From the Department of Molecular and Integrative Physiology and the Neuroscience Program, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801
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 µM, 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.
Key words: Pleurobranchaea; cyclic nucleotide-gated cation current; protein kinase A; H currentLigand-gated ion channels typically exhibit binding affinities for their ligands within the concentration range
they normally encounter. For the cAMP-gated Na+ currents (INa,cAMP) of molluscan neurons, maximal activation is not achieved at cAMP concentrations below 100 µM (Sudlow et al., 1993). Such high, naturally occurring concentrations of cyclic nucleotides have been inferred from conventional biochemical measurements in other cell types possessing cyclic nucleotide-gated
cation currents: photoreceptor cells of retina and peripheral sensory neurons of olfactory epithelium in vertebrates (Pace et al., 1985
; Stryer, 1986
; Breer et al.,
1990
; Koutalos et al., 1995
). These concentrations are
many times in excess of those causing maximal activation of cAMP-dependent protein kinase (Beavo et al.,
1974
). In molluscan neurons, cAMP mediates both short-
and long-term stimulation of excitability in both sensory neurons (Siegelbaum et al., 1982
; Ocorr et al.,
1985
; Walsh and Byrne, 1989
; Goldsmith and Abrams,
1991
; Byrne et al., 1993
) and neurons of motor networks (Gillette et al., 1982
; Gillette and Gillette, 1983
;
Connor and Hockberger, 1984
; Gillette and Green, 1987
;
Kirk et al., 1988
; Kehoe, 1990
; Huang and Gillette, 1991
;
Funte and Haydon, 1993
; Price and Goldberg, 1993
;
Sudlow and Gillette, 1995
); thus, it is of appreciable interest to know the levels of cAMP prevailing in both
resting and stimulated central neurons, and the levels of synthetic activity that maintain them.
Using INa,cAMP as a reporter of intracellular cAMP concentrations, we measured cAMP concentrations and adenylyl cyclase (AC)1 activities in neurons in the locomotor network of the predatory gastropod Pleurobranchaea californica, where we compared the values for resting state with those obtained where AC was stimulated by the neuromodulator serotonin, 5-hydroxytryptamine (5-HT). Our results indicate that stimulated levels of cAMP are indeed in the range of tens of micromolar, and that these are achieved through correspondingly high levels of AC stimulation.
Electrophysiology
Neurons of the "G" group of the pedal ganglia of the sea slug P. californica were used in all experiments. These cells are putative
locomotor neurons in which INa,cAMP is stimulated by 5-HT
through activation of AC (Sudlow and Gillette, 1995). Specimens (80-500 g) of Pleurobranchaea were obtained from Sea Life Supply (Sand City, CA) and Pacific BioMarine (Venice, CA) and maintained in 14°C artificial sea water. Animals were anesthetized by
chilling, the pedal ganglia were removed, and axotomized somata (110-240 µm diameter) were isolated and prepared for
voltage clamping as previously described (Sudlow and Gillette,
1995
). Artificial Pleurobranchaea saline consisting of (mM) 420 NaCl, 10 KCl, 25 MgCl2, 25 MgSO4, 10 CaCl2, 10 MOPS buffered
with NaOH, final pH 7.5, 14°C was constantly perfused over the
cells. Cells were clamped via two-electrode voltage clamp amplifier with total cellular current reported by virtual ground amplifier. The voltage-sensing electrode was filled with 3 M KCl. A double-barreled electrode was used for current passing and cAMP
iontophoresis. The current passing barrel was filled with 3 M KCl
and the second barrel was filled with 200 mM cAMP (adenosine
3
:5
cyclic monophosphate free acid; Sigma Chemical Co., St.
Louis, MO), 200 mM KOH, and 20 mM Tris, pH 7.4. Resistances
of both double-barreled and single pipettes were lowered by
breaking back the tips against a clean capillary tube. Typical resistances for voltage and current electrodes were 4-12 and 35-80 M
, respectively, for the cAMP iontophoresis barrel. The double-barreled electrode was positioned at the center of the soma with
a calibrated micromanipulator, as estimated from soma diameter
measured with a calibrated eyepiece micrometer. All current recordings were done at a holding potential of
50 mV. Iontophoretic injections of cAMP were made with a programmable
constant current source (260; WPI, New Haven, CT). Variation of
iontophoretic currents over multiple injections at the same programed delivery level was negligible.
