RESUMEN
BACKGROUND AND PURPOSE: The level of cell surface expression of the meningococcal vaccine antigen, Factor H binding protein (FHbp) varies between and within strains and this limits the breadth of strains that can be targeted by FHbp-based vaccines. The molecular pathway controlling expression of FHbp at the cell surface, including its lipidation, sorting to the outer membrane and export, and the potential regulation of this pathway have not been investigated until now. This knowledge will aid our evaluation of FHbp vaccines. EXPERIMENTAL APPROACH: A meningococcal transposon library was screened by whole cell immuno-dot blotting using an anti-FHbp antibody to identify a mutant with reduced binding and the disrupted gene was determined. KEY RESULTS: In a mutant with markedly reduced binding, the transposon was located in the lnt gene which encodes apolipoprotein N-acyl transferase, Lnt, responsible for the addition of the third fatty acid to apolipoproteins prior to their sorting to the outer membrane. We provide data indicating that in the Lnt mutant, FHbp is diacylated and its expression within the cell is reduced 10 fold, partly due to inhibition of transcription. Furthermore the Lnt mutant showed 64 fold and 16 fold increase in susceptibility to rifampicin and ciprofloxacin respectively. CONCLUSION AND IMPLICATIONS: We speculate that the inefficient sorting of diacylated FHbp in the meningococcus results in its accumulation in the periplasm inducing an envelope stress response to down-regulate its expression. We propose Lnt as a potential novel drug target for combination therapy with antibiotics. LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/farmacología , Neisseria meningitidis/efectos de los fármacos , Aciltransferasas/genética , Aciltransferasas/metabolismo , Ciprofloxacina/farmacología , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Mutación , Neisseria meningitidis/crecimiento & desarrollo , Neisseria meningitidis/metabolismo , Rifampin/farmacología , Relación Estructura-ActividadRESUMEN
Activation of the sarcolemmal Na(+)-H(+) exchanger (NHE) has been implicated as a mechanism of inotropic, arrhythmogenic, antiacidotic, and hypertrophic effects of alpha(1)-adrenoceptor (AR) stimulation. Although such regulation of sarcolemmal NHE activity has been shown to be selectively mediated through the alpha(1A)-AR subtype, distal signaling mechanisms remain poorly defined. We investigated the roles of various kinase pathways in alpha(1A)-AR-mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes. As an index of NHE activity, trans-sarcolemmal acid efflux rate (J(H)) was determined through microepifluorescence in single cells, during recovery from intracellular acidosis in bicarbonate-free conditions. Extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase (MAPK), and p90(rsk) activities were indexed on the basis of analysis of their phosphorylation status. In control cells, there was no change in J(H) in response to vehicle. Phenylephrine and A61603, an alpha(1A)-AR subtype-selective agonist, increased J(H), as well as cellular ERK and p90(rsk) activities. Neither agonist affected p38 activity, which was increased with sorbitol. The MAPK kinase inhibitor PD98059 abolished phenylephrine- and A61603-induced increases in J(H) and cellular ERK and p90(rsk) activities. In contrast, the PKC inhibitor GF109203X abolished phenylephrine- and A61603-induced increases in J(H) but failed to prevent the increases in ERK and p90(rsk) activities. Our findings suggest that alpha(1A)-AR-mediated stimulation of sarcolemmal NHE activity in rat ventricular myocytes requires activation of the ERK (but not the p38) pathway of the MAPK cascade and that the ERK-mediated effect may occur via p90(rsk). Activation of PKC is also required for alpha(1A)-AR-mediated NHE stimulation, but such regulation occurs through an ERK-independent pathway.