Ascher's syndrome: a rare cause of lip swelling.

Ascher's syndrome is a rare, benign entity with just over 100 reported cases. The condition is characterised by a 'double' upper lip, blepharochalasis and non-toxic thyroid enlargement. It presents before the age of 20 years in the majority of cases and shows no racial or gender differences. While the exact cause is unknown, hormonal dysfunction and autosomal dominant inheritance have been suggested as possible aetiological factors. We present two cases of Ascher's syndrome referred for investigation of lip swelling.

Phospholipids released from activated platelets improve platelet aggregation and endothelial cell migration.

This study was undertaken to isolate phospholipids released from activated platelets and to investigate their biological activities. Freshly washed platelets were activated with freezing/thawing, thrombin, ionophore 23187, and arachidonic acid. Thrombin was incubated with platelet-rich plasma to promote synthesis and release of phospholipids from platelets. Phospholipids in supernatants of activated platelets were extracted with butanol and separated by thin-layer chromatography. Release of phosphatidylserine (PS) and phosphatidic acid (PA) increased when platelets were treated with freezing/thawing, ionophore, and thrombin. The lysophosphatidyl ethanolamine (LPE) appeared not to be induced with freezing/thawing, but increased significantly by thrombin, ionophore, and arachidonic acid. The effects of platelet phospholipids on hemostasis and angiogenesis were studied with platelet aggregation and endothelium chemotaxis. Phospholipids isolated from thrombin-stimulated platelet-rich/platelet-poor plasmas were used as synergistic agonists in platelet aggregation and as chemotactic agents in endothelial cell migration. Several phospholipids increased chemotaxis and platelet aggregation; these were PS, PA, LPE, and sphingosine-1-phosphate. Also, chemotaxis of those phospholipids increased when combined with charcoal-stripped fetal bovine serum, suggesting that cofactors in serum enhanced phospholipid-induced cell migration. These observations suggest that activated platelets release biologically active phospholipids into the blood stream, where they may play an important role in thrombosis and angiogenesis.

Nitric oxide attenuates H(2)O(2)-induced endothelial barrier dysfunction: mechanisms of protection.

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that.NO attenuates H(2)O(2)-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H(2)O(2), and barrier function was measured as transmonolayer albumin clearance. H(2)O(2) caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular.NO generation.NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate.NO-mediated barrier protection. In contrast, H(2)O(2) decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of.NO. H(2)O(2)-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H(2)O(2)-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that.NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.

Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells.

Tyrosine phosphorylation of proteins, controlled by tyrosine kinases and protein tyrosine phosphatases, plays a key role in cellular growth and differentiating. A wide variety of hormones, growth factors, and cytokines modulate cellular tyrosine phosphorylation to transmit signals across the plasma membrane to the nucleus. Recent studies suggest that reactive oxygen species (ROS) also induce cellular protein tyrosine phosphorylation through receptor or nonreceptor tyrosine kinases. To determine whether protein tyrosine phosphorylation by ROS regulates endothelial cell (EC) metabolism and function, we exposed vascular ECs to H2O2 or H2O2 plus vanadate. This resulted in a time- and dose-dependent increase in protein tyrosine phosphorylation of several proteins (M(r) 21-200 kDa), as determined by immunoprecipitation and Western blot analysis with antiphosphotyrosine antibody. Immunoprecipitation with specific antibodies identified increased tyrosine phosphorylation of mitogen-activated protein kinases (42-44 kDa), paxillin (68 kDa), and FAK (125 kDa) by ROS. An immediate signaling response to increased protein tyrosine phosphorylation by ROS was activation of phospholipases such as A2, C, and D. Suramin pretreatment inhibited ROS stimulation of phospholipase D (PLD), suggesting a role for growth factor receptors in this activation. Further, PLD activation by ROS was attenuated by N-acetylcysteine, indicating that intracellular thiol status is critical to ROS-mediated signal transduction. These results provide evidence that ROS modulate EC signal transduction via a protein tyrosine phosphorylation-dependent mechanism.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate.

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

The enhancement by wortmannin of protein kinase C-dependent activation of phospholipase D in vascular endothelial cells.