Quantification of Iontophoretic Ejections of cAMP
A transport number for cAMP iontophoresis was measured by radioimmunoassay. cAMP was iontophoresed from double-barreled
electrodes (current barrel resistance 2-8 M) into the mouth of a
collecting siphon tube (PE 10 polyethylene tubing) flowing at 20 µl·min
1. Samples were collected in polypropylene microcentrifuge tubes and frozen for subsequent analysis. Saline in the reservoir from which the siphon collected was designed to mimic cytoplasmic cation composition of molluscan neurons, and contained
(mM) 400 KCl, 50 NaCl, and 1 Tris, pH 7.5. Frozen samples were
dried in a vacuum centrifuge (Speed-Vac; Savant Instruments, Inc.,
Farmingdale, NY) and radioimmunoassayed as below. cAMP delivery from three electrodes resulting from
20 and
100 nA, 50-s
iontophoretic currents yielded a linear relation extrapolating to
zero; an average transport number of 0.11 (0.01 SEM) was calculated from the
100 nA samples (5.79 pmol cAMP; 0.61 SEM).
Tissue Measures of cAMP
The G lobes of pedal ganglia were excised with small scissors as
near-spherical clumps of cell bodies whose volumes were estimated from the approximate diameters measured by a calibrated
ocular micrometer. The isolated G lobes were allowed to recover
for 1 h at 14°C, and then perfused either with normal saline or saline containing 100 µM 5-HT (serotonin creatinine sulfate complex; Sigma Chemical Co.) for 5 min, after which a cold (20°C)
antifreeze solution of 1:1 (vol/vol) propylene glycol:2 M NaCl
was flushed into the dish (Greenberg et al., 1987
). The frozen tissue was placed in a
20°C freezer for 1-2 h, and then homogenized in a glass microhomogenizer (Radnoti Glass Technology,
Inc., Monrovia, CA) in 50 µl ice-cold 65% ethanol with 10 turns
of the pestle. The homogenate was transferred to a clean microcentrifuge tube and the homogenizer was washed twice with 50-µl rinses of ice-cold 65% ethanol into the microcentrifuge tube.
Homogenate and rinses were mixed and immediately aliquotted
for both cAMP and protein assays. Aliquots were frozen at
20°C
and dried on a vacuum centrifuge. Freeze-dried aliquots were
stored at
20°C until assayed. Protein concentrations were determined via the Bradford method (Bradford, 1976
).
cAMP Radioimmunoassay
cAMP was measured by scintillation proximity assay (SPA), using
a modified RIA protocol (Amersham Corp., Arlington Heights, IL). The SPA measures cAMP via competition between cAMP
and adenosine 3:5
-cyclic phosphoric acid 2
-O-succinyl-3-[125I]iodotyrosine methyl ester for an antibody/scintillant microbead system. The dried samples were dissolved in 0.5 ml of 0.05 M acetate buffer immediately before assay. Samples were processed under the nonacetylation protocols for the kit. The range of sensitivity for the nonacetylation protocols of the kit was 0.2-12.8 pmol/ assay tube. Aliquots (100 µl) of the samples were transferred to
polypropylene scintillation vials (Sarstedt, Inc., Newton, NC). SPA
analysis of the samples was performed as per package instructions
and assay vials were counted on a
-scintillation counter. Data
were adjusted for percent recovery, typically 90%.
5-HT Delivery
5-HT (serotonin creatinine sulfate complex; Sigma Chemical Co.) was dissolved in Pleurobranchaea saline and pH was adjusted to 7.5 with NaOH. A gravity-fed perfusion pipette was placed upstream of the voltage-clamped somata.
Analysis of INa,cAMP Dynamics
Each soma was internally calibrated for its INa,cAMP responses to
iontophoretically injected cAMP. In addition to the INa,cAMP amplitudes, two additional sets of measures were made during
steady state injections of cAMP. First, for the pedal ganglia cells, depolarizing voltage command steps to inactivate INa,cAMP were delivered from 50 (holding) to +10 mV for 100 ms. The degree of inactivation was measured as the tail current (Itail) representing the net decrease in INa,cAMP amplitude measured 1 s after
the voltage step delivered during steady state cAMP injections or 5-HT application (Gillette and Green, 1987
; Huang and Gillette, 1993
; Sudlow and Gillette, 1995
). The second measure examined the exponential decay rate of INa,cAMP after 5-s pulsed iontophoretic injections of cAMP superimposed on the steady state injections. The data obtained during the decay were digitized and
were fit by a least-squares algorithm to a time-dependent exponential decay function (Y = X exp (
t · kh)) to obtain the exponential
term kh where kh is in the units of 1/s and t is time in s. The decay rate decreased as steady state iontophoretic current delivery increased. The basal levels of INa,cAMP could be calculated from either
the Itail or kh values obtained during the absence of exogenous cAMP injections using the measured slope of the inactivation/ INa,cAMP relation, or from the slope of the kh/INa,cAMP relation.
