Piezo1 integration of vascular architecture with physiological force.

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

Expression of a long variant of CRACR2A that belongs to the Rab GTPase protein family in endothelial cells.

CRACR2A protein is described in T cells as an EF-hand-containing modulator of calcium-release-activated calcium (CRAC) channels. Here we sought relevance to calcium entry of endothelial cells. Unexpectedly, short interfering RNA designed to deplete CRACR2A had no effect on CRAC channels in endothelial cells but reduced the abundance of a protein with about twice the mass of CRACR2A. Reference to gene sequence data indicated the potential for a variant transcript encoding a C-terminal Rab GTPase extension of CRACR2A. Full-length cloning demonstrated expression of the long variant in endothelial cells. It was designated CRACR2A-L. Sequence analysis suggested it to be a previously unrecognised member of the Rab GTPase family. It made a positive contribution to endothelial tube formation. The data suggest that endothelial cells contain a long variant of CRACR2A which is an EF-hand-containing Rab protein that lacks impact on CRAC channels.

Constitutively active TRPC channels of adipocytes confer a mechanism for sensing dietary fatty acids and regulating adiponectin.

RATIONALE: Calcium entry is pivotal in the heart and blood vessels, but its significance and mechanisms in adipose tissue are largely unknown. An important factor produced by adipocytes is adiponectin, which confers myocardial protection, insulin-sensitization, and antiatherosclerotic effects. OBJECTIVE: To investigate the relevance of calcium channels to adipocytes and the production of adiponectin. METHODS AND RESULTS: Microarray analysis led to identification of transient receptor potential canonical (TRPC)1 and TRPC5 as channel subunits that are induced when adipocytes mature. Both subunits were found in perivascular fat of patients with atherosclerosis. Intracellular calcium and patch-clamp measurements showed that adipocytes exhibit constitutively active calcium-permeable nonselective cationic channels that depend on TRPC1 and TRPC5. The activity could be enhanced by lanthanum or rosiglitazone, known stimulators of TRPC5 and TRPC5-containing channels. Screening identified lipid modulators of the channels that are relevant to adipose biology. Dietary ω-3 fatty acids (eg, α-linolenic acid) were inhibitory at concentrations that are achieved by ingestion. The adipocyte TRPC1/TRPC5-containing channel was functionally negative for the generation of adiponectin because channel blockade by antibodies, knock-down of TRPC1-TRPC5 in vitro, or conditional disruption of calcium permeability in TRPC5-incorporating channels in vivo increased the generation of adiponectin. The previously recognized capability of α-linolenic acid to stimulate the generation of adiponectin was lost when calcium permeability in the channels was disrupted. CONCLUSIONS: The data suggest that TRPC1 and TRPC5 contribute a constitutively active heteromultimeric channel of adipocytes that negatively regulates adiponectin and through which ω-3 fatty acids enhance the anti-inflammatory adipokine, adiponectin.

Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.

RATIONALE: Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown. OBJECTIVE: The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. METHODS AND RESULTS: In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release-activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry. CONCLUSIONS: The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.

Rapid and contrasting effects of rosiglitazone on transient receptor potential TRPM3 and TRPC5 channels.

The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 µM but was inhibited completely at higher concentrations (IC(50), ∼22.5 µM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1-1 µM) and full inhibition at higher concentrations (IC(50), 5-10 µM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 µM (EC(50), ∼30 µM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC(50), 12 µM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J(2), had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.

Neuropeptide modulation of a lumbar spinal reflex: potential implications for female sexual function.

