Autonomic regulation of pacemaker activity: role of heart nitric oxide synthases

In autonomic-blocked rats treated with NG-nitro-L-arginine methyl ester (L-NAME, 7.5 mg/kg), heart rate increased 18% and mean arterial pressure increased 48%. Thyroidectomy, along with autonomic blockade, hampered the chronotropic response but did not modify the effect on blood pressure. After 150 min of autonomic blockade, the experimental end point, total nitric oxide (NO) production by heart NO synthases (NOS) decreased 61%: from 54 to 21 nmol NO.min-1.g heart-1. Mitochondrial NOS (mtNOS) and sarcoplasmic reticulum endothelial NOS activities decreased 74% and 52%, respectively. Mitochondria isolated from whole heart showed a well-coupled oxidative phosphorylation with high respiratory control and ADP-to-O ratios, decreased mtNOS activity (55-60%), and decreased mtNOS protein expression (70%). Immunohistochemistry with anti-inducible NOS antibody linked to gold particles localized mtNOS at the inner mitochondrial membranes. Histochemical right atrial NOS (NADPH-diaphorase) decreased 55% after heart denervation. The effects of autonomic denervation on the NO system were partially prevented by thyroidectomy performed simultaneously with autonomic blockade. Western blot analysis indicated a very rapid mtNOS protein turnover (half time=120 min) with a process of protein expression that was upregulated by thyroidectomy and a degradation process that was downregulated by the autonomic nervous system. The observations suggest that NO-mediated pathways contribute to pacemaker heart activity, likely through the NO steady-state levels in the right atrium and the whole heart.     

Role of nitric oxide and prostanoids in attenuation of rapid baroreceptor resetting

Because the regulation of vascular function involves complex mutual interactions between nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) products, we examined the contribution of NO and prostanoids derived from the COX pathway in modulating aortic baroreceptor resetting during an acute (30 min) increase in arterial pressure in anesthetized rats. Increase in pressure was induced either by administration of the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or aortic coarctation (COA) with or without treatment with the COX inhibitor indomethacin (INDO) or the selective neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)imidazole (TRIM). The activity of the aortic depressor nerve and arterial pressure were simultaneously recorded, and the degree of resetting was determined by the shift of the pressure-nerve activity curve using the ratio [delta systolic pressure at 50% of maximum baroreceptor activity/delta systolic pressure] x 100. The magnitude of pressure rise was similar in the different groups (59 +/- 6, 53 +/- 5, 53 +/- 5, 45 +/- 5, 49 +/- 3, and 41 +/- 3 mmHg for COA, L-NAME, INDO+COA, INDO+L-NAME, TRIM+COA, and TRIM+INDO+COA, respectively, P = 0.27). The degree of resetting that occurred with L-NAME or COA combined with treatment with TRIM was attenuated compared with COA alone (7 +/- 4, 5 +/- 2, and 31 +/- 6%, respectively, P = 0.04). INDO failed to influence baroreceptor resetting to higher pressure but prevented L-NAME- and TRIM-induced effects (20 +/- 7, 21 +/- 8, and 32 +/- 6% for INDO+COA, INDO+L-NAME, and INDO+TRIM+COA, respectively; P = 0.38). Baroreceptor gain was affected only by l-NAME. These findings indicate that NO, probably from neuronal origin, may exert stimulatory influence on the degree of rapid baroreceptor resetting to hypertension that involves COX-derived prostanoids.     

Antiproliferative effects of tocopherols (vitamin E) on murine glioma C6 cells: homologue-specific control of PKC/ERK and cyclin signaling