Exponential decay slopes (kh) of INa,cAMP after 5-s pulsed injections of cAMP are directly proportional to the rate of change in
[cAMP]. The decrease in [cAMP] is directly attributable to degradation by phosphodiesterase (PDE) (Huang and Gillette,
1991). While some cellular binding of cAMP may occur, buffering effects are not evident and are presumed negligible. The
dose-response relationship between kh and steady state INa,cAMP
responses was used to calculate a kh for an INa,cAMP response to injected cAMP of equal amplitude to the 5-HT-induced INa,cAMP
(I5-HT). This equivalent kh and the iontophoretic current equivalent for I5-HT were then used in Eq. 5 to calculate a cAMP concentration at the submembrane space ([cAMP]memb). The kh and
[cAMP]memb obtained during 5-HT stimulation were used in Eq. 6 to calculate AC activity.
Distribution of cAMP Concentrations and AC Activity
Iontophoretic equivalents for basal and 5-HT-stimulated INa,cAMP
responses were calibrated on a molar basis by Eq. 1, which describes the rate of release of cAMP from an iontophoretic pipette tip (Purves, 1981):
![]() |
(1) |
where qi is the iontophoretic molar flux (mol · s1), I is the iontophoretic current, n is the transport number for cAMP empirically derived for these experimental conditions, z is the valence, and F
is the Faraday constant. The total amount of cAMP delivered during a steady state iontophoretic injection is equal to qi·t, where t is
time in s.
Two additional processes are involved in the movement of
cAMP out of the iontophoretic pipette: diffusional leak and bulk flow, which can both be significantly affected by the diameter of
the pipette tip. Passive diffusion can be described by the relation
(Purves, 1981):
![]() |
(2) |
where qd is the diffusional leak (mol·s1), D is the apparent diffusion coefficient (3.3 ·10
6 cm2 · s
1) for cAMP in Pleurobranchaea
neurons (Huang and Gillette, 1991
), Cpip is the concentration of
cAMP in the pipette (0.2 M), and
is the included angle in radians
of the pipette tip from the center axis of the pipette; typically for
our pipettes the angle was 1.5°. The term rpip is the inside radius
(cm) of the pipette. We assumed that breakage of the double-barreled electrodes broke both barrels equally. Effective tip diameters
were calculated from resistances for the current passing barrels of
the double-barreled electrodes using the relation (Purves, 1981
):
![]() |
(3) |
where describes the conductivity of the 3 M KCl electrolyte of
the voltage clamp current barrel, nominally 0.26 S·cm
1 (Purves,
1981
); and Rue is the pipette resistance in ohms. Using this relationship, the range of the resistances of the current passing electrodes corresponded to tip diameters of 0.08-0.23 µm.
Bulk flow arises from hydrostatic pressure effects imposed at
the pipette tip by gravity by the relation (Purves, 1981):
![]() |
(4) |
where qh is the hydrostatic bulk flow (mol·s1), p is the viscosity
(1,000 kg·m
3), g is gravitational attraction (9.8 m·s
2), h is the
height of cAMP solution in the iontophoretic barrel (m), and
is
the viscosity of the cAMP solution (0.001 Pa·s). The terms
, Cpip,
and rpip have the same meanings as above. Diffusional leak and
hydrostatic bulk flow of cAMP from the cAMP iontophoresis barrels were calculated to have contributed to <3 fmol·s
1 cAMP under these conditions given the pipette diameters involved (Krnjevi
et al., 1963
; Purves, 1981
). In radioimmunoassays of sample
aliquots taken after 300 s of diffusion with low resistance iontophoretic electrodes, cAMP levels did not register above the kit's
detection limits (0.2 pmol/aliquot).
The steady state distribution of a solute (such as cAMP) released from a point source (the injection electrode) in a finite spherical space with an impermeable boundary (the axotomized
neuron soma), in which a first-order decay occurs, is described by
Eq. 5 (Purves, 1976, 1977
):
![]() |
(5) |
where C(r) is cAMP concentration at radius r, qi is the iontophoretic injection flux described by Eq. 1, rn is the radius of the
cell membrane, kh is the relaxation rate constant for INa,cAMP elicited by I in Eq. 1, and D has the same value as above. We assumed
the simplest cases where PDE activity is distributed homogeneously in the cytoplasm and there are no significant internal diffusion barriers (see Huang and Gillette, 1991). The exponentially decreasing gradient from pipette tip to cell membrane described by Eq. 5 is largely influenced by the diameter of the
neuron and PDE, which is reflected in kh. Eq. 5 was simplified
from its original form since the radius of the release point has no
significant impact on the computations as long as the inside diameter of the pipette is <4 µm. In practice, our pipette tip diameters were <1 µm.
5-HT Depolarizes Neurons via INa,cAMP
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.