INTRODUCTION: Neuropeptides are known to modulate female receptivity. However, even though receptivity is a spinal reflex, the role of neuropeptides within the spinal cord remains to be elucidated. AIM: The aims were to (i) investigate neuropeptides in the lumbosacral region; and (ii) determine how neuropeptides modulate glutamate release from stretch Ia fibers, touch sensation Abeta fibers and Adelta/C pain fibers. MAIN OUTCOME MEASURES: Neuropeptide modulation of the lumbosacral dorsal-root ventral-root reflex in vitro. METHODS: Spinal cords were removed from Sprague-Dawley rats in compliance with UK Home Office guidelines. Hemisected cords were superfused with aCSF and the dorsal root (L4-S1) was stimulated to evoke glutamate release. A biphasic reflex response was evoked from the opposite ventral root consisting of a monosynaptic (Ia fibers) and polysynaptic (Abeta, Adelta/C fibers) component. RESULTS: The micro opioid receptor (MOR) agonist DAMGO inhibited the monosynaptic (EC(50) 0.02 +/- 0.02 nM) and polysynaptic area (EC(50) 125 +/- 167 nM) but not polysynaptic amplitude. Oxytocin and corticotrophin releasing factor (CRF) inhibited the monosynaptic amplitude (EC(50), 1.4 +/- 1.0 nM and EC(50) 4.3 +/- 3.5 nM, respectively), polysynaptic amplitude (EC(50) 18.2 +/- 28.0 nM and EC(50), 9.5 +/- 13.3 nM, respectively), and area (EC(50) 11.6 +/- 13.0 nM and EC(50), 2.8 +/- 3.3 nM, respectively); effects that were abolished by oxytocin and CRF(1) antagonists, L-368899 and 8w. Melanocortin agonists solely inhibited the monosynaptic component, which were blocked by the MC(3/4) receptor antagonist SHU9119. CONCLUSION: These data suggest endogenous neuropeptides are released within the lumbosacral spinal cord. Melanocortin agonists, oxytocin, CRF, and DAMGO via MC(4), oxytocin, CRF(1), and MOR inhibit glutamate release but with differing effects on afferent fiber subtypes. Melanocortins, oxytocin, CRF, and DAMGO have the ability to modulate orgasm whereas oxytocin, CRF and DAMGO can increase pain threshold. Oxytocin and CRF may dampen touch sensation.

Rab46 integrates Ca2+ and histamine signaling to regulate selective cargo release from Weibel-Palade bodies.

Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes the release of the proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory cargo away from the plasma membrane to the microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes to a subset of P-selectin-negative WPBs. After acute stimulation of the H1 receptor, GTP-bound Rab46 evokes dynein-dependent retrograde transport of a subset of WPBs along microtubules. Upon continued histamine stimulation, Rab46 senses localized elevations of intracellular calcium and evokes dispersal of microtubule organizing center-clustered WPBs. These data demonstrate for the first time that a Rab GTPase, Rab46, integrates G protein and Ca2+ signals to couple on-demand histamine signals to selective WPB trafficking.

Natural and synthetic flavonoid modulation of TRPC5 channels.

BACKGROUND AND PURPOSE: The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products. EXPERIMENTAL APPROACH: Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. KEY RESULTS: Through a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide-evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC50 0.45 µM). Galangin also inhibited lanthanide-evoked TRPC5-mediated current in whole-cell and outside-out patch recordings. In differentiated 3T3-L1 cells, it inhibited constitutive and lanthanide-evoked calcium entry through endogenous TRPC5-containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure-activity relationship studies with natural and synthetic flavonols, we designed 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12), which inhibited lanthanide-evoked TRPC5 activity with an IC50 of 0.28 µM. AM12 also inhibited TRPC5 activity evoked by the agonist (-)-Englerin A and was effective in excised outside-out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1-TRPC5 heteromeric channels was weaker. CONCLUSIONS AND IMPLICATIONS: The data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.

Urinalysis.

Urine analysis is an essential component of patient assessment, which is used for screening, diagnosis and planning care. This article discusses specimen collection and reagent strip testing.

Understanding bowel problems in older people: Part 2.

Bowel problems can be devastating for patients and those who care for them. Accurate assessment is essential in determining the cause of symptoms and deciding treatment and management strategies. Treatment can be complex, but with care and patience improvement or cure is possible. Part 1 of this two-part article offered an overview of bowel problems in older people and an introduction to assessment. Part 2 discusses specific components of assessment, treatment and management.

