Distinct potentiation of L-type currents and secretion by cAMP in rat chromaffin cells.
We have investigated the potentiating action of cAMP on L-currents of rat chromaffin cells and the corresponding increase of Ca(2+)-evoked secretory responses with the aim of separating the action of cAMP on Ca(2+) entry through L-channels and the downstream effects of cAMP/protein kinase A (PKA) on exocytosis. In omega-toxin-treated rat chromaffin cells, exposure to the permeable cAMP analog 8-(4-chlorophenylthio)-adenosine 3',5'-monophosphate (pCPT-cAMP; 1 mM, 30 min) caused a moderate increase of Ca(2+) charge carried through L-channels (19% in 10 mM Ca(2+) at +10 mV) and a drastic potentiation of secretion ( approximately 100%), measured as membrane capacitance increments (deltaC). The apparent Ca(2+) dependency of exocytosis increased with pCPT-cAMP and was accompanied by 83% enhancement of the readily releasable pool of vesicles with no significant change of the probability of release, as evaluated with paired-pulse stimulation protocols. pCPT-cAMP effects could be mimicked by stimulation of beta(1)-adrenoreceptors and reversed by the PKA inhibitor H89, suggesting strict PKA dependence. For short pulses to +10 mV (100 ms), potentiation of exocytosis by pCPT-cAMP was proportional to the quantity of charge entering the cell and occurred independently of whether L, N, or P/Q channels were blocked, suggesting that cAMP acts as a constant amplification factor for secretion regardless of the channel type carrying Ca(2+). Analysis of statistical variations among depolarization-induced capacitance increments indicates that pCPT-cAMP acts downstream of Ca(2+) entry by almost doubling the mean size of unitary exocytic events, most likely as a consequence of an increased granule-to-granule rather than a granule-to-membrane fusion.
Carabelli
V
V
Dipartimento di Neuroscienze, Unità di Ricerca, Instituto Nazionale Fisica della Materia, 10125 Turin, Italy. valentina.carabelli@unito.it
Giancippoli
A
A
Baldelli
P
P
Carbone
E
E
Artalejo
A R
AR
D000222:Adaptation, Physiological / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D020746:Calcium Channels, L-Type; D000242:Cyclic AMP; D002118:Calcium