Increasing plasmalogen levels protects human endothelial cells during hypoxia

Supplementation of cultured human pulmonary arterial endothelial cells (PAEC) with sn-1-O-hexadecylglycerol (HG) resulted in an approximately twofold increase in cellular levels of plasmalogens, a subclass of phospholipids known to have antioxidant properties; this was due, primarily, to a fourfold increase in the choline plasmalogens. Exposure of unsupplemented human PAEC to hypoxia (PO(2) = 20-25 mmHg) caused an increase in cellular reactive oxygen species (ROS) over a period of 5 days with a coincident decrease in viability. In contrast, HG-supplemented cells survived for at least 2 wk under these conditions with no evidence of increased ROS. Hypoxia resulted in a selective increase in the turnover of the plasmalogen plasmenylethanolamine. Human PAEC with elevated plasmalogen levels were also more resistant to H(2)O(2), hyperoxia, and the superoxide generator plumbagin. This protection was seemingly specific to cellular stresses in which significant ROS were generated because the sensitivity to lethal heat shock or glucose deprivation was not altered in HG-treated human PAEC. HG, by itself, was not sufficient for protection; HG supplementation of bovine PAEC had no effect upon plasmalogen levels and did not rescue these cells from the cytotoxic effects of hypoxia. This is the initial demonstration that plasmalogen content can be substantially enhanced in a normal cell. These data also demonstrate that HG can protect cells during hypoxia and other ROS-mediated stress, likely due to the resulting increase in these antioxidant phospholipids.     

Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice explant cultures prepared from specific rat forebrain regions

Five serially sectioned tissue slices (400 microns) from the preoptic area/hypothalamus of postnatal day 4 rats were cultured using a slice explant roller culture technique. After 18 days in culture, these slices thinned sufficiently to allow immunocytochemical and in situ hybridization histochemical assays for LHRH peptide and LHRH mRNA, respectively. Large numbers of neurons containing mRNA encoding LHRH were detected in these slices using in situ hybridization histochemistry (ISHH). These 35S-labeled cells were distributed in the cultured slices in a pattern similar to that found with LHRH immunocytochemistry and ISHH in vivo, indicating that LHRH neurons were maintained in these cultures in an organotypic manner. Densitometric single cell analyses after ISHH of the culture slices were performed using a Loats image analysis system, so as to provide a density value per cell (density/cell). Comparisons of these density values from the slice explants cultured in presence or absence of 10(-7) M estradiol found that: 1) under basal (control) culture conditions there were no consistent differences in the frequency distributions of the density/cell values between all the five slices derived from either male or female rats, 2) mean density/culture values under control conditions did not differ significantly between slices and sexes, 3) the presence of estradiol in the culture media resulted in an overall decrease in density/cell values, with the most significant decrease occurring in slice 3 which is comparable to the level of the organum vasculosum lamina terminalis/rostral preoptic area (OVLT/rPOA) in vivo, and 4) this decrease in density/cell values in slice 3 due to estradiol treatment, was greater in cultures derived from female vs. male tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation

Chinese hamster ovary cells (CHO-K1) photosensitized with 12-(1'-pyrene)dodecanoic acid (P12) are killed when exposed to long wavelength ultraviolet (UV) light (greater than 300 nm). Mutants deficient in plasmalogen biosynthesis are hypersensitive to this treatment. We now demonstrate that plasmenylethanolamine is rapidly and preferentially destroyed when CHO-K1 cells, photosensitized either with P12 or merocyanine 540, are irradiated with light of the appropriate wavelength. Using [2-14C]ethanolamine, [1-14C]hexadecanol, or [U-14C]hexadecanol to follow the turnover of plasmenylethanolamine, we show that 2-monoacylglycerophosphoethanolamine, formic acid, and pentadecanal are formed during P12/UV treatment of CHO-K1 cells, but not of mutant cells deficient in plasmalogen synthesis. The decomposition of plasmenylethanolamine is O2-dependent, is enhanced in D2O, and is reduced in the presence of sodium azide. The process may be explained, in part, by the cycloaddition of singlet oxygen to the vinyl ether linkage of plasmenylethanolamine, generating a dioxetane intermediate that would be expected to decompose under physiological conditions to the observed products. An additional possibility is the formation of an allylic hydroperoxide at the 1'-carbon of the alkyl moiety by an "ene" reaction of singlet oxygen, or by radical-mediated oxidation, followed by metabolism or chemical decomposition of the hydroperoxide. Given the P12/UV hypersensitivity of plasmalogen-deficient mutants, we suggest that plasmalogens might protect animal cell membranes from singlet oxygen and/or radical-initiated oxidation by functioning as scavengers and decomposing to products that can be reutilized.     

