Increase in Ca2+ current by sustained cAMP levels enhances proliferation rate in GH3 cells.

AIMS: Ca2+ and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca2+ currents and proliferation in pituitary tumor GH3 cells. MAIN METHODS: Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca2+ current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. KEY FINDINGS: Sustained forskolin treatment (24 and 48h) induced a significant increase in total Ca2+ current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca2+ current density. However, the maximum effect of dbcAMP occurred only after 72h incubation, whereas forskolin showed maximal effect at 48h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca2+ channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. SIGNIFICANCE: We conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca2+ current density and this phenomenon impacts proliferation rate in GH3 cells.

Angiotensin II increases excitability and inhibits a transient potassium current in vagal primary sensory neurons.

The octapeptide angiotensin II (ANG II) plays a pivotal role in the maintenance of blood pressure by activating ANG II receptors located in variety of cell types including neurons housed in the central nervous system (CNS) and in the peripheral nervous system (PNS). ANG II (100 nM) blocked spike frequency accommodation (SFA) recorded with whole-cell patch technique in acutely isolated nodose ganglion neurons (NGN) from adult rats. ANG II increased the frequency of action potentials (AP) produced by supramaximal 500 ms depolarizing currents recorded in both tonic (16 Hz vs. 58 Hz, control vs. ANG II perfusion respectively, n=9) and phasic (1Hz vs. 38 Hz, n=13) NGNs. ANG II produced no significant changes in: the resting membrane potential (-51 mV vs. -50 mV, n=65), AP overshoot (46 mV vs. 41 mV, n=25), AP undershoot (-65 mV vs. -61 mV, n=25), AP duration (1 ms vs. 1.2 ms, n=25), and AP threshold (-40 mV vs. -43 mV, n=19). CV-11974 (600 nM), a specific AT1 receptor antagonist, prevented ANG II-evoked changes SFA (n=10). ANG II (100 nM) had no significant effect on total outward potassium current (I(K)) but inhibited a fast activating and fast inactivating I(K) recorded in the presence of TEA. A kinetically similar I(K) was also inhibited by 4-AP (3mM). In phasic NGNs, 4-AP occluded the effects of 100 nM ANG II on SFA. Our results indicate that ANG II can block an A-type of I(K) and that this effect may underlie the ANG II-mediated change in SFA.

Angiotensin II inhibition of Ca2+ currents is independent of ATR1 angiotensin II receptor activation in rat adult vagal afferent neurons.

Angiotensin II (ANG II) has the ability to modulate the activity of neurons involved in the cardiovascular regulation. One effective way of doing that is by changing calcium currents. In the present study, we investigated the effects of ANG II on high-voltage-activated (HVA) Ca2+ currents measured in adult vagal afferent neurons using the whole-cell patch-clamp technique. In addition, we demonstrated the presence of ATR1 and ATR2 receptors mRNA at nodose neurons using conventional reverse transcriptase-polymerase chain reaction (RT-PCR). ANG II (100 nM) decreased the HVA Ca2+ current (peak current recorded at 0 mV: -60.9+/-8.7 pA/pF in control conditions versus -31.9+/-5.7 pA/pF in the presence of ANG II) and shifted the Ca2+ current activation to a more negative membrane potential (control V0.5=-12.5+/-1.5 mV versus -18.4+/-2.8 mV during perfusion with ANG II). Losartan (500 nM) was not able to prevent the ANG II effect on the HVA Ca2+ current making unlikely the involvement of the ATR1 receptor. When ANG II was perfused in the continuous presence of saralasin, a non-selective ANG II receptor antagonist, we observed a faster but transient inhibition of HVA Ca2+ current. The inhibition was not sustained as observed when we applied ANG II alone and the HVA Ca2+ current recovered with time reaching levels close to the control. Unexpectedly, treatment with the ATR2 blocker PD 123,319 (500 nM) caused a significant inhibition on the HVA Ca2+ current making rather difficult any further conclusions. The above results allow us to conclude that ANG II induced inhibition on the HVA Ca2+ current is probably not via ATR1 receptor activation.

Veratridine modifies the TTX-resistant Na+ channels in rat vagal afferent neurons.

A number of neurotoxins from venoms of invertebrates and plants are ligands for voltage-gated Na+ channels and are useful tools for studying Na+ channel function and structure. Using whole-cell recordings from vagal afferent nodose neurons, we studied neurotoxins that target Na+ channels. We asked whether Ts3 (an alpha-scorpion toxin) and/or veratridine (a lipid-soluble toxin), could modify the TTX-resistant Na+ current generated by vagal afferent nodose neurons. Nodose TTX-resistant current was not affected by Ts3, whereas Ts3 slowed inactivation of the current generated by TTX-sensitive current component. We found that veratridine inhibited the TTX-resistant Na+ currents on rat nodose neurons. Interestingly, veratridine-modified Na+ channels developed a persistent current that accounted for the large tail current observed. We propose that veratridine modifies TTX-resistant Na+ channels through a mechanism distinct from its actions on other voltage-gated Na+ channels.