Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen

Endogenous angiotensin (ANG) II and ANG-(1-7) act at the nucleus tractus solitarius (NTS) to differentially modulate neural control of the circulation. The role of these peptides endogenous to NTS on cardiovascular reflex function was investigated in transgenic rats with low brain angiotensinogen (Aogen) due to glial overexpression of an antisense to Aogen (ASrAOGEN) and in Sprague-Dawley (SD) rats. Arterial baroreceptor reflex sensitivity (BRS) for control of heart rate (HR) in response to increases in mean arterial pressure (MAP) was tested before and after bilateral microinjection of the angiotensin type 1 (AT(1)) receptor blocker candesartan or the ANG-(1-7) receptor blocker (d-Ala(7))-ANG-(1-7) into the NTS of urethane-chloralose-anesthetized ASrAOGEN and SD rats. Baseline MAP was higher in ASrAOGEN than in SD rats under anesthesia (P < 0.01). Injection of candesartan or (d-Ala(7))-ANG-(1-7) decreased MAP (P < 0.01) and HR (P < 0.05) in ASrAOGEN, but not SD, rats. The BRS at baseline was similar in ASrAOGEN and SD rats. Candesartan increased BRS by 41% in SD rats (P < 0.01) but was without effect in ASrAOGEN rats. In contrast, the reduction in BRS after (d-Ala(7))-ANG-(1-7) administration was comparable in SD (31%) and ASrAOGEN rats (34%). These findings indicate that the absence of glia-derived Aogen is associated with 1) an increase in MAP under anesthesia mediated via AT(1) and ANG-(1-7) receptors within the NTS, 2) the absence of an endogenous ANG II contribution to tonic inhibition of BRS, and 3) a continued contribution of endogenous ANG-(1-7) to tonic enhancement of BRS.     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

The proinflammatory phenotype of PECAM-1-deficient mice results in atherogenic diet-induced steatohepatitis

The severity of nonalcoholic steatohepatitis (NASH) is determined by environmental and genetic factors, the latter of which are incompletely characterized. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed on blood and vascular cells. In the present study, we provide data for the novel finding that genetic deficiency of PECAM-1 potentiates the development and progression of NASH. We found that the rate of development and severity of diet-induced NASH are markedly enhanced in PECAM-1-deficient [knockout (KO)] mice relative to wild-type (WT) mice, as measured by histological and biochemical evaluation. Livers from KO mice exhibited typical histological features of NASH, including macrovesicular fat accumulation, hepatocyte injury with infiltration of inflammatory cells, fibrosis, and heightened oxidative stress. Alanine aminotransferase, a marker for liver injury, was also significantly higher in KO compared with WT mice. Consistent with a role for PECAM-1 as a suppressor of proinflammatory cytokines, plasma levels of inflammatory cytokines, including TNF-alpha and monocyte chemoattractant protein-1 (MCP-1), were also significantly higher in KO compared with WT mice. These findings are the first to show that the PECAM-1-deficient mouse develops progressive nonalcoholic fatty liver disease (NAFLD), supporting a role for PECAM-1 as a negative regulator of NAFLD progression. Future examination of recently identified PECAM-1 allelic isoforms in humans as potential risk factors for developing NASH may be warranted.     

Uncoupling protein 2 modulates cell viability in adult rat cardiomyocytes

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane proton carrier that uncouples ATP synthesis. The aim of this study was to determine whether UCP2 plays a role in survival of adult rat cardiac myocytes. We first studied the effects of UCP2 overexpression in vitro. Overexpression of UCP2 in primary cardiomyocytes led to a significant decline in ATP level and the development of acidosis but had no observable effect on cell survival. When cardiomyocytes were challenged with hypoxia-reoxygenation, cells overexpressing UCP2 survived significantly less compared with control. This finding was associated with upregulation of proapoptotic protein Bcl-2 and 19-kDa interacting protein 3 (BNIP3). Furthermore, UCP2 short interfering RNA prevented both the increase in cell death and BNIP3 expression. To examine the in vivo role of UCP2 in the heart, we used the Dahl salt-sensitive rat heart-failure model. Northern blot analysis revealed that UCP2 mRNA level was significantly upregulated in rat heart failure along with BNIP3 protein level. In conclusion, UCP2 increases sensitivity of adult rat cardiac myocytes to hypoxia-reoxygenation by way of ATP depletion and acidosis, which in turn causes accumulation of prodeath protein BNIP3.     

Hair bundles are specialized for ATP delivery via creatine kinase

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.     

