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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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Relationships between iontophoretic current and  INa,cAMP, and among INa,cAMP and  inactivation, kh and occlusion for  the data of Fig. 1. (A) The dose– response relation for cAMP injection current and steady state  INa,cAMP response was linear at low  injection currents. (B) Inactivation tail current amplitudes were  plotted against steady state  INa,cAMP induced by iontophoretic  cAMP injection (▪) or I5-HT during bath addition of 10 μM 5-HT  (⋄). The linear slope of the relation represents the fractional inactivation of INa,cAMP by the depolarizing pulse and thus permits  calculation of basal INa,cAMP in the  absence of exogenous cAMP injection (Huang and Gillette, 1991;  Sudlow and Gillette, 1995) and  during 5-HT stimulation. The  INa,cAMP equivalent for the inactivation measured during 5-HT  treatment was 7.2 nA, corresponding to a 26.9 nA iontophoretic injection equivalent. (C) Exponential decay slopes (kh) were plotted  against steady state INa,cAMP elicited by tonic iontophoretic injection (▪) or by application of  5-HT (⋄). The linear slope of  the relation represents the proportional slowing of the kh associated with higher levels of INa,cAMP  and can allow the determination of basal INa,cAMP. (D) Occlusion of INa,cAMP test pulse responses were measured against steady state INa,cAMP  induced by tonic cAMP injection (see Fig. 1). Occlusion ratios were obtained from the relation (I − Io)/I (Huang and Gillette, 1993; Sudlow and Gillette, 1995), where I is the amplitude of INa,cAMP elicited by a 5-s pulse of cAMP delivered in the absence of steady state INa,cAMP induced by cAMP injection or by 5-HT. Io is the amplitude of INa,cAMP elicited by pulse injection of cAMP superimposed during tonic injections of cAMP or bath application of 5-HT. Most somata exhibited occlusion ratios during I5-HT similar to INa,cAMP of equal amplitude evoked  by iontophoretic injection of cAMP. The lines are the best fits from least-squares analysis of the data.
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Figure 2: Relationships between iontophoretic current and INa,cAMP, and among INa,cAMP and inactivation, kh and occlusion for the data of Fig. 1. (A) The dose– response relation for cAMP injection current and steady state INa,cAMP response was linear at low injection currents. (B) Inactivation tail current amplitudes were plotted against steady state INa,cAMP induced by iontophoretic cAMP injection (▪) or I5-HT during bath addition of 10 μM 5-HT (⋄). The linear slope of the relation represents the fractional inactivation of INa,cAMP by the depolarizing pulse and thus permits calculation of basal INa,cAMP in the absence of exogenous cAMP injection (Huang and Gillette, 1991; Sudlow and Gillette, 1995) and during 5-HT stimulation. The INa,cAMP equivalent for the inactivation measured during 5-HT treatment was 7.2 nA, corresponding to a 26.9 nA iontophoretic injection equivalent. (C) Exponential decay slopes (kh) were plotted against steady state INa,cAMP elicited by tonic iontophoretic injection (▪) or by application of 5-HT (⋄). The linear slope of the relation represents the proportional slowing of the kh associated with higher levels of INa,cAMP and can allow the determination of basal INa,cAMP. (D) Occlusion of INa,cAMP test pulse responses were measured against steady state INa,cAMP induced by tonic cAMP injection (see Fig. 1). Occlusion ratios were obtained from the relation (I − Io)/I (Huang and Gillette, 1993; Sudlow and Gillette, 1995), where I is the amplitude of INa,cAMP elicited by a 5-s pulse of cAMP delivered in the absence of steady state INa,cAMP induced by cAMP injection or by 5-HT. Io is the amplitude of INa,cAMP elicited by pulse injection of cAMP superimposed during tonic injections of cAMP or bath application of 5-HT. Most somata exhibited occlusion ratios during I5-HT similar to INa,cAMP of equal amplitude evoked by iontophoretic injection of cAMP. The lines are the best fits from least-squares analysis of the data.