Contractions of amphibian Wolffian duct in response to acetylcholine, norepinephrine, and arginine vasotocin

The regulation of sperm transport through the Wolffian duct of male amphibians is poorly understood. These experiments were conducted using rough-skinned newts (Taricha granulosa) to determine if Wolffian ducts are capable of contracting in vitro and, if so, to characterize the contractile responses to acetylcholine (ACh), norepinephrine (NE), and neurohypophysial hormones. Dose-response curves for NE and ACh, which were prepared by measuring isometric contractions, are similar to those reported for mammalian vas deferens. For NE, the minimum effective dose and ED50 were found to be 1 X 10(-5)M and 4.17 X 10(-5)M, respectively. For ACh, the minimum effective dose was 3.2 X 10(-8)M and the ED50 was 1.37 X 10(-5)M. Alpha-adrenoreceptors appear to mediate the contractile responses to NE because phentolamine (10(-5)M) blocked or attenuated the response to NE (10(-6)M, 10(-5)M or 10(-4) M). Beta-adrenoreceptors appear to mediate relaxation because dichloroisoproterenol (10(-5)M) enhanced the response to 10(-5)M NE. The contractile response to three neurohypophysial hormones were also investigated. Arginine vasotocin was more effective in eliciting contractions than oxytocin. The effect of lysine vasopressin was intermediate between arginine vasotocin and oxytocin. These experiments demonstrate that amphibian (Taricha) Wolffian ducts contract in vitro in response to neurotransmitters and neurohypophysial hormones. The contractile response to neurotransmitters occurs in a dose-dependent manner; the response to neurohypophysial hormones is hormone specific.     

Resistin polymorphisms are associated with muscle, bone, and fat phenotypes in white men and women

Objective: The biological function of resistin (RST) is unknown, although it may have roles in obesity, diabetes, and insulin resistance. The objective of this study was to examine the effects of single nucleotide polymorphisms (SNPs) in the human RST gene on muscle, bone, and adipose tissue phenotypes and in response to resistance training (RT). Research Methods and Procedures: Subjects were white and consisted of strength (n = 482) and size (n = 409) cohorts who had not performed RT in the previous year. Subjects completed 12 weeks of structured, unilateral upper arm RT aimed at increasing the size and strength of the non‐dominant arm, using their dominant arm as an untrained control. Strength measurements were taken pre‐ and post‐12‐week RT and consisted of elbow flexor isometric strength and one‐repetition maximum during a biceps curl using free weights. Whole muscle, subcutaneous fat, and cortical bone volumes were measured by magnetic resonance imaging. Six RST SNPs were identified. Analysis of covariance was used to test for effects of the SNPs on pre‐ and post‐muscle strength and whole muscle, fat, and bone volumes independent of gender, age, and body weight. Results: Five RST SNPs (?537 A>C, ?420 C>G, 398 C>T, 540 G>A, 980 C>G) were associated with measured phenotypes among subjects when stratified by BMI (<25, ≥25 kg/m²). Several gender‐specific associations were observed between RST SNPs and phenotypes among individuals with a BMI ≥ 25. Conversely, only two associations were observed among individuals with a BMI < 25. Discussion: These data support previous identified associations of RST with adipose tissue and demonstrate additional associations with bone and skeletal muscle that warrant further investigation.     

Effects of β-alanine supplementation on the onset of neuromuscular fatigue and ventilatory threshold in women

Summary. This study examined the effects of 28 days of β-alanine supplementation on the physical working capacity at fatigue threshold (PWC_FT), ventilatory threshold (VT), maximal oxygen consumption ( O_2-MAX), and time-to-exhaustion (TTE) in women. Twenty-two women (age ± SD 27.4 ± 6.1 yrs) participated and were randomly assigned to either the β-alanine (CarnoSyn™) or Placebo (PL) group. Before (pre) and after (post) the supplementation period, participants performed a continuous, incremental cycle ergometry test to exhaustion to determine the PWC_FT, VT, O_2-MAX, and TTE. There was a 13.9, 12.6 and 2.5% increase (p < 0.05) in VT, PWC_FT, and TTE, respectively, for the β-alanine group, with no changes in the PL (p > 0.05). There were no changes for O_2-MAX (p > 0.05) in either group. Results of this study indicate that β-alanine supplementation delays the onset of neuromuscular fatigue (PWC_FT) and the ventilatory threshold (VT) at submaximal workloads, and increase in TTE during maximal cycle ergometry performance. However, β-alanine supplementation did not affect maximal aerobic power ( O_2-MAX). In conclusion, β-alanine supplementation appears to improve submaximal cycle ergometry performance and TTE in young women, perhaps as a result of an increased buffering capacity due to elevated muscle carnosine concentrations.