A2-Cblk-B52-Bw4-DR15-DQ5. However, DNA typing demonstrated the presence of a DRB5 gene in the haplotype DRB1*1502- DRB5*0102-DQB1*05031. Complete DRB5 cDNA sequencing revealed a DRB5*0102 allele with a deletion of two nucleotides at exon 2 (239-240) in codon 80. This change generates a frame shift leading to a stop codon at position 86, and could explain the lack of DR51 protein at the cell surface. This is the second DRB5 null allele described together with DRB5*0108N, raising the number of HLA alleles with an expression disorder. [ABSTRACT FROM AUTHOR]