Mentions: We related quantitative cAMP injections to steady state INa,cAMP, its inactivation characteristics, exponential decay rates, and measurements of current saturation. INa,cAMP reaches a steady state condition during tonic iontophoretic cAMP injections (Fig. 1). At nonsaturating levels, the amplitude of elicited INa,cAMP increases linearly with the iontophoretic current (Figs. 1 and 2 A). Although most experiments were performed in the nonsaturating range of the iontophoretic current/INa,cAMP dose–response function, some experiments were designed to specifically examine the saturability of INa,cAMP by iontophoretic current. The INa,cAMP responses did saturate at high current (>−200 nA) iontophoretic injections of cAMP (Fig. 3). These iontophoretic current/ INa,cAMP saturation measures were not typically performed in most experiments, due to adverse long term consequences on the viability of the cell and on diminishing amplitude and lengthening duration of INa,cAMP after prolonged saturating injections 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)

Relationships between iontophoretic current and  INa,cAMP, and among INa,cAMP and  inactivation, kh and occlusion for  the data of Fig. 1. (A) The dose– response relation for cAMP injection current and steady state  INa,cAMP response was linear at low  injection currents. (B) Inactivation tail current amplitudes were  plotted against steady state  INa,cAMP induced by iontophoretic  cAMP injection (▪) or I5-HT during bath addition of 10 μM 5-HT  (⋄). The linear slope of the relation represents the fractional inactivation of INa,cAMP by the depolarizing pulse and thus permits  calculation of basal INa,cAMP in the  absence of exogenous cAMP injection (Huang and Gillette, 1991;  Sudlow and Gillette, 1995) and  during 5-HT stimulation. The  INa,cAMP equivalent for the inactivation measured during 5-HT  treatment was 7.2 nA, corresponding to a 26.9 nA iontophoretic injection equivalent. (C) Exponential decay slopes (kh) were plotted  against steady state INa,cAMP elicited by tonic iontophoretic injection (▪) or by application of  5-HT (⋄). The linear slope of  the relation represents the proportional slowing of the kh associated with higher levels of INa,cAMP  and can allow the determination of basal INa,cAMP. (D) Occlusion of INa,cAMP test pulse responses were measured against steady state INa,cAMP  induced by tonic cAMP injection (see Fig. 1). Occlusion ratios were obtained from the relation (I − Io)/I (Huang and Gillette, 1993; Sudlow and Gillette, 1995), where I is the amplitude of INa,cAMP elicited by a 5-s pulse of cAMP delivered in the absence of steady state INa,cAMP induced by cAMP injection or by 5-HT. Io is the amplitude of INa,cAMP elicited by pulse injection of cAMP superimposed during tonic injections of cAMP or bath application of 5-HT. Most somata exhibited occlusion ratios during I5-HT similar to INa,cAMP of equal amplitude evoked  by iontophoretic injection of cAMP. The lines are the best fits from least-squares analysis of the data.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Relationships between iontophoretic current and INa,cAMP, and among INa,cAMP and inactivation, kh and occlusion for the data of Fig. 1. (A) The dose– response relation for cAMP injection current and steady state INa,cAMP response was linear at low injection currents. (B) Inactivation tail current amplitudes were plotted against steady state INa,cAMP induced by iontophoretic cAMP injection (▪) or I5-HT during bath addition of 10 μM 5-HT (⋄). The linear slope of the relation represents the fractional inactivation of INa,cAMP by the depolarizing pulse and thus permits calculation of basal INa,cAMP in the absence of exogenous cAMP injection (Huang and Gillette, 1991; Sudlow and Gillette, 1995) and during 5-HT stimulation. The INa,cAMP equivalent for the inactivation measured during 5-HT treatment was 7.2 nA, corresponding to a 26.9 nA iontophoretic injection equivalent. (C) Exponential decay slopes (kh) were plotted against steady state INa,cAMP elicited by tonic iontophoretic injection (▪) or by application of 5-HT (⋄). The linear slope of the relation represents the proportional slowing of the kh associated with higher levels of INa,cAMP and can allow the determination of basal INa,cAMP. (D) Occlusion of INa,cAMP test pulse responses were measured against steady state INa,cAMP induced by tonic cAMP injection (see Fig. 1). Occlusion ratios were obtained from the relation (I − Io)/I (Huang and Gillette, 1993; Sudlow and Gillette, 1995), where I is the amplitude of INa,cAMP elicited by a 5-s pulse of cAMP delivered in the absence of steady state INa,cAMP induced by cAMP injection or by 5-HT. Io is the amplitude of INa,cAMP elicited by pulse injection of cAMP superimposed during tonic injections of cAMP or bath application of 5-HT. Most somata exhibited occlusion ratios during I5-HT similar to INa,cAMP of equal amplitude evoked by iontophoretic injection of cAMP. The lines are the best fits from least-squares analysis of the data.
Mentions: We related quantitative cAMP injections to steady state INa,cAMP, its inactivation characteristics, exponential decay rates, and measurements of current saturation. INa,cAMP reaches a steady state condition during tonic iontophoretic cAMP injections (Fig. 1). At nonsaturating levels, the amplitude of elicited INa,cAMP increases linearly with the iontophoretic current (Figs. 1 and 2 A). Although most experiments were performed in the nonsaturating range of the iontophoretic current/INa,cAMP dose–response function, some experiments were designed to specifically examine the saturability of INa,cAMP by iontophoretic current. The INa,cAMP responses did saturate at high current (>−200 nA) iontophoretic injections of cAMP (Fig. 3). These iontophoretic current/ INa,cAMP saturation measures were not typically performed in most experiments, due to adverse long term consequences on the viability of the cell and on diminishing amplitude and lengthening duration of INa,cAMP after prolonged saturating injections 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