Pectin induces apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure

Treatment options for androgen-independent prostate cancer cells are limited. Therefore, it is critical to identify agents that induce death of both androgen-responsive and androgen-insensitive cells. Here we demonstrate that a product of plant cell walls, pectin, is capable of inducing apoptosis in androgen-responsive (LNCaP) and androgen-independent (LNCaP C4-2) human prostate cancer cells. Commercially available fractionated pectin powder (FPP) induced apoptosis (approximately 40-fold above non-treated cells) in both cell lines as determined by the Apoptosense assay and activation of caspase-3 and its substrate, poly(ADP-ribose) polymerase. Conversely, citrus pectin (CP) and the pH-modified CP, PectaSol, had little or no apoptotic activity. Glycosyl residue composition and linkage analyses revealed no significant differences among the pectins. Mild base treatment to remove ester linkages destroyed FPP's apoptotic activity and yielded homogalacturonan (HG) oligosaccharides. The treatment of FPP with pectinmethylesterase to remove galacturonosyl carboxymethylesters and/or with endopolygalacturonase to cleave nonmethylesterified HG caused no major reduction in apoptotic activity, implicating the requirement for a base-sensitive linkage other than the carboxymethylester. Heat treatment of CP (HTCP) led to the induction of significant levels of apoptosis comparable to FPP, suggesting a means for generating apoptotic pectic structures. These results indicate that specific structural elements within pectin are responsible for the apoptotic activity, and that this structure can be generated, or enriched for, by heat treatment of CP. These findings provide the foundation for mechanistic studies of pectin apoptotic activity and a basis for the development of pectin-based pharmaceuticals, nutraceuticals, or recommended diet changes aimed at combating prostate cancer occurrence and progression.     

The stoichiometry of the two photosystems in higher plants revisited

The stoichiometry of Photosystem II (PSII) to Photosystem I (PSI) reaction centres in spinach leaf segments was determined by two methods, each capable of being applied to monitor the presence of both photosystems in a given sample. One method was based on a fast electrochromic (EC) signal, which in the millisecond time scale represents a change in the delocalized electric potential difference across the thylakoid membrane resulting from charge separation in both photosystems. This method was applied to leaf segments, thus avoiding any potential artefacts associated with the isolation of thylakoid membranes. Two variations of this method, suppressing PSII activity by prior photoinactivation (in spinach and poplar leaf segments) or suppressing PSI by photo-oxidation of P700 (the chlorophyll dimer in PSI) with background far-red light (in spinach, poplar and cucumber leaf segments), each gave the separate contribution of each photosystem to the fast EC signal; the PSII/PSI stoichiometry obtained by this method was in the range 1.5-1.9 for the three plant species, and 1.5-1.8 for spinach in particular. A second method, based on electron paramagnetic resonance (EPR), gave values in a comparable range of 1.7-2.1 for spinach. A third method, which consisted of separately determining the content of functional PSII in leaf segments by the oxygen yield per single turnover-flash and that of PSI by photo-oxidation of P700 in thylakoids isolated from the corresponding leaves, gave a PSII/PSI stoichiometry (1.5-1.7) that was consistent with the above values. It is concluded that the ratio of PSII to PSI reaction centres is considerably higher than unity in typical higher plants, in contrast to a surprisingly low PSII/PSI ratio of 0.88, determined by EPR, that was reported for spinach grown in a cabinet under far-red-deficient light in Sweden [Danielsson et al. (2004) Biochim. Biophys. Acta 1608: 53-61]. We suggest that the low PSII/PSI ratio in the Swedish spinach, grown in far-red-deficient light with a lower PSII content, is not due to greater accuracy of the EPR method of measurement, as suggested by the authors, but is rather due to the growth conditions.     

Proton transfer in water wires in proteins: modulation by local constraint and polarity in gramicidin a channels

The transfer of protons in membrane proteins is an essential phenomenon in biology. However, the basic rules by which H(+) transfer occurs in water wires inside proteins are not well characterized. In particular, the effects of specific atoms and small groups of atoms on the rate of H(+) transfer in water wires are not known. In this study, new covalently linked gramicidin-A (gA) peptides were synthesized, and the effects of specific atoms and peptide constraints on the rate of H(+) transfer were measured in single molecules. The N-termini of two gA peptides were linked to various molecules: S,S-cyclopentane diacid, R,R-cyclopentane diacid, and succinic acid. Single-channel proton conductances (g(H)) were measured at various proton concentrations ([H(+)]) and compared to previous measurements obtained in the S,S- and R,R-dioxolane-linked as well as in native gA channels. Replacing the S,S-dioxolane by an S,S-cyclopentane had no effects on the g(H)-[H(+)] relationships, suggesting that the constrained and continuous transition between the two gA peptides via these S,S linkers is ultimately responsible for the two- to fourfold increase in g(H) relative to native gA channels. It is likely that constraining a continuous transition between the two gA peptides enhances the rate of H(+) transfer in water wires by decreasing the number of water wire configurations that do not transfer H(+) at higher rates as in native gA channels (a decrease in the activation entropy of the system). On the other hand, g(H) values in the R,R-cyclopentane are considerably larger than those in R,R-dioxolane-linked gA channels. One explanation would be that the electrostatic interactions between the oxygens in the dioxolane and adjacent carbonyls in the R,R-dioxolane-linked gA channel attenuate the rate of H(+) transfer in the middle of the pore. Interestingly, g(H)-[H(+)] relationships in the R,R-cyclopentane-linked gA channel are quite similar to those in native gA channels. g(H) values in succinyl-linked gA channels display a wide distribution of values that is well represented by a bigaussian. The larger peaks of these distributions are similar to g(H) values measured in native gA channel. This observation is also consistent with the notion that constraining the transition between the two beta-helical gA peptides enhances the rate of H(+) transfer in water wires by decreasing the activation entropy of the system.