No abstract

Intracellular microelectrode recordings were used to determine whether nitric oxide (NO), affects the pacemaker events that initiate vasomotion in lymphatic vessels of the guinea pig mesentery. This pacemaker activity is recorded as spontaneous transient depolarizations (STDs) and is likely to arise through synchronized Ca2+ release from intracellular stores. We show here that acetylcholine-induced endothelium-derived NO and exogenous NO released by sodium nitroprusside (SNP; 100 microM) and DEA-NONOate (500 microM) reduced the frequency and amplitude of STDs. This inhibition of STD frequency and amplitude was independent of the NO-induced hyperpolarization of the smooth muscle. The SNP-induced inhibition of STD frequency and amplitude was abolished during superfusion with the soluble guanylyl cyclase inhibitor ODQ (10 microM) and was diminished in the presence of cGMP and cAMP-dependent protein kinase inhibitors. The data are consistent with the hypothesis that NO inhibits vasomotion primarily by production of cGMP and activation of both cGMP- and cAMP-dependent protein kinases, which reduce the size and frequency of STDs, probably by acting on the underlying synchronized Ca2+ release from intracellular stores.

This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP(3)R)-operated Ca(2+) stores interact as coupled oscillators to produce near-synchronous Ca(2+) release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP(3), to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca(2+) transients and/or intracellular Ca(2+) waves. Larger near-synchronous Ca(2+) transients then occurred leading to global synchronous Ca(2+) transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca(2+) channels with nifedipine prevented ET-1 from inducing near-synchronous Ca(2+) transients and resultant action potentials, leaving only asynchronous Ca(2+) transients and local Ca(2+) waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca(2+) release from IP(3)R-operated Ca(2+) stores, which causes depolarization; 2), L-type Ca(2+) channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca(2+) channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca(2+) store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca(2+) stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca(2+) store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca(2+) release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca(2+) release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.

Changes in the structural and gel textural properties were investigated in soy protein isolate (SPI) that was subjected to extreme acid pH-shifting and mild heating processes. The SPI was incubated up to 5 h in pH 1.5 solutions at room temperature or in a heated water bath (50 or 60 °C) to lead to protein structural unfolding, followed by refolding at pH 7.0 for 1 h. The combination of pH-shifting and heating treatments resulted in drastic increases in the SPI gel penetration force (p < 0.05). These treatments also significantly enforced the conversion of sulphydryl groups into disulfides, increased the particle size and hydrophobicity values, reduced the protein solubility (p < 0.05), and strengthened the disulfide-mediated aggregation of SPI. The intrinsic fluorescence spectroscopy results indicated structural unravelling when protein was subjected to acidic pH-shifting in combination with heating processes. The slight loss of secondary structure was observed by circular dichroism. These results suggested that pH-shifting combined with heating treatments provide great potential for the production of functionality-improved SPI, with the improved gelling property highly related to changes in the protein structure and hydrophobic aggregation.

1 In vitro experiments were performed to investigate the actions of endothelin-1 (ET-1) on vasomotion and vasospasm in guinea-pig mesenteric lymphatics. 2 ET-1 modulated lymphatic vasomotion independent of the endothelium, with lower concentrations (p10 nM) increasing lymphatic vasomotion and higher concentrations (X100 nM) causing vasospasm. 3 ET-1-induced increases in vasomotion were accompanied by an increase in tonic [Ca 2 þ ] i . 4 These actions were inhibited by the ET A receptor antagonist BQ-123 (1 mM), the phospholipase C (PLC) inhibitor U73122 (5 mM), removal of extracellular Ca 2 þ , chelation of intracellular Ca 2 þ with BAPTA/AM (10 mM), the store Ca 2 þ -ATPase inhibitor thapsigargin (1 mM), caffeine (10 mM) and the inositol 1,4,5-trisphosphate (IP 3 ) receptor blocker heparin and 2-APB (30 mM). In contrast, the ET B receptor antagonist BQ-788 (1 mM), ryanodine (1 & 20 mM), pertussis toxin (PTx) or Cs þ had no significant actions on vasomotion or the magnitude of increase in tonic [Ca 2 þ ] i . 5 ET-1-induced vasospasm was accompanied by a transient increase in smooth muscle [Ca 2 þ ] i followed by a sustained plateau, an action that was abolished by removal of extracellular Ca 2 þ , but only marginally inhibited by nifedipine (1 mM). 6 Caffeine (10 mM), SKF 96165 (30 mM) or U73122 (5 mM) together with nifedipine (1 mM) abolished ET-1-induced vasospasm and increase in [Ca 2 þ ] i . 7 These results indicate that ET-1 increases lymphatic vasomotion by acting on smooth muscle ET A receptors and activation of G-protein-PLC-IP 3 cascade, which is known to cause pacemaker Ca 2 þ release and resultant pacemaker potentials. High concentrations of ET-1 cause a failure in Ca 2 þ homeostasis causing vasospasm, triggered by excessive Ca 2 þ influx primarily through store-operated channels (SOCs) with L-Ca 2 þ voltage-operated channels (VOCs) also contributing, but to a much lesser extent.