Phosphatidic acid generation by phospholipase D (PLD) activation has been implicated in agonist- and oxidant-mediated endothelial cell signal transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. Pretreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100 microM), and bradykinin- (1 microM) induced [32P]PEt formation, an index of PLD activation. However, wortmannin by itself had no effect on PLD activity. The potentiating effect of wortmannin on TPA-induced PLD activation was dose- (1-10 microM) and time-dependent (5-30 min) and was inhibited by bisindoylmalemide, an inhibitor of protein kinase C (PKC). Furthermore, down-regulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuated the wortmannin effect. This effect of wortmannin was specific for TPA- or agonist-induced PLD activation as no potentiation of [32P]PEt formation was observed with H2O2 (1 mM) or ionomycin (1 microM). The effect of wortmannin was not due to activation of PKC alpha as determined by western blot analysis of PKC alpha in the cytosol and membrane fractions. Also, genistein, an inhibitor of tyrosine kinases, did not attenuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involvement of protein tyrosine phosphorylation. These results indicate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells.

Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle.

Evidence suggests that the mechanical behavior of smooth muscle tissues is regulated by Ca(2+)-dependent changes in the phosphorylation of the 20,000-Da light chain of myosin (MLC). However, alternative mechanisms activated by specific kinases may be involved in regulating the shortening velocity in some smooth muscle tissues. To determine how the activation of protein kinases A or C affects the regulation of the shortening velocity in canine tracheal smooth muscle, we evaluated the effects of forskolin (10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 3 x 10(-6) M) on active stress, intracellular Ca2+ ([Ca2+]i), MLC phosphorylation, and isotonic shortening velocity during contractions elicited by 60 mM KCl. Forskolin depressed and PDBu increased active stress, [Ca2+]i, MLC phosphorylation, and shortening velocity; thus the effects of these agents on the shortening velocity may result from changes in Ca(2+)-dependent MLC phosphorylation. In contrast, the decline in velocity that occurred with time during tonic contractions elicited by K+ depolarization was not associated with significant changes in MLC phosphorylation; thus the time-dependent changes in shortening velocity may be regulated by a mechanism other than MLC phosphorylation.

Differences in Ca2+ mobilization by muscarinic agonists in tracheal smooth muscle.

The muscarinic agonists acetylcholine (ACh) and McN-A-343 activate a homogeneous population of M3 receptors in canine tracheal smooth muscle. However, ACh is much more efficacious than McN-A-343 both at stimulating force development and active shortening and at antagonizing relaxation induced by isoproterenol. In other tissues, the same muscarinic receptor may be coupled to multiple subcellular pathways, but activate different pathways depending on the efficacy of the agonist. The present study investigated mechanisms of excitation-contraction coupling in canine tracheal smooth muscle by muscarinic agonists of different efficacy. ACh was more effective at stimulating inositol phosphate production and elicited a large initial transient increase in intracellular Ca2+ concentration ([Ca2+]i), whereas McN-A-343 elicited only a slow gradual rise in [Ca2+]i. Extracellular Ca2+ influx through voltage-operated channels played a minor role in contractions induced by either agonist. Results suggest that contractions by ACh and McN-A-343 may be mediated by the same subcellular pathways; however, the greater potency of ACh at stimulating those pathways results in very different kinetics of Ca2+ activation.

Ca2+ sensitivity of contractile activation during muscarinic stimulation of tracheal muscle.

The muscarinic agonists acetylcholine (ACh) and McN-A-343 act on a homogenous population of M3 receptors in canine tracheal smooth muscle; however, ACh is more effective at releasing stored Ca2+ and at stimulating inositol phosphate production. The effects of ACh and McN-A-343 on intracellular Ca2+ concentration ([Ca2+]i), myosin light chain (MLC) phosphorylation, active stress, and isotonic shortening velocity were compared to determine whether differences in their potency at stimulating second messenger pathways affected their ability to modulate the Ca2+ sensitivity of contractile filament activation. There were no differences in [Ca2+]i or isometric stress during the steady-state phase of submaximal contractions induced by ACh and McN-A-343. ACh produced slightly higher levels of MLC phosphorylation than McN-A-343; these levels were associated with much higher rates of isotonic shortening. This could indicate either an extremely high sensitivity of the shortening velocity to differences in MLC phosphorylation or that mechanisms other than MLC phosphorylation contribute to the regulation of shortening velocity. Results show that receptor-coupled pathways can modulate the relationship between [Ca2+]i and isotonic shortening velocity independently of the relationship between [Ca2+]i and isometric stress.