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

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.

Figure 1

Dose–response relation for iontophoretically injected cAMP vs. characteristics of the I_Na,cAMP response. Test pulse injections (short bars, 5 s, −10 nA) were delivered before and during tonic iontophoretic injections of cAMP and during bath application of 5-HT. The steady state I_Na,cAMP response, the exponential decay rate (k _h) after the 5-s test pulse injections, and degree of occlusion were obtained from these records. Depolarizing voltage steps (arrows, from −50 to +10 mV, 100 ms) were also delivered before and during tonic iontophoretic injections of cAMP to measure amplitudes of the slowly decaying tail currents. The level of inactivation was arbitrarily taken as the decrease of I_Na,cAMP measured 1 s after the voltage step. The tail current amplitude is a function of the summed inactivation of basal and induced I_Na,cAMP (Huang and Gillette, 1991; Sudlow and Gillette, 1995). Leakage current for this soma was −1.5 nA.

Figure 2

Relationships between iontophoretic current and I_Na,cAMP, and among I_Na,cAMP and inactivation, k _h and occlusion for the data of Fig. 1. (A) The dose– response relation for cAMP injection current and steady state I_Na,cAMP response was linear at low injection currents. (B) Inactivation tail current amplitudes were plotted against steady state I_Na,cAMP induced by iontophoretic cAMP injection (▪) or I_5-HT during bath addition of 10 μM 5-HT (⋄). The linear slope of the relation represents the fractional inactivation of I_Na,cAMP by the depolarizing pulse and thus permits calculation of basal I_Na,cAMP in the absence of exogenous cAMP injection (Huang and Gillette, 1991; Sudlow and Gillette, 1995) and during 5-HT stimulation. The I_Na,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 (k _h) were plotted against steady state I_Na,cAMP elicited by tonic iontophoretic injection (▪) or by application of 5-HT (⋄). The linear slope of the relation represents the proportional slowing of the k _h associated with higher levels of I_Na,cAMP and can allow the determination of basal I_Na,cAMP. (D) Occlusion of I_Na,cAMP test pulse responses were measured against steady state I_Na,cAMP induced by tonic cAMP injection (see Fig. 1). Occlusion ratios were obtained from the relation (I − I _o)/I (Huang and Gillette, 1993; Sudlow and Gillette, 1995), where I is the amplitude of I_Na,cAMP elicited by a 5-s pulse of cAMP delivered in the absence of steady state I_Na,cAMP induced by cAMP injection or by 5-HT. I _o is the amplitude of I_Na,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 I_5-HT similar to I_Na,cAMP of equal amplitude evoked by iontophoretic injection of cAMP. The lines are the best fits from least-squares analysis of the data.

Figure 3

Saturability of the iontophoretic current/steady state I_Na,cAMP relation. Voltage clamped somata (n = 4) were injected with iontophoretic currents ranging from −2 to −500 nA (90–180 s) and steady state I_Na,cAMP response characteristics were determined. For this representative cell, steady state I_Na,cAMP increased in a dose- dependent manner up to the −100 nA injections, above which I_Na,cAMP exhibited saturation.

Figure 4

Frequency distribution histogram of whole cell cAMP concentrations in unstimulated (□) and 5-HT-stimulated (▪) somata. Bins were set 0.1-μM wide for the range 0–1 μM, 1-μM for 1–10 μM, and 10 μM for 10–100 μM.

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.

Figure 5

Saturability of I_Na,cAMP by increasing levels of [cAMP]_memb. Measures of I_Na,cAMP, inactivation, and k _h were obtained during tonic injections ranging from −2 to −500 nA. The results were used in Eq. 5 to calculate [cAMP]_memb. The I_Na,cAMP response and the calculated [cAMP]_memb were used in Eq. 7 via a least-squares algorithm to examine the I _max (maximum I_Na,cAMP available) and K _c (the apparent dissociation constant of the channels for cAMP, in micromolar). The filled squares represent the experimental I_Na,cAMP and the calculated [cAMP]_memb. The line represents the least-squares analysis best fit of Eq. 7.

Figure 6

Cells with lower basal AC rates showed greater stimulation by 5-HT than those with higher rates. The histogram shows the relationship of AC activation to basal (unstimulated) rates. Activation ratios for 5-HT-stimulated AC activity were compared with their prestimulation basal AC activity.