INa,cAMP in pedal neurons is inactivated by Ca2+ influx
caused by depolarizing voltage steps (Fig. 1); after such
a step, inactivation is manifested as a slowly decaying
tail current (Itail; Gillette and Green, 1987; Huang and
Gillette, 1993
). When brief depolarizing voltage steps
are delivered to otherwise unstimulated cells, small tail
currents are observed resulting from inactivation of
basal INa,cAMP. The Itail amplitude increases linearly with
the inward current induced during either tonic iontophoretic injections of cAMP or bath application of
5-HT, indicating the origin in INa,cAMP (Figs. 1 and 2 B).
Exponential decay slopes (kh) of INa,cAMP after 5-s
pulse injections of cAMP were also dependent on the
steady state level of INa,cAMP in the cell (Fig. 1), with kh
decreasing with increasing injections of cAMP (Fig. 2
C). This relationship between kh and steady state INa,cAMP
was linear when the iontophoretic injections were below saturation levels for INa,cAMP. The relaxation rate kh
is highly sensitive to PDE inhibitors (Aldenhoff et al.,
1983; Connor and Hockberger, 1984
; Huang and Gillette,
1991
) and is a quantitative index of PDE-mediated degradation of cAMP (Huang and Gillette, 1991
). PDE inhibitors, such as 3-isobutylmethylxanthine, cause dose-
dependent decreases in the decay rate kh and increases in the cAMP levels in the soma without change in adenylyl cyclase activities (Sudlow and Gillette, manuscript
in preparation).
The steady inward current stimulated by 5-HT (I5-HT)
occludes the INa,cAMP response to iontophoretic pulsed
injections of cAMP (Figs. 1 and 2 D). I5-HT in the pedal
neurons is largely composed of INa,cAMP, whose levels
can be calculated from inactivation (Sudlow and Gillette,
1995) or measures of kh saturation (see further). The
degree of occlusion was also indicative of the degree of
saturation of INa,cAMP by iontophoretic current. As the
iontophoretically elicited INa,cAMP approached saturation, the degree of occlusion also increased.
INa,cAMP Responses Report on AC Stimulation and [cAMP]
Since INa,cAMP is a direct function of the cAMP concentration, the amplitude of the current is the balanced result of the activities of the synthetic and degradative enzymes, AC and PDE, respectively (Huang and Gillette,
1991; also see Hodgkin and Nunn, 1988
). Knowledge
of the cAMP concentration and of the activity of at least
one of the enzymes would allow the calculation of the
remaining enzyme's activity at steady state. However, in
the living cell, unlike the biochemist's reaction vessel,
such a calculation must take into account the compartmentalization and geometry of the cell and the kinetics
of cAMP diffusion and degradation. In the neuron, AC is
bound at the plasma membrane, while PDE is distributed in the cytoplasm in both soluble and membrane-bound
fractions (Fell, 1980
; Shakur et al., 1993
; Bolger, 1994
).
Thus, we took into account the existence of a gradient of
cAMP concentrations. During neurotransmitter stimulation of the soma, this cAMP concentration gradient is
highest next to the membrane and falls exponentially with distance towards the center of the cell (Fell, 1980
;
Bacskai et al., 1993
; Shakur et al., 1993
; Bolger, 1994
).
AC activity was calculated from the INa,cAMP records
obtained before and during bath applications of 5-HT.
The responses of the cell were calibrated using three
relationships based upon direct experimental measures:
(a) the dose-response relation of tonic cAMP iontophoretic current to the induced steady state INa,cAMP (see Fig. 2 A); (b) the dose-response relation of steady
state INa,cAMP current amplitudes to depolarization-
induced inactivation (see Fig. 2 B), which allowed estimation of INa,cAMP in the absence of cAMP injection; and (c)
the dose-response relation of the steady state INa,cAMP to kh
(see Fig. 2 C) obtained during steady state INa,cAMP, which
also allowed for the estimation of basal INa,cAMP (Huang and
Gillette, 1991). Resting and 5-HT-stimulated levels of
INa,cAMP were calculated using the resting and 5-HT-stimulated inactivation measures and the linear regression coefficients of the inactivation/steady state INa,cAMP
relation (see Fig. 2 B). The kh values associated with the
steady state INa,cAMP equivalents for resting and 5-HT-stimulated states were determined from the INa,cAMP
equivalents and the linear regression coefficients of the
kh/steady state INa,cAMP dose-response relation (see Fig. 2
C). The steady state INa,cAMP equivalents were translated
into iontophoretic cAMP injection equivalents using
the linear regression coefficients of the dose-response
relation of tonic injection current to induced INa,cAMP
(see Fig. 2 A). These iontophoretic equivalents, along
with their equivalent kh values, were adjusted for diffusion and hydrolysis by Eq. 5 to estimate [cAMP]memb. Last, AC activities were calculated from the [cAMP]memb
and the kh during 5-HT-stimulated state using Eq. 6. We
derived Eq. 6 (appendix) to explicitly describe the active synthesis, degradation, and inward diffusion of an
enzymatic product from the boundary of an impermeable sphere:
![]() |
(6) |
where C(r) is [cAMP] at radius r, taken here as the compartment 100 nm subjacent to the cell membrane. QAC
is the flux of the membrane-bound AC expressed in
terms of mol·s1. The variables r, rn, kh, and D have the
same meanings as in Eq. 5. The equation was derived
with the assumption that PDE is homogeneously distributed in the soma. After calculating [cAMP]memb with Eq. 5, Eq. 6 is solved for QAC. With the soma diameter, AC activity is normalized to the surface area of the
soma (mol·s
1·cm
2) to allow ready comparison of different cells.
