RBC NOS: regulatory mechanisms and therapeutic aspects

Nitric oxide (NO), one of the most important vascular signaling molecules, is primarily produced by endothelial NO synthase (eNOS). eNOS is tightly regulated by its substrate l-arginine, cofactors and diverse interacting proteins. Interestingly, an NO synthase (NOS) was described within red blood cells (RBC NOS), and it was recently shown to significantly contribute to the intravascular NO pool and to regulate physiologically relevant mechanisms. However, the regulatory mechanisms and clinical implications of RBC NOS are unknown. The aim of this review is to highlight intracellular RBC NOS interactions and the role of RBC NOS in RBC homeostasis. Furthermore, macro- and microvascular diseases affected by RBC-derived NO are discussed.     

Efficiency of facultative frugivory in the nectar-feeding bat Glossophaga commissarisi: the quality of fruits as an alternative food source

The efficiency of food exploitation correlates positively with the extent of dietary specialization. Neotropical nectar-feeding bats (Glossophaginae) have one of the most specialized diets among mammals, as floral nectar constitutes a sugar-rich and highly digestible but protein and fiber depleted food source. However, dietary constraints, such as a temporary scarcity of nectar, or protein demands may sometimes require the uptake of alternative food items. We investigated the influence of a diet switch from nectar to fruit on intestinal morphology, body mass, and energy budget in the nectar-feeding bat Glossophaga commissarisi and quantified feeding efficiency. We hypothesized that these nectar specialists depend on a constant supply of nectar, if they were lacking the ability for morphological and physiological plasticity in response to a fiber-rich diet. Although capable of harvesting infructescences of Piper hispidum, G. commissarisi was less efficient in extracting energy from fruits (48% digestive efficiency of total fruit energy content) than from nectar (c. 99% digestive efficiency). The intestinal morphology and organ masses did not change after bats were switched from nectar to fruits. Captive bats exhibited lower daily energy expenditures and flight activity when feeding on fruits than during nectarivory. Possibly, this may have been a deliberate regulation to balance reduced feeding efficiency, or simply the consequence of extended digestive pauses. The low digestibility of Piper, in combination with slow digestion and the bats’ inability for morphological and physiological plasticity may cause nectar-feeders to reduce their maximum energy expenditure when feeding on fruits. We argue that although fruits may substitute for nectar, they may cause restricted maximum energy assimilation compared with nectar.     

Identification of phosphorylation sites in the polo-like kinases Plx1 and Plk1 by a novel strategy based on element and electrospray high resolution mass spectrometry

A novel strategy for the determination of protein phosphorylation sites is described and applied to the polo-like kinases Plx1 (Xenopus laevis) and Plk1 (Homo sapiens). The strategy comprises the sequential application of the following techniques: proteolytic digestion, capillary liquid chromatography (LC)-inductively coupled plasma mass spectrometry with phosphorus detection, capillary LC-electrospray mass spectrometry and electrospray tandem mass spectrometry. In this approach, phosphopeptides are generated, their elution time in capillary LC is determined, candidate phosphopeptides at the corresponding elution times are identified, and positive identification and sequencing of phosphopeptides is performed in the last step of the analysis. Using this technique, Ser25/26, Ser326, and Ser340 were identified as phosphorylation sites in recombinant Plx1, and Ser340 was identified as the major phosphorylation site in a kinase-dead mutant of Plx1 expressed in okadaic acid-treated Sf9 insect cells. A site corresponding to Ser326 in Plx1 was also shown to be phosphorylated in the human polo-like kinase Plk1 (Ser335). Element mass spectrometry with phosphorus detection provides a quantitative phosphorylation profile of all phosphorylation sites accessible by LC.     

Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1

Polo-like kinases (Plks) control multiple important events during M phase progression, but little is known about their activation during the cell cycle. The activities of both mammalian Plk1 and Xenopus Plx1 peak during M phase, and this activation has been attributed to phosphorylation. However, no phosphorylation sites have previously been identified in any member of the Plk family. Here we have combined tryptic phosphopeptide mapping with mass spectrometry to identify four major phosphorylation sites in Xenopus Plx1. All four sites appear to be phosphorylated in a cell cycle-dependent manner. Phosphorylations at two sites (Ser-260 and Ser-326) most likely represent autophosphorylation events, whereas two other sites (Thr-201 and Ser-340) are targeted by upstream kinases. Several recombinant kinases were tested for their ability to phosphorylate Plx1 in vitro. Whereas xPlkk1 phosphorylated primarily Thr-10, Thr-201 was readily phosphorylated by protein kinase A, and Cdk1/cyclin B was identified as a likely kinase acting on Ser-340. Phosphorylation of Ser-340 was shown to be responsible for the retarded electrophoretic mobility of Plx1 during M phase, and phosphorylation of Thr-201 was identified as a major activating event.     

Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues

The formation of nitric oxide (NO) has been linked to many regulatory functions in mammalian cells. With the appreciation that NO-mediated nitrosation reactions are involved in cell signaling and pathology there is a need to elucidate and better characterize the different biochemical pathways of NO in vivo. Despite significant methodological advances over the years one major obstacle in assessing the significance of nitrosated species and other NO-related metabolites remains: their reliable measurement in complex biological matrices. In this review we briefly discuss the major routes of NO metabolism and transport in the mammalian circulation, considering plasma, red blood cell, and tissue compartments separately. In addition, we attempt to give a recommendation as to the most appropriate analytical technique and sample processing procedures for the reliable quantification of either species.     

Thiols enhance NO formation from nitrate photolysis

Nitrate is generally considered an inert oxidative breakdown product of nitric oxide (NO). Whereas it has been shown that limited amounts of NO are produced during the photolysis of nitrate in aqueous solution, the photochemistry of nitrate in biological matrices such as plasma is unknown. We hypothesized that thiols, which are ubiquitously present in biological systems, may significantly enhance NO-quantum yields from nitrate photolysis. Exposure of fresh human plasma to high-intensity UV-light resulted in NO-formation (19 +/- 3 nmol/l/min) as measured by gas phase chemiluminescence, and this signal was almost completely abolished by the removal of plasma N-oxides (2 +/- 1 nmol/l/min). Reconstitution of NOx-depleted plasma samples with a physiological concentration of nitrate, but not nitrite, restored photolytic NO-generation to values comparable to na?ve plasma. Addition of the thiol-reducing agent, dithiothreitol or the sulfhydryl-bearing amino acid, L-cysteine increased NO-formation above control levels. Thiol-blockade by either N-ethylmaleimide (NEM) or mercuric chloride (HgCl2) reduced basal NO formation from 19 +/- 3 to 7 +/- 2 and 4 +/- 1 nmol/l/min, respectively. Exposure of plasma to UV-light increased NO-adduct concentrations from 18 +/- 5 to 1662 +/- 658 nmol/l. Collectively, our results show that thiols facilitate photolytic conversion of nitrate to NO and NO-adducts such as S-nitrosothiols. This may lead to substantial overestimation of the latter when photolysis-based methodologies are used for their determination. Whether this novel reaction channel also has in vivo relevance remains to be investigated.