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/enzimología , Proteína Quinasa C/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Sarcolema/enzimología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Ventrículos Cardíacos/química , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/enzimología , Imidazoles/farmacología , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Maleimidas/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/enzimología , Miocardio/química , Miocardio/citología , Presión Osmótica , Fenilefrina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Wistar , Sarcolema/química , Tetrahidronaftalenos/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: Polarized arrest, induced by tetrodotoxin (TTX) at an optimal concentration of 22 micromol/L, has been shown to reduce ionic imbalance and improve myocardial preservation compared with hyperkalemic (depolarized) arrest. Additional pharmacologic manipulation of ionic changes (involving inhibition of Na+ influx by the Na+/H+ exchanger [HOE694] and Na+/K+/2Cl- cotransporter [furosemide], and calcium desensitization [BDM]) may further improve long-term preservation. In this study, we (i) established optimal concentrations of each drug, (ii) determined additive effects of optimal concentrations of each drug and (iii) compared our optimal preservation solution to an established depolarizing cardioplegia (St Thomas' Hospital solution No 2: STH2) used during long-term hypothermic storage for clinical transplantation. METHODS: The isolated working rat heart, perfused with Krebs Henseleit (KH) buffer was used; cardiac function was measured after 20 min aerobic working mode perfusion. The hearts (n=6/group) were arrested with a 2 ml infusion (for 30 sec) of the polarizing (control) solution (22 micromol/L TTX in KH) or control+drug and subjected to 5 hr or 8 hr of storage at 7.5 degrees C in the arresting solution. Postischemic function during reperfusion was measured (expressed as percentage of preischemic function). RESULTS: Dose-response studies established optimal concentrations of HOE694 (10 micromol/L), furosemide (1.0 micromol/L) and BDM (30 mmol/L) in the polarizing (control) solution. Sequential addition to the control solution (Group I) of optimal concentrations of HOE694 (Group II), furosemide (Group III), and BDM (Group IV) were compared with STH2 (Group V); postischemic recovery of aortic flow was 29+/-7%, 49+/-6%*, 56+/-2%*, 76+/-3%*, and 25+/-6%, respectively (*P<0.05 vs. I and V). Creatine kinase leakage was lowest, and myocardial ATP content was highest in Group IV. CONCLUSIONS: A polarizing preservation solution (KH+TTX) containing HOE694, furosemide, and BDM significantly enhanced long-term preservation compared with an optimized depolarizing solution (STH2) used clinically for long-term donor heart preservation.
Asunto(s)
Soluciones Cardiopléjicas , Corazón , Miocardio/metabolismo , Preservación de Órganos/métodos , Nucleótidos de Adenina/metabolismo , Animales , Bicarbonatos , Cloruro de Calcio , Creatina Quinasa/metabolismo , Metabolismo Energético , Guanidinas/farmacología , Corazón/efectos de los fármacos , Paro Cardíaco/inducido químicamente , Magnesio , Masculino , Potasio , Cloruro de Potasio , Ratas , Ratas Wistar , Cloruro de Sodio , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Sulfonas/farmacología , TetrodotoxinaRESUMEN
The cardiac sarcolemmal Na(+)/H(+) exchanger (NHE) extrudes one H(+) in exchange for one Na(+) entering the myocyte, utilizing for its driving force the inwardly directed Na(+) gradient maintained by the Na(+), K(+)-ATPase. The exchanger is quiescent at physiological values of intracellular pH but becomes activated in response to intracellular acidosis. Recent evidence suggests that a variety of extracellular signals (e.g., adrenergic agonists, thrombin, endothelin, and oxidant stress) also modulate sarcolemmal NHE activity by altering its sensitivity to intracellular H(+). Because sarcolemmal NHE activity is believed to be an important determinant of the extent of myocardial injury during ischemia and reperfusion, regulation of exchanger activity by factors that are associated with ischemia is likely to be pathophysiological importance.
