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

Cell Calcium. Nov-Dec 2009;46(5-6):303-12. doi: 10.1016/j.ceca.2009.08.007. Epub 2009 Oct 8.

Abstract

Using immortalized hypothalamic GT1-7 neurons, which express the CB1 cannabinoid receptor (CB1R) and three Ca2+ channel types (T, R and L), we found that the CB1R agonist WIN 55,212-2 inhibited the voltage-gated Ca2+ currents by about 35%. The inhibition by WIN 55,212-2 (10 microM) was reversible and prevented by nifedipine (3 microM), suggesting a selective action on L-type Ca2+ channels (LTCCs). WIN 55,212-2 action exhibited all the features of voltage-independent Ca2+ channel modulation: (1) no changes of the activation kinetics, (2) equal depressive action at all potentials and (3) no facilitation following strong prepulses. At variance with WIN 55,212-2, the CB1R inverse agonist AM-251 (10 microM) caused 20% increase of Ca2+ currents. The inhibition of LTCCs by WIN 55,212-2 was prevented by overnight PTX-incubation and by intracellular perfusion with GDP-beta-S. The latter caused also a 20% Ca2+ current up-regulation. WIN 55,212-2 action was also prevented by application of the PKA-blocker H89 or by loading the neurons with 8-CPT-cAMP. Our results suggest that LTCCs in GT1-7 neurons are partially inhibited at rest due to a constitutive CB1R activity removed by AM-251 and GDP-beta-S. Activation of CB1R via PTX-sensitive G proteins and cAMP/PKA pathway selectively depresses LTCCs that critically control the synchronized spontaneous firing and pulsatile release of gonadotropin-releasing hormone in GT1-7 neurons.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoxazines / pharmacology
  • Calcium Channels, L-Type / physiology*
  • Calcium Signaling / physiology*
  • Cell Line
  • Cyclic AMP / analogs & derivatives
  • Cyclic AMP / metabolism*
  • Cyclic AMP / pharmacology
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Down-Regulation
  • Electrophysiology
  • GTP-Binding Proteins / metabolism*
  • Guanosine Diphosphate / analogs & derivatives
  • Guanosine Diphosphate / pharmacology
  • Hypothalamus / metabolism*
  • Isoquinolines / pharmacology
  • Mice
  • Morpholines / pharmacology
  • Naphthalenes / pharmacology
  • Neuroendocrine Cells / metabolism*
  • Piperidines / pharmacology
  • Pyrazoles / pharmacology
  • Receptor, Cannabinoid, CB1 / agonists
  • Receptor, Cannabinoid, CB1 / antagonists & inhibitors
  • Receptor, Cannabinoid, CB1 / metabolism*
  • Signal Transduction / physiology
  • Sulfonamides / pharmacology
  • Thionucleotides / pharmacology

Substances

  • Benzoxazines
  • Calcium Channels, L-Type
  • Isoquinolines
  • Morpholines
  • Naphthalenes
  • Piperidines
  • Pyrazoles
  • Receptor, Cannabinoid, CB1
  • Sulfonamides
  • Thionucleotides
  • Guanosine Diphosphate
  • AM 251
  • 8-((4-chlorophenyl)thio)cyclic-3',5'-AMP
  • (3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone
  • guanosine 5'-O-(2-thiodiphosphate)
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • GTP-Binding Proteins
  • N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide