Abstract
The role of cAMP/cAMP-dependent protein kinase (PKA) on the late phase of exocytosis has been studied by amperometry on Ba2+-stimulated single bovine chromaffin cells. Forskolin (FSK) increases the intracellular cAMP levels in a concentration-dependent manner. Forskolin (100 nM) does not increase the number of exocytotic events, although it significantly increases the net granule content of catecholamines (CA), which is accompanied by a slowing of the process of degranulation. These effects are reversible, occur within 15 to 60 s, and are not due to newly synthesized CA. Isoprenaline, pituitary adenylate cyclase-activating polypeptide-38 or dB-cAMP reproduce FSK effects as does cholera toxin. The inhibition of phosphodiesterases with 3-isobutyl-1-methylxanthine mimics and potentiates the effect of FSK and isoprenaline. Rolipram and okadaic acid also produce a drastic increase in net granule content of CA, whereas H-89 attenuates the FSK response. These data indicate that cyclic AMP/PKA might favor the granule aggregation before its fusion with cell membrane and slow the late step of the exocytotic process.
Footnotes
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This work was supported in part by the Spanish Ministerio de Ciencia y Technologı́a, Dirección General de Investigación Cientı́fica y Tecnológica Grant PB97–1483 and Le Fonds Européen de Développement Régional Grant 1FD97–1065-C03–01. It was supported in part by Eli Lilly S. A. (Madrid, Spain), Zeneca Pharmaceuticals plc (Madrid, Spain), and Cepsa (Tenerife, Spain). J.D.M. is recipient of a fellowship from Instituto Tecnológico de Canarias, A.M from Spanish Ministerio de Ciencia y Tecnologı́a, and J.F.G. from Consejerı́a de Educación del Gobierno de Canarias.
- Abbreviations:
- CA
- catecholamines
- FSK
- forskolin
- PKG
- cGMP-dependent protein kinase
- PKC
- Ca2+/phospholipid-dependent protein kinase
- PKA
- cAMP-dependent protein kinase
- C-PTIO
- 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium
- t1/2
- spike width at half height
- Q
- spike net charge
- m
- ascending slope of spike
- tP
- time to reach the spike maximum
- IBMX
- 3-isobutyl-1-methylxanthine
- HPLC
- high pressure liquid chromatography
- VMAT
- vesicular monoamine transporter
- dB-cAMP
- N6,2′-O-dybutyril-3′:5′-cyclic monophosphate
- PACAP
- pituitary adenylate cyclase-activating polypeptide-38
- Received January 16, 2001.
- Accepted May 16, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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