Biomedical subjects

Delrae M Eckman

Publications and source records attributed to Delrae M Eckman.

7 recordsLinked to original sources

PubMed2006

Nicotine upregulates the expression of P2Y12 on vascular cells and megakaryoblasts.

BACKGROUND: P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression. METHODS: Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed. RESULTS: Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. CONCLUSION: Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. CONDENSED ABSTRACT: The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.

Adult↗

PubMed2005

Piglet pial arteries respond to N-methyl-D-aspartate in vivo but not in vitro.

Controversy exists concerning whether activation of N-methyl-D-aspartate (NMDA) receptors exerts direct dilator effects on cerebral arteries. The purpose of this study was to examine the responses of isolated piglet arteries to NMDA to determine whether isolated arteries, apart from surrounding neuronal tissue, are capable of responding to NMDA. Piglet arteries (100-200 microm) were isolated from branches of the middle cerebral artery and carefully dissected free of adherent tissue. Arteries were then mounted in an arteriograph system and pressurized to either 30 mm Hg (n=8), 60 mm Hg (n=10), 80 mm Hg (n=6), or 100 mm Hg (n=5). After development of spontaneous tone, NMDA (10(-5) to 10(-3) M) was administered abluminally to the vessels, and no appreciable response was noted (for example; 10(-4) M, 30 mm Hg: 3+/-3% change in active diameter; 60 mm Hg: -4+/-3% change in active diameter). Following a thorough washout, vessels were treated with bradykinin (10(-9) to 10(-7) M), and the arteries did respond (10(-7) M, 30 mm Hg: 26+/-3% change in active diameter; 60 mm Hg: 65+/-10% change in active diameter). In contrast, 10(-5) M and 10(-4) M NMDA dilated arteries in vivo by 9+/-2% and 29+/-6% change in active diameter, respectively (n=6). These results demonstrate that isolated cerebral arteries do not respond directly to NMDA receptor activation. This work confirms our previous in vivo data and is consistent with the hypothesis that cerebral arteries respond to NMDA through a secondary interaction mediated by neuronal release of NO and not to NMDA directly.

Animals↗

PubMed2004

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling.

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.

Amino Acid Sequence↗

PubMed2004

Novel PPARgamma agonists GI 262570, GW 7845, GW 1929, and pioglitazone decrease calcium channel function and myogenic tone in rat mesenteric arteries.

Novel non-thiazolidinedione, tyrosine-derived peroxisome proliferator-activated receptor gamma agonists, GI 262570, GW 7845, GW 1929, developed by GlaxoSmithKline (GSK) along with pioglitazone and nisoldipine, were studied on currents through L-type voltage-dependent calcium channels (VDCC) in freshly isolated smooth muscle cells from mesenteric arteries, and on the diameter of pressurized mesenteric arteries in vitro. Using Ba2+ (10 mmol/l) as the charge carrier through VDCC, the half-inhibition constants (IC50) for GI 262570, GW 7845, GW 1929, and pioglitazone were 2.0 +/- 0.5, 3.0 +/- 0.5, 5.0 +/- 0.7, and 10.0 +/- 0.8 mumol/l, respectively. For arterial diameter measurements the IC50 values for GI 262570, GW 7845, GW 1929, and pioglitazone were 2.4, 4.1, 6.3, and 13.9 mumol/l, respectively. Each GSK compound and pioglitazone was effective at inhibiting VDCC and relaxing pressurized arteries, suggesting that the vasodilation of resistance arteries could be explained by the inhibition of calcium entry through VDCC.

Algorithms↗

PubMed2004

20-hydroxyeicosatetraenoic acid is a vasoconstrictor in the newborn piglet pulmonary microcirculation.

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome p-450 metabolite of arachidonic acid, is a vasoconstrictor in the systemic circulation and a vasodilator in the adult pulmonary circulation. Little is known about the vasoactive properties of 20-HETE in the newborn pulmonary circulation. The objectives of this study were to determine the vascular effects of 20-HETE and to explore the signaling mechanism(s) that mediate these effects in newborn pulmonary resistance-level arteries (PRA). Our findings demonstrate that, in contrast to the adult pulmonary circulation where 20-HETE mediates vasodilation, it causes constriction in newborn PRA at resting tone. Furthermore, inhibition of cyclooxygenase (COX) with indomethacin augments 20-HETE-induced constriction. The enhanced constrictor response to 20-HETE under conditions of COX inhibition is abolished in endothelium-disrupted PRA, suggesting that 20-HETE either stimulates endothelium-derived COX to release a counteracting vasodilator or is rapidly metabolized by COX to a less potent vasoconstrictor. 20-HETE-induced constriction is significantly inhibited by blocking calcium-dependent K(+) (K(Ca)) channels and the thromboxane-PGH(2) receptor. Altogether, our data indicate that the vascular actions of 20-HETE are partially mediated via the activation of K(Ca) channels and are significantly modulated by interactions with the COX-prostaglandin pathway.

Animals↗

PubMed2003

Intraluminal pressure is a stimulus for NFATc3 nuclear accumulation: role of calcium, endothelium-derived nitric oxide, and cGMP-dependent protein kinase.

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle we have found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Here, we demonstrate that physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. We further show that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2(-/-) mice. Collectively, our results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. Our findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation.

Active Transport, Cell Nucleus↗

PubMed2003

Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates arterial tone and blood pressure.

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca2+-activated K+ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure- and phenylephrine-induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure.

Animals↗