Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+) measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, omega-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of approximately 50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.     

Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin

Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation.     

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> metalloproteinase TvMP50 is a monomeric Aminopeptidase P-like enzyme

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a “pseudo-homodimer” array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a “pita bread” fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.     

Differences in regulation of Ca2+-activated Cl- channels in colonic and parotid secretory cells

We investigatedthe regulation of Ca²⁺-activatedCl channels in cells fromthe human colonic cell line T84 and acinar cells from rat parotidglands. The participation of multifunctional Ca²⁺- and calmodulin-dependentprotein kinase (CaM kinase) II in the activation of these channels wasstudied using selective inhibitors of calmodulin and CaM kinase II.Ca²⁺-dependentCl currents were recordedusing the whole cell patch-clamp technique. Direct inhibition of CaMkinase II by 40 µM peptide 281-302 or by 10 µM KN-62, anotherCaM kinase inhibitor, did not block the Cl current in parotidacinar cells, whereas in T84 cells KN-62 markedly inhibited theCa²⁺-dependentCl current. We also usedthe calmodulin-binding domain peptide 290-309 (0.5 µM), whichcompetitively inhibits the activation of CaM kinase II. This peptidereduced the Cl current inT84 cells by ~70% but was without effect on the channels in parotidacinar cells. We conclude that theCa²⁺-dependentCl channels in T84 cellsare activated by CaM kinase II but that the channels in parotid acinarcells must be regulated by a fundamentally differentCa²⁺-dependent mechanism that doesnot utilize CaM kinase II or any calmodulin-dependent process.

     

Infection of J774A.1 with different Mycobacterium species induces differential immune and miRNA‐related responses

Macrophages act as a reservoir for Mycobacterium tuberculosis, producing latent infection in approximately 90% of infected people. In this study, J774A.1 mouse macrophage cell line response and microRNA (miRNA) expression during infection with the most relevant mycobacterial strains for humans (M. tuberculosis, M. bovis and M. bovis BCG) was explored. No significant differences in bacillary loads were observed between activate and naive macrophages infected with M. tuberculosis and M. bovis. Nitrite production inhibition and infection control were in accordance with the virulence of the strain. Expression of let‐7e, miR‐21, miR‐155, miR‐210 and miR‐223 was opposite in the two species and miR‐146b* and miR‐1224 expression seemed to be part of the general response to infection.     

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.