Exercise training enhances baroreflex sensitivity by an angiotensin II-dependent mechanism in chronic heart failure.

Exercise training (EX) has become an important modality capable of enhancing the quality of life and survival of patients with chronic heart failure (CHF). Although 4 wk of EX in animals with CHF evoked a reduction in renal sympathetic nerve activity and ANG II plasma levels and an enhancement in baroreflex sensitivity at rest (Liu JL, Irvine S, Reid IA, Patel KP, Zucker IH, Circulation 102: 1854-1862, 2000; Liu JL, Kulakofsky J, Zucker IH, J Appl Physiol 92: 2403-2408, 2002), it is unclear whether these phenomena are causally related. CHF was induced in rabbits by ventricular pacing (360-380 beats/min) for 3 wk. CHF rabbits were EX for 4 wk at 15-18 m/min, 6 days/wk, 30-40 min/day. Three groups of rabbits were studied: CHF (with no EX), CHF-EX, and CHF-EX + ANG II infusion [in which ANG II levels were kept at or near levels observed in CHF (non-EX) rabbits by subcutaneous osmotic minipump infusion]. EX prevented the increase in plasma ANG II levels shown in CHF rabbits. CHF and CHF-EX + ANG II infusion rabbits had significantly depressed baroreflex sensitivity slopes (P < 0.01 for sodium nitroprusside and P < 0.001 for phenylephrine) and higher baseline renal sympathetic nerve activities than CHF-EX animals. EX downregulated mRNA and protein expression of ANG II type 1 receptors in the rostral ventrolateral medulla in CHF rabbits. This was prevented by ANG II infusion. These data are consistent with the view that the reduction in sympathetic nerve activity and the improvement in baroreflex function in CHF after EX are due to the concomitant reduction in ANG II and angiotensin receptors in the central nervous system.     

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.     

Identification and characterization of a cytosolic 30 kDa histamine stimulated phosphoprotein in parietal cell cytosol

Histamine stimulated acid secretion is mediated by an increase in intracellular cAMP. Cytosolic protein phosphorylation stimulated by histamine was investigated in isolated rabbit parietal cells. Histamine stimulated the phosphorylation of a 30 kDa phosphoprotein with an isoelectric point of 5.6. Cimetidine completely inhibited histamine-stimulated pp30 phosphorylation. However, omeprazole had no effect on the phosphorylation of pp30. Forskolin and 8-bromo-cAMP also stimulated the phosphorylation of pp30. The results suggest that pp30 is a histamine-stimulated, cAMP-dependently phosphorylated protein substrate in parietal cell cytosol.     

Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.     

The efficiency of a weighed log-rank test under a percent error misspecification model for the log hazard ratio.

For comparison of two survival distributions, it is natural to use a weighted log-rank test with weight function given by the log hazard ratio function that is anticipated a priori. This paper investigates the efficiency of this test when the a priori estimate of the log hazard ratio is subject to a specified percentage error. The test is shown to be the maximum efficiency robust test over the class of alternatives in question and a simple expression for the maximum efficiency is established.     

Thymus subpopulations: density gradients and volume distributions studies.

The size and density of thymus cells was studied during development and after in vivo hydrocortisone, cyclophosphamide, and radiation treatment. Four populations classified by size and density were shown to exist in the thymus. The smallest thymocytes were most sensitive to destruction by both hydrocortisone and immunosuppressive treatment. The combination of density gradient separations with electronic size distributions revealed that many size populations comprised the HC resistant cell population.