AI Article Synopsis
- The study investigated how cAMP, calcium ions (Ca(2+)), and ATP influence exocytosis and the release of adipokines in white adipocytes, particularly using 3T3-L1 cells.
- Exocytosis occurred without intracellular Ca(2+), provided cAMP was present, but plateaued after about 10 minutes, likely due to vesicle depletion.
- Inclusion of ATP and elevated Ca(2+) significantly enhanced the duration and rate of cAMP-triggered exocytosis, and similar stimulation was observed using the Epac agonist, while inhibiting PKA did not affect the process.
Article Abstract
We examined the effects of cAMP, Ca(2+) and ATP on exocytosis and adipokine release in white adipocytes by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of adipokine secretion. 3T3-L1 adipocyte exocytosis proceeded even in the complete absence of intracellular Ca(2+) ([Ca(2+)]i; buffered with BAPTA) provided cAMP (0.1 mm) was included in the intracellular (pipette-filling) solution. Exocytosis typically plateaued within ∼10 min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3 mm ATP in combination with elevation of [Ca(2+)]i to ≥700 nm augmented the rate of cAMP-evoked exocytosis ∼2-fold and exocytosis proceeded for longer periods (≥20 min) than with cAMP alone. Exocytosis was stimulated to a similar extent upon substitution of cAMP by the Epac (exchange proteins activated by cAMP) agonist 8-Br-2'-O-Me-cAMP (1 mm included in the pipette solution). Inhibition of protein kinase A (PKA) by addition of Rp-cAMPS (0.5 mm) to the cAMP-containing pipette solution was without effect. A combination of the adenylate cyclase activator forskolin (10 μm) and the phosphodiesterase inhibitor IBMX (200 μm; forsk-IBMX) augmented adiponectin secretion measured over 30 min 3-fold and 2-fold in 3T3-L1 and human subcutaneous adipocytes, respectively. This effect was unaltered by pre-loading of cells with the Ca(2+) chelator BAPTA-AM and 2-fold amplified upon inclusion of the Ca(2+) ionophore ionomycin (1 μm) in the extracellular solution. Adiponectin release was also stimulated by the membrane-permeable Epac agonist 8-Br-2'-O-Me-cAMP-AM but unaffected by inclusion of the membrane-permeable PKA inhibitor Rp-8-Br-cAMPS (200 μm). The adipokines leptin, resistin and apelin were present in low amounts in the incubation medium (1-6% of measured adiponectin). Adipsin was secreted in substantial quantities (50% of adiponectin concentration) but release of this adipokine was unaffected by forsk-IBMX. We propose that white adipocyte exocytosis is stimulated by cAMP/Epac-dependent but Ca(2+)- and PKA-independent release of vesicles residing in a readily releasable pool and that the release is augmented by a combination of Ca(2+) and ATP. We further suggest that secreted vesicles chiefly contain adiponectin.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262332 | PMC |
| http://dx.doi.org/10.1113/jphysiol.2014.280388 | DOI Listing |
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