Exposure to cAMP and β‐adrenergic stimulation recruits Ca_V3 T‐type channels in rat chromaffin cells through Epac cAMP‐receptor proteins

Abstract

T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3-5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca(2+) channels: (1) steep voltage-dependent activation from -50 mV in 10 mm Ca(2+), (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to -30 mV, (4) effective block of Ca(2+) influx with 50 microM Ni(2+), (5) comparable permeability to Ca(2+) and Ba(2+), and (6) insensitivity to common Ca(2+) channel antagonists. The action of exogenous pCPT-cAMP (200 microM) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 microM) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of beta-adrenoceptors. Stimulation of beta-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of 'chromaffin cell plasticity', which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation.

T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis

“…As reported by various groups, T-type channels are either absent or weakly expressed in bovine and rat chromaffin cells (RCCs) [15,23,24,31] but can be expressed when RCCs are exposed to cAMP for several days [25,49], after overnight incubation in hypoxic conditions (3% O 2 ) or in the presence of the hypoxia mimicking factor deferoxamine (DFX) ( Fig. 1; see also [9] and [19]).…”

“…Thus, exposure to cAMP, chronic hypoxia, and β 1 -adrenergic stimulations (which raise the level of cAMP) appear to be the suitable factors for mimicking prolonged stress conditions in vitro. We found that all these factors induce a common response in RCCs: a marked recruitment of α 1H T-type channels through cAMPreceptor proteins (Epac) [49]. This lowers the threshold of cell excitability and increases the quantity of catecholamine secreted near resting potentials [25].…”

“…A possible explanation for the absence of T-type channel secretion coupling is that in immature RCCs, LVA channels are not sufficiently localized to secretory vesicles to induce secretion even when extracellular Ca 2+ is raised to produce Ca 2+ entry in the order of 60 pC. A reason for this could be due to the lower density of T-type channel expression in immature vs mature RCCs (6.5 pA/pF at −10 mV in 10 mM Ba 2+ vs 15 pA/pF at −10 mV in 10 mM Ca 2+ , [49]), but it cannot be excluded the absence of critical factors associated to secretion or an 2+ . ΔC increases vs the corresponding quantity of charge for cAMP-treated RCCs expressing T-and R-type channels and incubated in a medium containing ω-toxins and nifedipine (empty circles, solid line) compared to ΔC increases induced by L-and Rtype channels expressed in control RCCs treated with ω-toxins (empty triangles, dashed line).…”

“…a, b Whole-cell Ca 2+ currents recorded in 10 mM Ca 2+ during a ramp command from −60 to +60 mV from −80 mV holding potential in control RCCs and from RCCs pretreated with 200 μM pCPT-cAMP and incubated for 5 days. Notice the second "lowthreshold peak" associated to the α 1H T-type currents recruited after cAMP treatment (see [49]). c, d Ca 2+ currents recorded in patchperforated conditions (5 mM Ca 2+ ) during a ramp command from −60 to +60 mV (−80 mV holding potential) in RCCs exposed overnight to a 5% CO 2 and 3% O 2 atmosphere (hypoxia) or to 300 μM deferoxamine that activates hypoxia-inducible transcription factors.…”

“…Overexpression of α 1H T-type channels during exposure to cAMP [25], β 1 -adrenergic stimulation [49], and chronic hypoxia [9,19], and their effective coupling to catecholamine secretion suggest that T-type channels of chromaffin cells may be conditional in the regulation of adrenal medulla response to stress conditions. Chromaffin cells play a key role in the response to stress and are an excellent model for studying the effects of stress on gene expression.…”