Chemoprevention strategies for brain tumors (specifically gliomas) are few and surprisingly poorly investigated. We have studied the effects of tocopherols (TOCs; vitamin E) on proliferation and death processes of murine glioma C6 cells. These vitamers showed different cell uptake and concentration- and time-dependent inhibitory effects on cell growth that were significant at the lowest concentrations tested (1-10 microM). However, the inhibitory potency of TOCs seemed to reflect at least in part their actual cell concentrations at steady state, with the order of magnitude gamma-TOC >or= alpha-TOC > delta-TOC approximately or = beta-TOC. Moreover, for extracellular concentrations >or=10 microM, TOCs also showed a significant cytotoxic effects due mainly to necrosis, while apoptosis was negligible. Gamma-TOC (the form showing preferential cell uptake and lowest unspecific cytotoxicity) was the most effective inhibitor of cell cycle progression (arrest in G0/G1 phase) leading to lowered expression of cyclin E and cyclin-dependent kinases 2 and 4 and overexpression of p27 (specific inhibitor of S-phase entering). According to these signals, activated ERK1/2 and PKC upstream and Rb phosphorylation downstream were decreased. In conclusion, within TOCs the gamma form exerts the most potent and specific control of cell cycle progression in C6 cells (cytostatic effect). This suggests a chemopreventive role of this form of vitamin E in gliomas.     

Pre-treatment of central venous catheters with the cathelicidin BMAP-28 enhances the efficacy of antistaphylococcal agents in the treatment of experimental catheter-related infection

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of the 27 residues cathelicidin peptide BMAP-28, quinupristin/dalfopristin (Q/D), linezolid, and vancomycin. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with BMAP-28. Thirty minutes later rats were challenged via the CVC with 1.0x10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC at a concentration equal to the MBC observed using adherent cells, or at a much higher concentration (1024 microg/mL) began 24 h later. The inhibition activities of all antibiotics against adherent bacteria were at least two-four-fold lower that against freely growing cells. When antibiotics were used in BMAP-28 pre-treated wells, they showed higher activities. The in vivo studies showed that when CVCs were pre-treated with BMAP-28 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated with both BMAP-28 and antibiotics, biofilm bacterial load was further decreased to 10(1) CFU/mL and bacteremia was not detected. These results suggest that CVC pre-treated with BMAP-28 represents an attractive choice for the treatment of device-related infections caused by staphylococci.     

An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells

Human embryonic stem (ES) cells are usually co-cultivated with supporting cells consisting of short-term cultures of fibroblasts (not an immortalized line) in a medium lacking serum. This method has promoted important progress in the field, but suffers from certain disadvantages. By serial cultivation for 27 consecutive transfers and about 63 cell generations, we have evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports the multiplication of H9 cells better than the 3T3 line. It supports the growth of H9 cells as well as do available short-term fibroblast cultures, but maintains more effectively the stem cell character of the H9 cells, judging by their better retention of Oct4. We have made MMM cells resistant to blasticidin and zeocin, the most efficient antibiotics for selection of stable transformants. In the presence of zeocin, the resistant MMM were able to support multiplication and selection of ES cells transfected with an exogenous gene encoding zeocin resistance.     

Central angiotensin AT1-receptor blockade affects thermoregulation and running performance in rats

The effect of central angiotensin AT1-receptor blockade on thermoregulation in rats during exercise on a treadmill (18 m/min, 5% inclination) was investigated. Core (Tb) and skin tail temperatures were measured in rats while they were exercising until fatigue after injection of 2 microl of losartan (Los; 20 nmol, n = 4; 30 nmol, n = 4; 60 nmol, n = 7), an angiotensin II AT1-receptor antagonist, or 2 microl of 0.15 mol/l NaCl (Sal; n = 15) into the right lateral cerebral ventricle. Body heat rate (BHR), heat storage rate, threshold Tb for tail vasodilation (TTbV), time to fatigue, and workload were calculated. During exercise, the BHR and heat storage rate of Los-treated animals were, respectively, 40 and 53% higher (P < 0.01) than in Sal-treated animals. Additionally, rats injected with Los showed an increased TTbV (38.59 +/- 0.19 degrees C for Los vs. 38.12 +/- 0.1 degrees C for Sal, P < 0.02), a higher Tb at fatigue point (39.07 +/- 0.14 degrees C Los vs. 38.66 +/- 0.07 degrees C Sal, P < 0.01), and a reduced running performance (27.29 +/- 4.48 min Los vs. 52.47 +/- 6.67 min Sal, P < 0.01), which was closely related to the increased BHR. Our data suggest that AT1-receptor blockade attenuates heat dissipation during exercise due to the higher TTbV, leading to a faster exercise-induced increase in Tb, thus decreasing running performance.