The results of measurements on 30 neurons are summarized in Table I. In unstimulated cells, the distribution of basal [cAMP]memb was markedly skewed to lower
values with a few rare higher values (Fig. 4); AC activities were distributed similarly. Stimulation of AC by
5-HT shifted the distribution of responses towards
markedly higher values (Fig. 4). The non-Gaussian distributions of [cAMP]memb and AC activities contributed
to wide variation among the calculated means and standard deviations. When cAMP concentrations and AC activities in bilaterally homologous neurons from the
pedal ganglia of the same animal were examined (see
Table I, cells A and B, E and F, and K and L), the levels
were very comparable. However, when cAMP concentrations and AC activities in different, nonhomologous neurons of the same animal were compared (see Table I,
cells F vs. G, I vs. J, and P vs. Q), levels varied substantially.
Thus, variability of the cAMP and AC measures is likely
to arise from phenotypic variation in the cell population
under study. 5-HT stimulation augmented [cAMP]memb
from 1.16- to 82.60-fold, averaging 23.35- ± 21.96-fold
(µM ± SD), and AC from 1.17- to 89.68-fold, with an
average stimulation of 18.56- ± 18.79-fold. The average resting [cAMP]memb was 3.64 ± 8.59 (µM ± SD). Adjusting for the PDE-induced gradient in cell bodies
yields an integrated average whole-cell concentration
of 2.54 ± 2.61 µM. 5-HT stimulation raised [cAMP]memb
to 27.68 ± 30.12 µM and whole-cell integrated [cAMP] to 20.60 ± 23.61 µM. Similarly, resting AC activities averaged 2.44 ± 5.26 × 1012 mol·s
1·cm
2 and were elevated by 5-HT stimulation to 15.96 ± 16.21 × 10
12
mol·s
1·cm
2. When these AC activities are expressed
in terms of cell volume, as µM·s
1 (micromol·liter
1·s
1),
basal AC was 0.83 ± 1.64 µM·s
1 and stimulated AC rates
(basal + 5-HT stimulated) were 5.55 ± 6.92 µM·s
1.
Table I. Basal and 5-HT-stimulated [cAMP] and AC Activities in Single Cells |
Tissue Measures of [cAMP]
Radioimmunoassays of basal levels of cAMP in the G
lobes yielded an average value of 0.82 ± 0.45 × 1012
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.
INa,cAMP Binding Affinity
We next examined the apparent binding affinity of
cAMP in activating INa,cAMP. The apparent binding affinity for cAMP is an inverse function of intracellular
[Ca2+] in the G cells (Huang and Gillette, 1993), and is
a minimum estimate of actual binding affinity. The
INa,cAMP elicited for each tonic iontophoretic injection
and the [cAMP]memb determined for that injection were
fit by least-squares analysis to the modified Hill equation (Huang and Gillette, 1991
):
![]() |
(7) |
where Imax is the maximum available INa,cAMP and Kc is the
apparent cAMP dissociation constant for the INa,cAMP
channels. The average Imax was 20.92 ± 16.53 nA (n = 17) within a range of 6.84 -90.62 nA. The average Kc for
the INa,cAMP current was 18.05 ± 8.95 µM (n = 17) within
a range of 4.32-34.05 µM. For experiments designed to
examine the interaction between 5-HT and INa,cAMP, the
calculated [cAMP]memb and nonsaturating INa,cAMP levels
were fit by a least-squares algorithm of Eq. 7. Additional experiments were designed to specifically examine the
entire dose-response relation between iontophoretic
current and INa,cAMP (see Fig. 3). The relation, presented
in terms of the [cAMP]memb/INa,cAMP response, was fitted
well by Eq. 7 (Fig. 5). The Imax and Kc for the data in Fig.