Asunto(s)
Corazón/fisiología , Sarcolema/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Humanos , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/ultraestructura , Estrés OxidativoRESUMEN
OBJECTIVES: We previously demonstrated improved myocardial preservation with polarized (tetrodotoxin-induced), compared with depolarized (hyperkalemia-induced), arrest and hypothermic storage. This study was undertaken to determine whether polarized arrest reduced ionic imbalance during ischemic storage and whether this was influenced by Na+/K +/2Cl- cotransport inhibition. METHODS: We used the isolated crystalloid perfused working rat heart preparation (1) to measure extracellular K+ accumulation (using a K+-sensitive intramyocardial electrode) during ischemic (control), depolarized (K+ 16 mmol/L), and polarized (tetrodotoxin, 22 micromol/L) arrest and hypothermic (7.5 degrees C) storage (5 hours), (2) to determine dose-dependent (0.1, 1.0, 10 and 100 micromol/L) effects of the Na +/K+/2Cl- cotransport inhibitor, furosemide, on extracellular K+ accumulation during polarized arrest and 7.5 degrees C storage, and (3) to correlate extracellular K+ accumulation to postischemic recovery of cardiac function. RESULTS: Characteristic triphasic profiles of extracellular K+ accumulation were observed in control and depolarized arrested hearts; a significantly attenuated profile with polarized arrested hearts demonstrated reduced extracellular K+ accumulation, correlating with higher postischemic function (recovery of aortic flow was 54% +/-4% [P =.01] compared with 39% +/-3% and 32% +/-3% in depolarized and control hearts, respectively). Furosemide (0.1, 1.0, 10, and 100 micromol/L) modified extracellular K+ accumulation by -18%, -38%, -0.2%, and +9%, respectively, after 30 minutes and by -4%, -27%, +31%, and +42%, respectively, after 5 hours of polarized storage. Recovery of aortic flow was 53% +/-4% (polarized arrest alone), 56% +/-8%, 70% +/-2% (P =.04 vs control), 69% +/-4% (P =.04 vs control), and 65% +/-3% ( P =. 04 vs control), respectively. CONCLUSIONS: Polarized arrest was associated with a reduced ionic imbalance (demonstrated by reduced extracellular K+ accumulation) and improved recovery of cardiac function. Further attenuation of extracellular K + accumulation (by furosemide) resulted in additional recovery.
Asunto(s)
Canales de Cloruro/efectos de los fármacos , Diuréticos/farmacología , Espacio Extracelular/efectos de los fármacos , Furosemida/farmacología , Paro Cardíaco Inducido/métodos , Trasplante de Corazón , Hiperpotasemia/complicaciones , Miocardio/metabolismo , Preservación de Órganos/métodos , Canales de Sodio/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Tetrodotoxina/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Glucosa/química , Glucosa/farmacología , Paro Cardíaco Inducido/efectos adversos , Hiperpotasemia/metabolismo , Masculino , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/metabolismo , Preservación de Órganos/efectos adversos , Ratas , Ratas Wistar , Factores de Tiempo , Trometamina/química , Trometamina/farmacologíaRESUMEN
BACKGROUND: Hypothermic hyperkalemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalemia-induced depolarization of the resting membrane potential (Em) may predispose the heart to Na+ and Ca2+ loading via voltage-dependent "window currents," thereby exacerbating injury and limiting the safe storage duration. Alternatively, maintaining the resting Em with a polarizing solution may reduce ionic movements and improve postischemic recovery; we investigated this concept with the reversible sodium channel blocker tetrodotoxin (TTX) to determine (1) whether polarized arrest was more efficacious than depolarized arrest during hypothermic long-term myocardial preservation and (2) whether TTX induces and maintains polarized arrest. METHODS AND RESULTS: The isolated crystalloid-perfused working rat heart preparation was used in this study. Preliminary studies determined an optimal TTX concentration of 22 micromol/L and an optimal storage temperature of 7.5 degrees C. To compare depolarized and polarized arrest, hearts were arrested with either Krebs-Henseleit (KH) buffer (control), KH buffer containing 16 mmol/L K+, or KH buffer containing 22 micromol/L TTX and then stored at 7.5 degrees C for 5 hours. Postischemic recovery of aortic flow was 13+/-4%, 38+/-2%, and 48+/-3%* (*P<.05 versus control and 16 mmol/L K+), respectively. When conventional 3 mol/L KCl-filled intracellular microelectrodes were used, Em gradually depolarized during control unprotected ischemia to approximately -55 mV before reperfusion, whereas arrest with 16 mmol/L K+ caused rapid depolarization to approximately -50 mV, where it remained throughout the 5-hour storage period. In contrast, in 22 micromol/L TTX-arrested hearts, Em remained more polarized, at approximately -70 mV, for the entire ischemic period. CONCLUSIONS: Blockade of cardiac sodium channels by TTX during ischemia maintained polarized arrest, which was more protective than depolarized arrest, possibly because of reduced ionic imbalance.