Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries

Hypertension is a well‐established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1^Ht) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1^Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age‐matched wild‐type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.     

Purification and partial characterization of a sperm motility-inhibitory protein of ram cauda epididymal plasma

Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE-sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility-inhibitory factor of sheep I and II (MIFS-I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS-I and II, respectively. Specific activities of the partially purified MIFS-I and II were 563 and 261 U/mg of protein, while the fold-purification of the activity were 5119 and 2373, respectively. Both the proteins were heat-labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS-I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS-I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross-reactivity of MIFS-I with polyclonal anti-human SEMA3D antibody. It was concluded that the MIFS-I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.     

Validation of a field‐ready handheld meter for plasma β‐hydroxybutyrate analysis

Plasma metabolite analysis is frequently used to assess the energetic state and energy intake of birds. Plasma β‐hydroxybutyrate (BUTY) is a common metabolite used in these studies, and is correlated with fasting and mass loss. BUTY is typically quantified in laboratory assays that are costly, time‐consuming, and prone to human error. We tested the accuracy and precision of a field‐ready handheld BUTY meter. We compared BUTY concentration values obtained in the laboratory and with the handheld meter in plasma samples from 19 Grasshopper Sparrows (Ammodramus savannarum), and assessed precision with repeat analysis of a single sample. The handheld meter reported BUTY concentrations in < 2 min, was highly precise, and as accurate as the laboratory assay method—all ideal for field conditions. Collecting blood samples for laboratory analysis, particularly from remote field sites, involves a series of risks and challenges for permitting or the logistics of storage and shipping, as well as the associated costs. Analyzing samples on site, whether with the unit we tested or other similar handheld meters, makes plasma metabolite analysis practical and possible in field conditions and for taxa where this technique has been underused due to permitting, transport, or other logistical constraints of laboratory analysis methods. The cost of analysis is similar on a per sample basis, but, without the need to store and transport samples, using handheld meters in the field may be cheaper.     

Maize ROP7 GTPase contains a unique, CaaX box-independent plasma membrane targeting signal

Signals in the carboxy-terminal hypervariable region (HVR) of Rho and Ras GTPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Rops (a plant-specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C-terminal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potential sites for post-translational modification that leads to membrane association; one is in an internal CaaX motif, which would be at the C-terminus if the final intron in both genes were not removed. Transient expression of a GFP-ROP7 fusion revealed its near-total association with the plasma membrane (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Surprisingly, the cysteine in the terminal CAA is not required for PM targeting of GFP-ROP7. In contrast, an internal HVR cysteine is essential for proper targeting of the fusion, and the cysteine in the internal CaaX is required for complete membrane association. Interestingly, this CaaX motif can also direct PM association when placed at the fusion C-terminus by addition of an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that the ROP7 HVR directs PM localization by a mechanism independent of a C-terminal CaaX motif; this mechanism may have evolved through addition of 3' intron/exon sequences to a rop progenitor.     

Acute inhibition of PMCA4, but not global ablation,reduces blood pressure and arterial contractility via a nNOS‐dependent mechanism

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild‐type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl‐l ‐NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.     

Determination of the enantiomers of citalopram,its demethylated and propionic acid metabolites in human plasma by chiral HPLC

A stereoselective HPLC assay has been developed to analyze the enantiomers of citalopram and of its three main metabolites in plasma after their separation on a Chiracel OD column. Using a fluorescence detector, the limit of quantification in plasma samples was 15, 4, 5, and 2 ng/ml for the enantiomers of citalopram (CIT), desmethylcitalopram (DCIT), didesmethylcitalopram (DDCIT), and for the citalopram propionic acid derivative (CIT-PROP), respectively. Except for CIT, all metabolites were derivatized with achiral reagents. Identification of the enantiomers was realized with an optical rotation detector which showed that the enantiomers invert their rotation depending on the polarity and nature of the solvent. Under varying conditions, a racemization study has shown that the pure enantiomers of CIT and its demethylated metabolites are configurationally stable. Preliminary results obtained with five patients treated with CIT show a mean S/R ratio of 0.7 for both CIT and its active metabolite DCIT and of 3.6 for CIT-PROP in plasma. This suggests that the pharmacologically relevant (+)-(S)-isomers of CIT and DCIT could be preferentially and steroselectively metabolized to CIT-PROP. © 1995 Wiley-Liss, Inc.     

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.