3, calculated as per above, were 16.86 nA and 22.12 µM,
respectively. These values are consistent with cAMP dissociation constants for INa,cAMP channels thought to be at least several tens of micromolar or greater (Aldenhoff et
al., 1983; Green and Gillette, 1983
; Connor and Hockberger, 1984
; Sudlow et al., 1993
).
The net increase in AC activation stimulated by 5-HT
was inversely proportional to resting AC rates (Fig. 6).
In general, cells that exhibited comparatively high basal
AC rates (see Table I) showed appreciably less stimulation by 5-HT than cells with low basal activity. Collectively, these data indicate that cyclase activity varies in
its basal levels of activation among different cells and
preparations, perhaps due to effects of dissection, endogenous neuromodulators, or other unknown factors.
5-HT elicited similar concentration-dependent effects in pairs of bilaterally homologous neurons (Fig. 7). Measured values of AC activation with increasing 5-HT concentrations for bilateral homologs (Table I, cells K and L) were similar, reaching a plateau phase above 10 µM 5-HT (Fig. 7).
The present findings document high average resting
levels of submembrane cAMP in unstimulated neurons,
in excess of 3.64 µM, within a wide range of 0.13-44
µM. The whole cell levels of cAMP were 2.54 µM within
a range of 0.07-50 µM, which corresponded well with
the radioimmunoassay measures of cAMP in Pleurobranchaea G lobes homogenates of 6.97 µM. These values
are also similar to the results of radioimmunoassays of
homogenates of large peripheral effector neurons of
Aplysia, neurons similar in function to those studied
here, that yielded resting levels of cAMP of 1-6 µM
(Hockberger and Yamane, 1987). These values in Pleurobranchaea, even at the lower limit, are considerably
higher than the resting levels of <50 nM in the somata
of cultured Aplysia sensory neurons, as estimated from
cAMP binding-induced fluorescence changes in fluorophore-labeled subunits of PKA (Bacskai et al., 1993
).
This is unlikely to result from a species difference between Aplysia and Pleurobranchaea, since our unpublished observations on identified effector neurons of
Aplysia (R2 and L10 of the abdominal ganglion) exhibiting INa,cAMP show that they require a similar range of
iontophoretic current to activate the current. The differences in values for [cAMP] between sensory and effector neurons may well be adaptively related to the expression of INa,cAMP in the effector neurons. INa,cAMP is
probably not significant in the sensory neurons; they
are well studied and a prominent INa,cAMP would doubtless have been reported previously. Thus, these results
suggest that cAMP levels tend to be higher in neurons
that express INa,cAMP than in those that lack it. This interpretation is consistent with observations on vertebrate retina and olfactory tissues, where cyclic nucleotide levels in cells expressing similar cGMP- or cAMP-gated cation currents attain similarly high values.
In 5-HT-stimulated somata the average whole cell
cAMP levels rose to nearly 21 µM. Because of the concentration gradient for cAMP caused by asymmetric
distribution of AC and PDE, the actual average submembrane values exceeded 27 µM and in several cases approached 100 µM (Table I). Observations on single
INa,cAMP channels in excised patches showed that the
probable cAMP dissociation constant was probably in
excess of some tens of micromolar (Sudlow et al.,
1993), a conclusion in accord with observations on the macro-current (Aldenhoff et al., 1983
; Green and
Gillette, 1983
; Connor and Hockberger, 1984
; Sudlow
et al., 1993
). In the present measures, we found that
the average dissociation constant for cAMP binding in
activation of INa,cAMP was about 18 µM, within a range of
4.32-34.05 µM. The wide variation in the apparent
binding affinity for cAMP is likely to arise in large part
from the fact that it is a function of intracellular [Ca2+]
in the G cells, which acts like a competitive inhibitor of
cAMP binding to the channel (Huang and Gillette,
1993
); other modes of channel regulation, such as
phosphorylation (see Wilson and Kaczmarek, 1993
),
are also possible. The present observations support the
expectation for the relatively low binding affinity for
cAMP in activation of the macro-INa,cAMP in the intact
neuron. They also show that the high endogenous levels of cAMP needed to activate the current are actually
attained during stimulation with the natural neuromodulator.
The system of equations presented in this study incorporates an assumption of homogeneous distribution of PDE in the soma. This assumption stems from
observations of Huang and Gillette (1991), who varied
the depth of the iontophoretic pipette and examined
the exponential decay rates of INa,cAMP after pulse injections of cAMP; they found no variation of kh with depth.
If PDE were significantly excluded from the nuclear
core of the soma, and PDE were concentrated in the
rind of the cytoplasm much like the distribution of regulatory subunit of PKA observed by Bacskai et al.
