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Cyclic AMP levels, adenylyl cyclase activity, and their stimulation by serotonin quantified in intact neurons.

Sudlow LC, Gillette R - J. Gen. Physiol. (1997)

Bottom Line: In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity.These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP.Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Integrative Physiology and the Neuroscience Program, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
In molluscan central neurons that express cAMP-gated Na+ current (INa,cAMP), estimates of the cAMP binding affinity of the channels have suggested that effective native intracellular cAMP concentrations should be much higher than characteristic of most cells. Using neurons of the marine opisthobranch snail Pleurobranchaea californica, we applied theory and conventional voltage clamp techniques to use INa,cAMP to report basal levels of endogenous cAMP and adenylyl cyclase, and their stimulation by serotonin. Measurements were calibrated to iontophoretic cAMP injection currents to enable expression of the data in molar terms. In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity. Serotonin stimulation of adenylyl cyclase and [cAMP] was inversely proportional to cells' resting adenylyl cyclase activity. Average cAMP concentration at the membrane rose from 3.6 to 27.6 microM, levels consistent with the expected cAMP dissociation constants of the INa,cAMP channels. These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP. Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

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5-HT dose-dependent increase in AC activities. The  dose–response relationship was compared in a pair of bilaterally homologous G cell neurons from a single animal, identifiable by position and size. 5-HT concentration-dependent increases in AC activation ratios reached plateaus above 10 μM 5-HT.
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Figure 7: 5-HT dose-dependent increase in AC activities. The dose–response relationship was compared in a pair of bilaterally homologous G cell neurons from a single animal, identifiable by position and size. 5-HT concentration-dependent increases in AC activation ratios reached plateaus above 10 μM 5-HT.

Mentions: Radioimmunoassays of basal levels of cAMP in the G lobes yielded an average value of 0.82 ± 0.45 × 10−12 mol·(μg protein)−1 (μM ± SD; n = 2). We used a presumedly maximal stimulatory concentration of 5-HT (100 μM; see Fig. 7) to observe the upper end of cAMP production. Such stimulation elevated cAMP to 9.08 ± 1.95 × 10−12 mol·(μg protein)−1 (n = 5) within a range of 6.38–11.46 × 10−12 mol·(μg protein)−1. The protein concentration per unit volume, measured by Bradford assay, was 8.5 μg·μl−1. With this conversion factor, the electrophysiological measures of whole cell [cAMP] (Table I) could be directly compared with the radioimmunoassays. The radioimmunoassayed levels of unstimulated G lobes were 6.97 ± 3.84 μM and levels during stimulation with 100 μM 5-HT were 77.15 ± 16.86 μM within a range of 54.2–97.38 μM. This compares with the electrophysiological measures of [cAMP] in unstimulated somata of 3.64 ± 8.59 μM (n = 30), within a range of 0.08–28.88 μM, and 10 μM 5-HT-stimulated whole cell [cAMP] of 20.60 ± 23.61 μM, within a range of 1.73–96.90 μM. Results of both biochemical and electrophysiological assays agree that 5-HT stimulates high levels of cAMP.


Cyclic AMP levels, adenylyl cyclase activity, and their stimulation by serotonin quantified in intact neurons.

Sudlow LC, Gillette R - J. Gen. Physiol. (1997)

5-HT dose-dependent increase in AC activities. The  dose–response relationship was compared in a pair of bilaterally homologous G cell neurons from a single animal, identifiable by position and size. 5-HT concentration-dependent increases in AC activation ratios reached plateaus above 10 μM 5-HT.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229365&req=5

Figure 7: 5-HT dose-dependent increase in AC activities. The dose–response relationship was compared in a pair of bilaterally homologous G cell neurons from a single animal, identifiable by position and size. 5-HT concentration-dependent increases in AC activation ratios reached plateaus above 10 μM 5-HT.
Mentions: Radioimmunoassays of basal levels of cAMP in the G lobes yielded an average value of 0.82 ± 0.45 × 10−12 mol·(μg protein)−1 (μM ± SD; n = 2). We used a presumedly maximal stimulatory concentration of 5-HT (100 μM; see Fig. 7) to observe the upper end of cAMP production. Such stimulation elevated cAMP to 9.08 ± 1.95 × 10−12 mol·(μg protein)−1 (n = 5) within a range of 6.38–11.46 × 10−12 mol·(μg protein)−1. The protein concentration per unit volume, measured by Bradford assay, was 8.5 μg·μl−1. With this conversion factor, the electrophysiological measures of whole cell [cAMP] (Table I) could be directly compared with the radioimmunoassays. The radioimmunoassayed levels of unstimulated G lobes were 6.97 ± 3.84 μM and levels during stimulation with 100 μM 5-HT were 77.15 ± 16.86 μM within a range of 54.2–97.38 μM. This compares with the electrophysiological measures of [cAMP] in unstimulated somata of 3.64 ± 8.59 μM (n = 30), within a range of 0.08–28.88 μM, and 10 μM 5-HT-stimulated whole cell [cAMP] of 20.60 ± 23.61 μM, within a range of 1.73–96.90 μM. Results of both biochemical and electrophysiological assays agree that 5-HT stimulates high levels of cAMP.

Bottom Line: In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity.These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP.Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Integrative Physiology and the Neuroscience Program, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
In molluscan central neurons that express cAMP-gated Na+ current (INa,cAMP), estimates of the cAMP binding affinity of the channels have suggested that effective native intracellular cAMP concentrations should be much higher than characteristic of most cells. Using neurons of the marine opisthobranch snail Pleurobranchaea californica, we applied theory and conventional voltage clamp techniques to use INa,cAMP to report basal levels of endogenous cAMP and adenylyl cyclase, and their stimulation by serotonin. Measurements were calibrated to iontophoretic cAMP injection currents to enable expression of the data in molar terms. In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity. Serotonin stimulation of adenylyl cyclase and [cAMP] was inversely proportional to cells' resting adenylyl cyclase activity. Average cAMP concentration at the membrane rose from 3.6 to 27.6 microM, levels consistent with the expected cAMP dissociation constants of the INa,cAMP channels. These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP. Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

Show MeSH
Related in: MedlinePlus