Acceleration of AMPA receptor kinetics underlies temperature-dependent changes in synaptic strength at the rat calyx of Held.

It is well established that synaptic transmission declines at temperatures below physiological, but many in vitro studies are conducted at lower temperatures. Recent evidence suggests that temperature-dependent changes in presynaptic mechanisms remain in overall equilibrium and have little effect on transmitter release at low transmission frequencies. Our objective was to examine the postsynaptic effects of temperature. Whole-cell patch-clamp recordings from principal neurons in the medial nucleus of the trapezoid body showed that a rise from 25 degrees C to 35 degrees C increased miniature EPSC (mEPSC) amplitude from -33 +/- 2.3 to -46 +/- 5.7 pA (n=6) and accelerated mEPSC kinetics. Evoked EPSC amplitude increased from -3.14 +/- 0.59 to -4.15 +/- 0.73 nA with the fast decay time constant accelerating from 0.75 +/- 0.09 ms at 25 degrees C to 0.56 +/- 0.08 ms at 35 degrees C. Direct application of glutamate produced currents which similarly increased in amplitude from -0.76 +/- 0.10 nA at 25 degrees C to -1.11 +/- 0.19 nA 35 degrees C. Kinetic modelling of fast AMPA receptors showed that a temperature-dependent scaling of all reaction rate constants by a single multiplicative factor (Q10=2.4) drives AMPA channels with multiple subconductances into the higher-conducting states at higher temperature. Furthermore, Monte Carlo simulation and deconvolution analysis of transmission at the calyx of Held showed that this acceleration of the receptor kinetics explained the temperature dependence of both the mEPSC and evoked EPSC. We propose that acceleration in postsynaptic AMPA receptor kinetics, rather than altered presynaptic release, is the primary mechanism by which temperature changes alter synaptic responses at low frequencies.

Growth inhibitory activity of indapamide on vascular smooth muscle cells.

Abnormal vascular smooth muscle cell proliferation has a fundamental role in the pathogenesis of vascular diseases. Indapamide is an oral diuretic antihypertensive drug effective for patients with mild or moderate essential hypertension. We now investigated the effects of indapamide on the growth of aortic vascular smooth muscle cells (A10 cell line). Indapamide inhibited cell proliferation as measured by the tetrazolium salt XTT (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) test. The increase in cell number was significantly reduced in the presence of indapamide 10(-6) and 5 x 10(-4) M (P < 0.05 n = 3 and P < 0.01, n = 3, respectively). Serum-induced DNA synthesis, determined as the incorporation of 5-bromo-2'-deoxyuridine (BrdU), was concentration-dependently inhibited by indapamide. BrdU incorporation was 47.2+/-1.6% (10% foetal calf serum). Indapamide treatment markedly prevented BrdU incorporation (37.2+/-2.1%, 29.2+/-4.8%, 15.0+/-1.8%, 8.7+/-2.1%) indapamide 10(-6), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. Cell-cycle progression was also evaluated. Flow cytometry analysis of DNA content in synchronised cells revealed blocking of the serum-inducible cell-cycle progression by indapamide. This inhibition was abolished when the drug was added 2 h after serum repletion, indicating that indapamide must act at the early events of a cell cycle to be fully effective against DNA synthesis. In addition, serum-induced intracellular Ca2+ movements and also p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation were studied in the presence or absence of indapamide. Indapamide 10(-5) and 5 x 10(-5) M decreased significantly cytosolic free calcium, and the p44/p42 mitogen-activated protein kinase phosphorylation (5 x 10(-5) M) stimulated by 10% foetal calf serum. In accordance with this finding, indapamide (5 x 10(-4) M) caused a 95% to 99% decrease in the early elevation of c-fos expression as evaluated by northern blot analysis of mRNA induced after serum addition. In conclusion, our results indicate that indapamide reduces vascular smooth muscle cell proliferation by a mechanism which involves a decrease in the intracellular Ca2+ movements that might link with the mitogen-activated protein kinase (MAPK) pathway, altering cell-cycle progression.

Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca2+ channels in smooth muscle cells.

There is an ongoing scientific debate concerning the potential threat of environmental estrogenic pollutants to animal and human health (1-5). Pollutants including the detergents 4-octylphenol and p-nonylphenol and chlorinated insecticides have recently been reported to modulate sexual differentiation by interacting with nuclear steroid receptors (6-8). So far, the focus has been on reproductive organs, but sex steroids have far more widespread actions. The lower incidence of cardiovascular disease in women has been attributed to estrogens (9-14), yet no information is available on the vascular actions of environmental estrogenic pollutants. In the present study we have investigated the effects of acute exposure to 17beta-estradiol, the antiestrogen ICI 182,780, and estrogenic pollutants on coronary vascular tone as well as on intracellular Ca2+ levels ([Ca2+]i) and Ca2+ and K+ channel activity in vascular smooth muscle cells. We report here that 4-octylphenol, p-nonylphenol, o.p'-DDT, and the antiestrogen ICI 182,780 inhibit L-type Ca2+ channels in vascular smooth muscle cells and evoke a rapid and endothelium-independent relaxation of the coronary vasculature similar to that induced by 17beta-estradiol. Thus, inhibition of Ca2+ influx via L-type Ca2+ channels in vascular smooth muscle cells may explain the acute, nongenomic vasodilator actions of environmental estrogenic pollutants.