Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins.
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.
Novara
M
M
Department of Neuroscience, INFM Research Unit, 10125 Torino, Italy.
Baldelli
P
P
Cavallari
D
D
Carabelli
V
V
Giancippoli
A
A
Carbone
E
E
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D001464:Barium / Q000493:pharmacokinetics; D002118:Calcium / Q000493:pharmacokinetics; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000502:physiology*; D019439:Chromaffin Cells / Q000502:physiology*; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D013564:Sympathetic Nervous System / Q000502:physiology
C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D000318:Adrenergic beta-Agonists; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D001464:Barium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D002118:Calcium