Understanding bowel problems in older people: part 1.

Bowel problems can be devastating for patients and those who care for them. Accurate assessment is essential to determine the cause of symptoms and to decide treatment and management strategies. Treatment and management can be complex but with care and patience improvement or cure is possible. Part 1 of this two-part article offers an overview of bowel problems in older people and an introduction to assessment.

Assessment of performance of laboratories in determining acrylamide in crispbread.

A proficiency testing round was undertaken to assess the performance of laboratories to measure acrylamide in a sample of crispbread. Retail samples of crispbread were ground to a fine powder and after thorough mixing were packed in 40 g units for distribution. Ten samples were selected at random and analyzed in duplicate for acrylamide by liquid chromatography/mass spectrometry (LC/MS). Standard statistical tests showed that the material was homogeneous for the purposes of proficiency testing. Test samples were distributed to 55 laboratories in 16 countries in Europe, North America, Australia, and the Middle East. The results were analyzed by standard proficiency testing statistical procedures, and laboratories were awarded z-scores on the basis of their reported results. Based on a target standard deviation (sigmap value) taken from the Horwitz equation, for a robust mean value of 1.2 mg/kg acrylamide, satisfactory results (z-score within +/- 2 for those between 0.8 and 1.6 mg/kg) were obtained by 86% of the 37 laboratories that returned results. Only 1 laboratory was unsatisfactory and 4 had questionable results. About equal numbers of laboratories used gas chromatography (GC)/MS and LC/MS procedures with about 25% using MS/MS and one using GC with electron capture detection. There was no evident trend in performance or bias in results. GC/MS and LC/MS data were evenly distributed across the population of laboratories reporting results.

Inhibition of endothelial cell Ca²âº entry and transient receptor potential channels by Sigma-1 receptor ligands.

BACKGROUND AND PURPOSE: The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels. EXPERIMENTAL APPROACH: Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies. KEY RESULTS: In endothelial cells, 10-100 µM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3. CONCLUSIONS AND IMPLICATIONS: The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.

cDNA cloning, functional expression, and characterization of chicken sulfotransferases belonging to the SULT1B and SULT1C families.

A search of the chicken expressed sequence tag (EST) database identified 2 cDNA clones that appeared to represent members of the SULT1B and SULT1C enzyme families. These cDNAs were fully sequenced and found to contain full-length inserts. Phylogenetic analysis of the derived amino acid sequences clearly placed them as the first members of the chicken SULT1B and SULT1C families, respectively, to be identified, and we propose they be named SULT1B1 and SULT1C1. (CHICK)SULT1B1 shares approximately 60% amino acid sequence identity with mammalian SULT1B enzymes, whereas the closest neighbor to (CHICK)SULT1C1 was the ortholog (RAT)SULT1C1, with 68% identity. We cloned these cDNAs into the bacterial expression vectors from the pET series. Transformed Escherichia coli cells strongly expressed the recombinant proteins. Purification of the recombinant enzymes from E. coli was accomplished by a three-step procedure involving ammonium sulfate precipitation, anion exchange chromatography, and affinity chromatography. The purified enzymes displayed subunit molecular weights of approximately 35,000Da on SDS-PAGE, as predicted, and were both able to sulfate a wide range of compounds, including xenobiotics and endogenous substrates such as iodothyronines. Detailed kinetic analysis showed SULT1C1 was more prolific in that it was able to sulfate dopamine, tyramine, and apomorphine, which SULT1B1 was not. 2-Bromophenol was the best substrate for both enzymes. We also raised antibodies against these proteins, which were able to detect the SULTs by ELISA, and which were able to strongly inhibit the recombinant enzymes. This is the first detailed characterization of sulfotransferases from the chicken, and it demonstrates that the avian and mammalian SULT1 enzymes are closely related in both structure and function.