(1993)
, then the exponential decay after a pulse injection of cAMP should have slowed as the pipette penetrated further to the center of the cell, where the PDE-free zone would have acted as a cAMP reservoir. Instead, the modeling of the time course of the response
of INa,cAMP to a pulse injection was best fit by a homogenous distribution of PDE, where depth and PDE activity accounted for the response's characteristics and time
course (Huang and Gillette, 1991
). We are not aware of
direct immunocytochemical documentation for the distribution of molluscan PDE in the soma. If in the future it should be found that Pleurobranchaea G neurons have substantial regions of the soma devoid of PDE, the
results presented in this report will be underestimations
of the cAMP levels of the cells in Table I. Conversely, a
second nonhomogenous distribution of PDE could involve a thin region of high PDE activity subjacent to the
neurolemma. Careful examination of the performance of the equations indicated that as long as the region of
high PDE activity (i.e., 2·kh) involved <14% of the total
volume of the cell, the consequences to the cAMP calculations amounted to <1% variations in the results. The
equations function not only due to the effects of kh and
diffusion, but through the distance that the cAMP has to
diffuse. If the region of high PDE activity is only a few microns thick near the neurolemma, the consequences to
the computations are negligible.
The [cAMP] and AC activities presented in Table I exhibit fairly large variation reflected in the means and standard deviations calculated for the sample. Some of the variability during resting measures can be accounted for by the extremely high levels of cAMP and AC in the two cells T and V, which appeared to be maximally activated in the basal state. The high levels associated with those cells are indicative of significant AC activation at the time of the experimental measures. During 5-HT stimulation, the measures associated with cells R and DD contributed to the variance of cAMP and AC measures. The levels of cAMP associated with cells R and DD, while high, are similar to the radioimmunoassay measures of cAMP obtained during 5-HT treatment, and thus appear to be within physiological range. It is highly unlikely that errors in the measures of kh would account for the large sums of squares associated with the measures in Table I. We examined the performance of the equations and found that for a given model cell, iontophoretic current, and kh, kh would have to be underestimated by >60% to reach cAMP values in excess of the mean [cAMP]memb, 27.6 µM. Typically, <2% variability was observed in repeated exponential slope measurements of kh of the same INa,cAMP response to cAMP pulse injection. The production of cAMP during 5-HT treatment falls into two major categories, cells that respond with cAMP levels between 2.97 and 13.91 µM with a mean and standard deviation of 7.63 ± 3.82 µM (n = 18) and those cells that respond with cAMP production between 31.46 and 119.35 µM with a mean and standard deviation of 57.97 ± 26.92 µM (n = 12) (P < 0.0001, Mann-Whitney U test). It appears that different Pleurobranchaea G neurons vary greatly in cAMP production in response to 5-HT and it is this phenotypic variation that accounts for the variability of the individual cell measures. This was borne out by the observations on nonhomologous neurons from pedal ganglia of the same animal (Table I: cells F vs. G, I vs. J, and P vs. Q).
The radioimmunoassay measures of cAMP of whole
Pleurobranchaea G lobe homogenates were higher than
most of the cellular measures of Table I. These homogenates involved not only somata, but neuropil as well.
cAMP levels in dendrites have been shown to be significantly higher than that observed in the somata in Aplysia sensory neurons (Bacskai et al., 1993). The higher
levels of cAMP measured in the RIA as compared with
the cellular measures may reflect, in part, the contributions of a higher neuropilar stimulation of cAMP production by 5-HT. Additionally, the neurons of the G
lobes of the animals used in the RIA as a group may
have been prone to produce cAMP at higher levels, like
that observed in Table I, cells R, Y, and DD.
The average 18-fold stimulation of AC activity, and
the increases shown by some neurons up to nearly 90-fold, are appreciably higher than values reported for in
vitro measures of molluscan nervous tissue, which may
vary from 0.75- to 7-fold in homogenates of Aplysia sensory neurons (Yovell et al., 1987; Eliot et al., 1989
;
Abrams et al., 1991
; Goldsmith and Abrams, 1991
; Yovell and Abrams, 1992
). These differences could result
from variation in AC activity among cells on the basis of
whether or not they express INa,cAMP. Alternatively, or in
addition, the considerable differences between in vitro
and in vivo stimulation may well arise from the disruption of the intracellular environment inherent to in
vitro assays. In particular, homogenization may greatly reduce the capacity for AC activity by dilution of the G
protein cofactors and disruption of the tubulin that interacts with the G proteins. This interpretation is suggested from observations on adrenergic stimulation of
AC in an assay system where cells are detergent permeabilized, but remain otherwise intact; AC stimulation in
these cases can exceed 15-fold (Rasenick et al., 1993
;
Popova et al., 1994
).
