Search

Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Cα activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Cα activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.

We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+] i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability.

The effects of moricizine on Na+ channel currents (INa) were investigated in guinea-pig atrial myocytes and its effects on INa in ventricular myocytes and on cloned hH1 current were compared using the whole-cell, patch-clamp technique. Moricizine induced the tonic block of INa with the apparent dissociation constant (Kd,app) of 6.3 microM at -100 mV and 99.3 microM at -140 mV. Moricizine at 30 microM shifted the h infinity curve to the hyperpolarizing direction by 8.6 +/- 2.4 mV. Moricizine also produced the phasic block of INa, which was enhanced with the increase in the duration of train pulses, and was more prominent with a holding potential (HP) of -100 mV than with an HP of -140 mV. The onset block of INa induced by moricizine during depolarization to -20 mV was continuously increased with increasing the pulse duration, and was enhanced at the less negative HP. The slower component of recovery of the moricizine-induced INa block was relatively slow, with a time constant of 4.2 +/- 2.0 s at -100 mV and 3.0 +/- 1.2 s at -140 mV. Since moricizine induced the tonic block of ventricular INa with Kd,app of 3.1 +/- 0.8 microM at HP = -100 mV and 30.2 +/- 6.8 microM at HP = -140 mV, and cloned hH1 with Kd,app of 3.0 +/- 0.5 microM at HP = -100 mV and 22.0 +/- 3.2 microM at HP = -140 mV, respectively, either ventricular INa or cloned hH1 had significantly higher sensitivity to moricizine than atrial INa. The h infinity curve of ventricular INa was shifted by 10.5 +/- 3.5 mV by 3 microM moricizine and that of hH1 was shifted by 5.0 +/- 2.3 mV by 30 microM moricizine. From the modulated receptor theory, we have estimated the dissociation constants for the resting and inactivated state to be 99.3 and 1.2 microM in atrial myocytes, 30 and 0.17 microM in ventricular myocytes, and 22 and 0.2 microM in cloned hH1, respectively. We conclude that moricizine has a higher affinity for the inactivated Na+ channel than for the resting state channel in atrial myocytes, and moricizine showed the significant atrioventricular difference of moricizine block on INa. Moricizine would exert an antiarrhythmic action on atrial myocytes, as well as on ventricular myocytes, by blocking Na+ channels with a high affinity to the inactivated state and a slow dissociation kinetics.

1. Ro 22-9194 reduced the Na current in ventricular myocytes in either a tonic block or phasic block manner. 2. Ro 22-9194 had a higher affinity to the inactivated state ( K di =10.3 μM) than to the rested state ( K drest =180 μM). 3. Extracellular acidification enhanced the tonic block but reduced the phasic block. 4. Elevation of extracellular Ca 2+ inhibited the enhancing effects of extracellular acidification. 5. These findings suggest that Ro 22-9194 strongly inhibits Na + channels of the ventricular myocytes of the diseased hearts, characterized by the depolarized cell membranes and by acid conditions.

We have reported that thyroid K+ channel is activated by extracellular application of the thyroid-stimulating hormone (TSH) using single channel recording method performed on cloned normal rat thyroid cell (FRTL-5) membrane. Treatment of dibutyryladenosine cyclic monophosphate (Bt2 cAMP) also activated the TSH-dependent K+ channel. These findings indicate that the thyroid K+ channel is activated through the TSH-adenosine cyclic monophosphate (cAMP)-protein kinase A system. We examined the effects of amitriptyline on TSH-guanosine triphosphate binding protein (G protein)-adenylate cyclase-cAMP-K+ channel system in the cloned normal rat thyroid cell line FRTL-5. Amitriptyline inhibited the cAMP production induced by TSH. Amitriptyline also inhibited the cAMP production induced by cholera toxin, indicating that amitriptyline inhibited the thyroid G protein. Amitriptyline had no effect on TSH-receptor binding and cAMP production by forskolin (adenylate cyclase stimulator). Amitriptyline inhibited the K+ channel activation by cAMP, indicating that the suppressing mechanism is not the inhibition of TSH receptor or G protein but the direct suppression of K+ channel. It was concluded that amitriptyline inhibited the thyroid G protein and K+ channel.

Background: Neurohormonal activation is present and neurohormonal responses to dynamic exercise are altered in congestive heart failure (CHF). Responses of plasma norepinephrine in various degrees of heart failure have been investigated, but the responses of the renin-angiotensin-aldosterone system have not been studied in relation to the severity of CHF. The aim of this study was to determine if the responses of the renin-angiotensin-aldosterone system to exercise are augmented according to the severity of CHF. Methods and Results: Ventilatory and neurohormonal responses were assessed in 38 patients with CHF (New York Heart Association class: I, 13 patients; II, 14 patients; III, 11 patients) and 11 normal subjects during symptom-limited cardiopulmonary exercise testing. Plasma norepinephrine, renin activity, angiotensin II, and aldosterone were measured at rest and at peak exercise. The increments in neurohormones were divided by peak oxygen consumption, and these ratios (norepinephrine exercise ratio, plasma renin activity-exercise ratio, angiotensin II-exercise ratio, aldosterone-exercise ratio) were compared among groups. Peak oxygen consumption and anaerobic threshold decreased progressively with the severity of CHF. Neurohormonal profiles at rest showed that plasma norepinephrine levels were significantly higher, and the renin-angiotensin-aldosterone system was augmented only in patients with class III CHF. Neurohormones increased during exercise both in patients with CHF and in normal subjects, but patients with class III CHF had significantly higher plasma renin activity (10.11 ± 2.32 ng/mL/h), angiotensin II (73.9 ± 14.2 pg/mL), and aldosterone (265.2 ± 61.1 pg/mL) than did normal subjects. Plasma renin activity-exercise ratio, angiotensin II-exercise ratio, and aldosterone-exercise ratio in patients with class III CHF were significantly higher compared to normal subjects. This augmentation of the renin-angiotensin-aldosterone system was not observed in class I or II patients. Peak plasma norepinephrine levels were not different among normal subjects and subgroups of CHF patients, but the norepinephrine-exercise ratio was significantly higher in classes II and III CHF compared to normal subjects. Conclusions: These data suggest that neurohormonal excitation during exercise increasesalong with the severity of CHF when normalized for peak exercise level.