PDE type-4 inhibition increases L-type Ca2+ currents, action potential firing, and quantal size of exocytosis in mouse chromaffin cells

Abstract

We studied the effects of the cAMP-hydrolyzing enzyme phosphodiesterase type-4 (PDE4) on the L-type Ca(2+) channels (LTCCs) and Ca(2+)-dependent secretion in mouse chromaffin cells (MCCs). The selective PDE4 inhibitor rolipram (3 microM) had a specific potentiating action on Ca(2+) currents of MCCs (40% increase within 3 min). A similar effect was produced by the selective beta(1)-AR agonist denopamine (1 microM) and by the unselective PDEs inhibitor IBMX (100 microM). Rolipram and denopamine actions were selective for LTCCs, and the Ca(2+) current increase remained unchanged if the two compounds were applied simultaneously. This suggests that at rest, LTCCs in MCCs are down-regulated by the low levels of cAMP determined by PDE4 activity and that LTCCs can be up-regulated by either inhibiting PDE4 or activating beta(1)-AR. No other PDEs are likely involved in this specific action. PDE4 inhibition had also a marked effect on the spontaneous firing of resting MCCs and catecholamine secretion. Rolipram up-regulated the LTCCs contributing to the "pace-maker" current underlying action potential (AP) discharges and accelerated the firing rate, with no significant effects on AP waveform. Acceleration of AP firing was also induced by the LTCC-agonist Bay K (1 microM), while nifedipine (3 microM) reduced the firing frequency, suggesting that LTCCs and intracellular cAMP play a key role in setting the pace-maker current regulating MCCs excitability. Rolipram increased also the size of the ready-releasable pool and the quantal content of secretory vesicles without affecting their probability of release. Thus, rolipram acts on MCCs by up-regulating both exocytosis and AP firings. These two processes are effectively down-regulated by PDE4 at rest and can dramatically increase the quantity of released catecholamines when PDE4 is inhibited and/or cAMP is raised.

Nanocrystalline diamond microelectrode arrays fabricated on sapphire technology for high-time resolution of quantal catecholamine secretion from chromaffin cells

“…The mean RRP was estimated by means of the double-pulse protocol shown in Fig. 3C (85 ± 6 fF; n = 5), in agreement with values reported by Marcantoni et al (2009). The two pulses (1 and 2) were delivered to produce the same quantity of Ca 2+ charge.…”

“…Patch-pipettes were made of thin borosilicate glass (Kimax 51; Witz Scientific, Holland, OH, USA) and filled with an internal solution containing (in mM): 135 CsMeSO 3 , 8 NaCl, 2MgCl 2 , 20 HEPES, pH 7.3, with CsOH plus amphotericin B. Ca 2+ currents were sampled at 10 kHz and filtered at 2 kHz. Depolarization-evoked exocytosis was measured as membrane capacitance increases by applying a sinusoidal wave function on the holding potential, as previously described (Carabelli et al, 2007;Marcantoni et al, 2009). Fast capacitive transients due to depolarizing pulses were minimized online by the patch-clamp analog compensation.…”

“…Due to the different culture conditions with respect to our standard protocols in which amperometric signals are measured from cells adherent to a substrate layer (Carabelli et al, 2007;Marcantoni et al, 2009), we first tested the functionality of non-adherent (floating) mouse chromaffin cells by evaluating the size of voltage-gated Ca 2+ channel currents and their coupling to the Ca 2+ -dependent secretion of catecholamines. Recordings were performed in the perforated-patch configuration to avoid the dialysis of the cell interior.…”

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

“…[12][13][14][15] In rat (RCCs) and mouse chromaffin cells (MCCs) they are responsible for nearly half of the total Ca 2+ current and the corresponding exocytosis. 12,[16][17][18][19][20] Second, compensatory and excess endocytosis are strongly attenuated when LTCCs are blocked. 21 Third, LTCC gating can be either up or downregulated by autocrinally released neurotransmitters coupled to membrane-delimited G protein dependent receptors or cGMP/PKG and cAMP/PKA pathways.…”

L-type channel inhibition by CB1 cannabinoid receptors is mediated by PTX-sensitive G proteins and cAMP/PKA in GT1-7 hypothalamic neurons

“…Electrophysiological recordings were performed either using an EPC-9 patch-clamp amplifier and PULSE software (HEKA Electronic, Lambrecht, Germany) [25] or using an Axopatch 200-A amplifier and pClamp 10.0 software programs (Axon Instruments Inc., Foster City, CA, USA) [32]. Currents were sampled at 10 kHz and filtered at 1-5 kHz.…”