Nucleotide cyclase activity in vertebrate photoreceptors, which use cyclic nucleotide-gated currents in sensory transduction, appears to be regulated 5-40-fold between light- and dark-adapted conditions, similar to the
range of stimulation of AC observed here (Pace et al.,
1985; Koutalos et al., 1995
). Use of the protein per unit
volume conversion factor allows comparison of the activities of AC in Pleurobranchaea and cyclase activity from vertebrate olfactory receptors and photoreceptors. Unstimulated levels of Pleurobranchaea AC were 5.86 ± 2.12 × 10
9
mol·min
1·(mg protein)
1. 5-HT-stimulated levels of AC
were 39.18 ± 7.62 × 10
9 mol·min
1·(mg protein)
1.
These ranges of cellular cAMP concentrations and cyclase activity are similar to vertebrate tissues that also express cyclic nucleotide-gated currents (Pace et al., 1985
;
Stryer, 1986
; Breer et al., 1990
; Koutalos et al., 1995
).
Cyclic nucleotide-gated currents have been identified in a wide variety of tissues including vertebrate
photoreceptors, vertebrate and invertebrate olfactory
receptors, cochlear hair cells, cardiac myocytes, sperm
cells, and cultured hippocampal neurons (Haynes and
Yau, 1985; Fesenko et al., 1985
; Nakamura and Gold,
1987
; Firestein et al., 1991
; Zufall et al., 1991
; Kolesnikov et al., 1991
; DiFrancesco and Tortora, 1991
; Hatt
and Ache, 1994
; Weyand et al., 1994
; Leinders-Zufall et
al., 1995
). Many other large, identified neurons of gastropod central nervous systems also express INa,cAMP
(Liberman et al., 1975
; Green and Gillette, 1983
; Aldenhoff et al., 1983
; Kononenko et al., 1983
; Connor
and Hockberger, 1984
, 1985
; Gillette and Green, 1987
;
McCrohan and Gillette, 1988
; Kehoe, 1990
; Huang and
Gillette, 1991
, 1993
; Price and Goldberg, 1993
; Sudlow
et al., 1993
). For other cell types where cyclic nucleotide-gated ion current is prominent, the analytical approach introduced here with suitable modifications
may complement conventional measures of cyclic nucleotides, particularly for the case of single cells that
are too small to assay by presently available biochemical techniques. The general method may be adaptable by
the use of caged cyclic nucleotides and several other
possible indices of baseline current. Introduction of cyclic nucleotide-gated channels either genetically or directly to act as reporter ion channels may be practical
in many types of cells. Aside from studies of neuromodulator-neurohormone actions as performed here, these
methods are applicable to inquiries of receptor-G protein-AC coupling, interactions of other intracellular
messenger pathways with cAMP metabolism, and the
time course of cAMP fluctuations within which cAMP-dependent phosphorylation might be influenced.
Address correspondence to Dr. L.C. Sudlow, Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. Fax: 217-333-1133; E-mail rhanor{at}uiuc.edu. Address reprint requests to Dr. R. Gillette, Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801.
Received for publication 24 March 1997 and accepted in revised form 25 June 1997.
1 Abbreviations used in this paper: 5-HT, serotonin, 5-hydroxytryptamine; AC, adenylyl cyclase; PDE, phosphodiesterase.The derivation of the differential equation follows from
that established for solutions in three dimensional electrotonus theory (Purves, 1976, 1977
). The following
normalized variables are used: B = rn(kh/D)0.5, R = r(kh/D)0.5, where r, rn, D, and kh have the same meanings as in the text.
The general form of the differential equation describing movement of potential in a three-dimensional
syncytium (Purves, 1976, 1977
) as recast in terms of
movement of ions. Since at steady state dC/dt = 0, the
differential equation simplifies to:
![]() |
(A1) |
The general solution to Eq. A1 is:
![]() |
(A2) |
The following boundary conditions apply to Eq. A2:
(A3)the initial conditions are: 0 R
B
(A4)impermeable cellular membrane at B: R B
![]() |
(A5) |
Solving the subsidiary equations with the boundary conditions yields:
![]() |
(A6) |
The restriction X1 = X2 must also be applied to Eq. A2 to prevent [cAMP] from approaching infinity as R
0 (Purves, 1976
, 1977
). Using the relationship from Eq. A6 and the restriction that X1 =
X2, Eq. A2 simplifies
to Eq. 3.
We thank Mr. Robert Moats for assistance in performing the Bradford assays, Dr. Lia Faiman for her kind assistance with the biochemical measures, and Drs. Martha Gillette and Jonathan Sweedler for careful reading of the manuscript.
This work was supported by National Institutes of Health (RO1 NS26838) and National Science Foundation (IBN 88-21219).
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